Organelle-Specific Pharmaceutical Nanotechnology

ORGANELLE-SPECIFIC PHARMACEUTICAL NANOTECHNOLOGY Edited by

Volkmar Weissig Gerard G. M. D’Souza

A JOHN WILEY & SONS, INC., PUBLICATION

ffirs.indd iii

7/30/2010 2:20:06 PM

ffirs.indd ii

7/30/2010 2:20:06 PM

ORGANELLE-SPECIFIC PHARMACEUTICAL NANOTECHNOLOGY

ffirs.indd i

7/30/2010 2:20:06 PM

ffirs.indd ii

7/30/2010 2:20:06 PM

ORGANELLE-SPECIFIC PHARMACEUTICAL NANOTECHNOLOGY Edited by

Volkmar Weissig Gerard G. M. D’Souza

A JOHN WILEY & SONS, INC., PUBLICATION

ffirs.indd iii

7/30/2010 2:20:06 PM

Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. ISBN 978-0-470-63165-2 Library of Congress Cataloging-in-Publication Data is available. Printed in the United States of America 10

ffirs.indd iv

9

8

7

6

5

4

3

2

1

7/30/2010 2:20:06 PM

CONTENTS

Preface

ix

Contributors

xi

1. An Introduction to Subcellular Nanomedicine: Current Trends and Future Developments

1

Gerard G. M. D’Souza and Volkmar Weissig

2. Delivery of Nanonsensors to Measure the Intracellular Environment

15

Paul G. Coupland and Jonathan W. Aylott

3. Cytoplasmic Diffiusion of Dendrimers and Dendriplexes

35

Alexander T. Florence and Pakatip Ruenraroengsak

4. Endocytosis and Intracellular Trafficking of Quantum Dot– Ligand Bioconjugates

55

Tore-Geir Iversen, Nadine Frerker, and Kirsten Sandvig

5. Synthesis of Metal Nanoparticle-Based Intracellular Biosensors and Therapeutic Agents

73

Neil Bricklebank

6. Subcellular Fate of Nanodelivery Systems

93

Dusica Maysinger, Sebastien Boridy, and Eliza Hutter

7. Intracellular Fate of Plasmid DNA Polyplexes

123

Kevin Maier and Ernst Wagner

8. Intracellular Trafficking of Membrane Receptor-Mediated Uptake of Carbon Nanotubes

143

Bin Kang and Yaodong Dai

9. Real-Time Particle Tracking for Studying Intracellular Transport of Nanotherapeutics

161

Clive Chen and Junghae Suh

10. Tracking Intracellular Polymer Localization Via Fluorescence Microscopy

177

Simon C. W. Richardson v

ftoc.indd v

8/2/2010 11:18:04 AM

vi

CONTENTS

11. Can QSAR Models Describing Small-Molecule Xenobiotics Give Useful Tips for Predicting Uptake and Localization of Nanoparticles in Living Cells? And If Not, Why Not?

193

Richard W. Horobin

12. Self-Unpacking Gene Delivery Scaffolds

207

Millicent O. Sullivan

13. Cellular Trafficking of Dendrimers

231

Yunus Emre Kurtoglu and Rangaramanujam M. Kannan

14. Endolysosomolytically Active pH-Sensitive Polymeric Nanotechnology

247

Han Chang Kang and You Han Bae

15. Uptake and Intracellular Dynamics of Proteins Internalized by Cell-Penetrating Peptides

263

Arwyn T. Jones

16. Cargo Transport by Teams of Molecular Motors: Basic Mechanisms for Intracellular Drug Delivery

289

Melanie J. I. Müller, Florian Berger, Stegan Klumpp, and Reinhard Lipowsky

17. The Potential of Photochemical Internalization (PCI) for the Cytosolic Delivery of Nanomedicines

311

Kristian Berg, Anette Weyergang, Anders Høgset, and Pål Kristian Selbo

18. Peptide-Based Nanocarriers for Intracellular Delivery of Biologically Active Proteins

323

Seong Loong Lo and Shu Wang

19. Organelle-Specific Pharmaceutical Nanotechnology: Active Cellular Transport of Submicro- and Nanoscale Particles

337

Galya Orr

20. Subcellular Targeting of Virus-Envelope-Coated Nanoparticles

357

Jia Wang, Mohammad F. Saeed, Andrey A. Kolokoltsov, and Robert A. Davey

21. Mitochondria-Targeted Pharmaceutical Nanocarriers

385

Volkmar Weissig and Gerard G.M. D’Souza

22. Cell-Penetrating Peptides for Cytosolic Delivery of Biomacromolecules

403

Camilla Foged, Xiaona Jing, and Hanne Moerck Nielsen

23. Therapeutic Nano-object Delivery to Subdomains of Cardiac Myocytes

433

Valeriy Lukyanenko

ftoc.indd vi

8/2/2010 11:18:04 AM

CONTENTS

24. Design Parameters Modulating Intracellular Drug Delivery: Anchoring to Specific Cellular Epitopes, Carrier Geometry, and Use of Auxiliary Pharmacological Agents

vii

449

Silvia Muro and Vladimir R. Muzykantov

25. Uptake Pathways Dependent Intracellular Trafficking of DNA Carrying Nanodelivery Systems

475

Ikramy A. Khalil, Yuma Yamada, Hidetaka Akita, and Hideyoshi Harashima

26. Cellular Interactions of Plasmon-Resonant Gold Nanorods

507

Qingshan Wei and Alexander Wei

27. Quantum Dot Labeling for Assessment of Intracellular Trafficking of Therapeutically Active Molecules

535

Diane J. Burgess and Mamta Kapoor

Index

ftoc.indd vii

569

8/2/2010 11:18:04 AM

ftoc.indd viii

8/2/2010 11:18:04 AM

PREFACE

Pharmaceutical nanotechnology as applied to the level of cell organelles, that is, to the subcellular level, is emerging as a new field in biomedical research. While efforts aiming at the targeted delivery of biologically active molecules date back to the early 1970s, the successful delivery of such agents to particular cell organelles has only in recent times gained broader recognition. Without exaggeration it can be stated that the subcellular organelle-specific targeting of therapeutic and diagnostic agents has become the new frontier for drug delivery. A variety of pharmaceutical nanocarrier platforms like liposomes, carbon nanotubes, quantum dots, micelles, and dendrimers have undergone testing for their ability to control the subcellular disposition of drugs. Potential improvements in therapy through the use of organelle-targeted nanocarriers have been demonstrated. Two major strategies are the basis for achieving organelle-specific targeting of pharmaceutical nano carriers. The first is based on the inherent predisposition of the nanocarrier for a particular cellular compartment and the second is based on an appropriate surface modification of the carrier with organelle-specific ligands. Despite encouraging preliminary findings, or maybe even because of them, many questions begin to emerge or still remain unanswered. Principally, our knowledge about the cellular internalization of nanocarriers and about the impact of intracellular morphology and dynamics on the fate and disposition of nanocarriers is very limited. Do nanocarriers remain intact upon cell entry and subsequent disposition? Naturally, significant differences between solid nanoparticles and liposomes can be expected. What is the impact of size and architecture of nanoparticles on their intracellular trafficking and distribution? Can the drug release from nanocarriers be controlled in a timely and spatial manner? Can or do cells actively transport nanocarriers in cell-membrane-derived vesicles? Can intracellular membrane trafficking be utilized for organelle-specific drug delivery? How do nanocarriers interact with different organelles? Do liposomal phospholipids become part of cellular vesicles? If so, what then is the fate of the lipophilic drugs originally incorporated into liposomal membranes? Can liposomes fuse with organellar membranes such as the mitochondrial outer or inner membrane? How do nonbiodegradable nanocarriers affect cellular transport and metabolism? Our ability to answer those and many more questions largely depends on technological advances in imaging technology. It can be hoped that improvements in real-time fluorescence confocal microscopy of ix

fpref.indd ix

7/30/2010 2:20:08 PM

x

PREFACE

live cells as well as the emergence of new imaging techniques together with the design of novel nanoprobes will help to more satisfactorily answer some of the questions raised above. This book is the result of our efforts to bring together the best of current knowledge in this new and emerging field of subcellular pharmaceutical nanotechnology. All chapters were written by leading experts in their particular fields, and we are extremely grateful to them for having spent part of their valuable time to contribute to this book. It is our hope that together we have succeeded in providing an essential source of state-of-the-art knowledge for any investigator, young and seasoned alike, whose research area involves the application of new nanotechnology to the subcellular level. VOLKMAR WEISSIG Glendale, Arizona GERARD G. M. D’SOUZA Boston, Massachusetts

fpref.indd x

7/30/2010 2:20:08 PM

CONTRIBUTORS

Hidetaka Akita, Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan and CREST, Japan Science and Technology Agency (JST), Japan Jonathan W. Aylott, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom You Han Bae, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah Kristian Berg, Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway Florian Berger, Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany Sebastian Boridy, Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada Neil Bricklebank, Faculty of Health and Wellbeing, Biomedical Research Center, Sheffield Hallam University, Sheffield, United Kingdom Diane Burgess, School of Pharmacy, University of Connecticut, Storrs, Connecticut Clive Chen, Department of Bioengineering, Rice University, Houston, Texas Paul G. Coupland, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom Gerard G. M. D’Souza, Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts Yaodong Dai, Nanjing University of Aeronautics and Astronautics, Nanjing, Peoples Republic of China Robert A. Davey, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas Alexander T. Florence, School of Pharmacy, University of London, London, United Kingdom xi

flast.indd xi

7/30/2010 2:20:07 PM

xii

CONTRIBUTORS

Camilla Foged, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark Nadine Frerker, Centre for Cancer Biomedicine, Faculty Division, Norwegian Radium Hospital, University of Oslo and Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Hideyoshi Harashima, Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido, University, Hokkaido, Japan and CREST, Japan Science and Technology Agency (JST), Japan Anders Hgset, PCI Biotech AS, Oslo, Norway Richard W. Horobin, Division of Integrated Biology, FBLS, The University of Glasgow, Glasgow, Scotland, United Kingdom Eliza Hutter, Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada Tore-Geir Iversen, Centre for Cancer Biomedicine, Faculty Division, Norwegian Radium Hospital, University of Oslo, and Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Xiaona Jing, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark Arwyn T. Jones, Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, United Kingdom Bin Kang, Nanjing University of Aeronautics and Astronautics, Nanjing, Peoples Republic of China Han Chang Kang, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah Rangaramanujam M. Kannan, Department of Chemical Engineering and Materials Science, NICHD Perinatology Research Branch, Wayne State University, Detroit, Michigan Mamta Kapoor, School of Pharmacy, University of Connecticut, Storrs, Connecticut Ikramy A. Khalil, Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido, University, Hokkaido, Japan and CREST, Japan Science and Technology Agency (JST), Japan Stefan Klumpp, Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

flast.indd xii

7/30/2010 2:20:07 PM

CONTRIBUTORS

xiii

Andrey A. Kolokoltsov, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas Yunus Emre Kurtoglu, Department of Chemical Engineering and Materials Science, NICHD Perinatology Research Branch, Wayne State University, Detroit, Michigan Reinhard Lipowsky, Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany Seong Loong Lo, Institute of Bioengineering and Nanotechnology, Singapore Valeriy Lukyanenko, Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland Kevin Maier, Pharmaceutical Biotechnology, Munich Center for SystemBased Drug Research, and Center for NanoScience, Ludwig-MaximiliansUniversität, Munich, Germany Dusica Maysinger, Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada Melanie J. I. Müller, Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany Silvia Muro, Center for Biosystems Research, University of Maryland Biotechnology Institute and Fischell Department of Bioengineering, University of Maryland, College Park, Maryland Vladimir R. Muzykantov, Department of Pharmacology and Targeted Therapeutics Program of Institute of Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Hanne Moerck Nielson, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark Galya Orr, Chemical and Materials Sciences Division, Pacific Northwest National Laboratory, Richland, Washington Simon C. W. Richardson, University of Greenwich School of Science, Kent, England Pakatip Ruenraroengsak, Lung Cell Biology, Respiratory Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom Mohammad F. Saeed, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas Kirsten Sandvig, Centre for Cancer Biomedicine, Faculty Division, Norwegian Radium Hospital, University of Oslo, and Department of Biochemistry,

flast.indd xiii

7/30/2010 2:20:07 PM

xiv

CONTRIBUTORS

Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Pål Kristian Selbo, Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway Junghae Suh, Department of Bioengineering, Rice University, Houston, Texas Millicent O. Sullivan, Department of Chemical Engineering, University of Delaware, Newark, Delaware Ernst Wagner, Pharmaceutical Biotechnology, Munich Center for SystemBased Drug Research, and Center for NanoScience, Ludwig-MaximiliansUniversität, Munich, Germany Jia Wang, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas Shu Wang, Institute of Bioengineering and Nanotechnology, and Department of Biological Sciences, National University of Singapore, Singapore Alexander Wei, Department of Chemistry, Purdue University, West Lafayette, Indiana Qingshan Wei, Department of Chemistry, Purdue University, West Lafayette, Indiana Volkmar Weissig, Department of Pharmaceutical Sciences, Midwestern University College of Pharmacy, Glendale, Arizona Anette Weyergang, Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway Yuma Yamada, Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan and CREST, Japan Science and Technology Agency (JST), Japan

flast.indd xiv

7/30/2010 2:20:07 PM

CHAPTER 1

An Introduction to Subcellular Nanomedicine: Current Trends and Future Developments GERARD G. M. D’SOUZA Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts

VOLKMAR WEISSIG Department of Pharmaceutical Sciences, Midwestern University College of Pharmacy Glendale, Glendale, Arizona

Drug therapy is based largely on the paradigm that an ideal drug will selectively exert a desired pharmacological activity free from negative side effects to modulate either the symptoms or the underlying biochemical cause of a disease to provide a benefit to the patient. In order to have such selective action, the drug molecule should ideally interact with only the disease-associated biochemical pathway but have no activity with respect to any normal biochemical pathway. This principle was famously explored by Paul Ehrlich in his search for agents with selective toxicity toward bacteria. Ehrlich’s work is widely accepted to have given rise to the concept of the ideal drug molecule as a “magic bullet,” a term that he used for the first time in his Harben Lectures [1]. Finding such selective molecules is relatively easy when there are significant differences between the disease-causing process and normal human biochemical pathways, as in the case of infectious diseases. Not surprisingly, in the decades since Ehrlich’s work infectious diseases have become much easier to treat but it does bear consideration that the lack of activity in nondisease cells is dose dependent and not absolute. Most drugs that are considered to be selectively toxic to invading pathogens are in fact toxic to human cells as well but just at higher doses. However, given that the new challenges in drug therapy lie in the treatment of diseases associated with malfunctions Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

1

c01.indd 1

7/30/2010 2:17:25 PM

2

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

of normal human biochemical pathways in certain tissues, the concept of the magic bullet perhaps needs to be redefined or at least clarified. In the infectious disease example it is essential to understand that the so called magic bullet did not necessarily have to home in on the disease agent but could in fact accumulate to the same level in both host and pathogen cells. Just as long as the agent was toxic only to the pathogen it was considered by many to be a magic bullet. In fact, Ehrlich’s Nobel Prize Lecture from December 11, 1908 speaks of “outlining the principles of selective toxicity” rather than selective accumulation. Therefore one could argue that for a molecule to be a magic bullet, it does not have to selectively accumulate at its intended site of action but just that it should not exert its action anywhere but at that intended site of action. This is different from what is now referred to as drug targeting. The term “targeting” is most often meant to imply that the molecule is in some way able to selectively accumulate at an intended site of action and that the selective accumulation is associated with its selective action as a magic bullet. Unfortunately (perhaps due to the widespread use of the noun target to describe a potential molecular site of action), there is often the misconception that a drug that is believed to act at a molecular target (noun) is by default also able to target (verb) or “home in” on that target (noun). It would therefore be more appropriate to define a true magic bullet as a drug molecule that is specific in its activity for a molecular target but that is also able to selectively accumulate at this molecular target and exert a selective therapeutic action by virtue of both its specific activity and its selective accumulation. This distinction is important when we consider the daunting challenge of developing magic bullets for diseases like cancer and neurodegenerative diseases like Alzheimer’s, as well as hormone imbalance diseases like diabetes that are becoming more widespread. Unless unique molecular targets found exclusively (or at sufficiently higher levels) in the diseased state and not in normal state are discovered, magic bullets by the traditional definition or the compromise of dose-dependent activity at the site of action may not be feasible. Strategies to effectively control the disposition of drug molecules thus represent an important tool for successful therapy. At a very basic level, selective accumulation is influenced by bioavailability and subsequent biodistribution. In the context of drug molecules, biodistribution is primarily related to physicochemical properties. Many potent drug candidates exhibit low bioavailability due to their limited water solubility. On the other hand, water-soluble compounds display a very limited ability to cross biological membranes, which essentially can exclude them from the cell interior. To overcome the limitations that a compound’s physicochemical nature can impose on its potential pharmaceutical application, the process of largescale screening of chemical libraries has been extended beyond just identifying desired bioactivity. Screening approaches routinely incorporate selection for desirable physicochemical properties that might confer high bioavailability as well. On the down side, this approach leads to many potent molecules being

c01.indd 2

7/30/2010 2:17:25 PM

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

3

excluded from further development because they aren’t true magic bullets; that is, while they may have a potent pharmacological action at a desired molecular target they aren’t able to find their way exclusively to that target. There is most certainly a growing list of such molecules that are in essence potential drugs if only a delivery strategy can be devised to get them to their molecular target in the human body. As the search for the perfect magic bullet continues there is a significant effort to improve the action of currently available molecules by using targeted delivery approaches. It is not surprising that the field of drug targeting has grown significantly in the effort to develop better therapy. Based on the experience over the decades since Ehrlich first introduced the magic bullet concept, it seems more reasonable to separate the functions of pharmacological action and selective accumulation into properties desired in a drug molecule and in a delivery system, respectively, rather than the traditional expectation that the drug molecule alone possess both properties. Generally, drug disposition may be modulated via three broad approaches First, the drug molecule might be modified subtly to change its physicochemical properties without adversely affecting its inherent pharmacological action. This is essentially the intent of medicinal chemistry approaches and the concept of structure–activity relationship (SAR) studies that have now become standard practice and often aren’t even considered a means of achieving targeting. The second approach might be considered to be an extension of the first but is different in that it involves using chemistry to conjugate ligands that are often larger than simple organic functional groups to change the biodistribution of a molecule. Again this approach works as long as the conjugation does not adversely affect the desired pharmacological activity of the molecule. Conjugation using selectively cleavable linkers is an extension of this strategy. The third strategy involves the use of some sort of delivery system or a carrier system and does not involve chemical modifications to the pharmacologically active molecule. Pharmaceutical nanocarriers fall into this category and several such technologies are being developed that are fast becoming applicable to a variety of pharmacologically active molecules. Pharmaceutical nanocarriers offer what might be viewed as a nonchemical approach to modify the disposition of drug molecules. All chemistry can be performed on the components of the nanocarrier system that can then be loaded with the drug to afford targeted delivery [2–5]. Most pharmaceutical nanocarriers can be modified for some level of targeting to specific tissues if not specific cell types. Long circulating liposomes and nanoparticles are able to passively target areas of leaky vasculature by virtue of the EPR effect and can additionally be modified with antibodies or other targeting ligands to afford cell specific recognition [6–10]. However, despite such advances, the improvement in drug action is not always dramatic. This is likely because many drugs act at molecular targets inside the cells and these molecular targets are often in well-organized subcellular structures inside the mammalian cell.

c01.indd 3

7/30/2010 2:17:25 PM

4

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

The interior of a cell is very different from an aqueous buffer solution, in which small drug molecules can freely diffuse and randomly interact with potential cosolutes. In addition to the presence of the cytoskeletal network and various dispersed organelles, the cytoplasm contains a large amount of dissolved macromolecules. The concentration of dissolved macromolecules in the nucleoplasm and cytoplasm of living cells has been determined to be between 50 and 400 g/L [11, 12]. Subsequently, transport or diffusion events in such a crowded solution cannot be expected to be the same as those in buffer solutions. Generally, intracellular diffusion has been characterized as hindered diffusion, reflecting among other factors the high level of molecular crowding [13, 14]. Additionally, the fluid-phase viscosity of the cytoplasm and binding to intracellular components are believed to influence the diffusion of solutes inside a cell [15, 16]. While efforts aimed at thoroughly understanding cellular material properties such as cytoplasmic viscosity are currently underway [17], it is generally accepted that the physicochemical properties of the drug also play a major role in determining the subcellular fate of the drug molecule. Consequently, the ability to predict the influence of various properties of the drug molecule on the likely site of accumulation within the cell could prove to be a powerful tool in drug design to either select molecules with a desired subcellular accumulation or identify molecules that would benefit from subcellular targeting strategies. There is apparent fractal symmetry between the case of drug delivery to a cell and drug delivery to a molecular target inside a subcellular compartment. The cell could be viewed as being a small, slightly simpler but nonetheless highly organized “body” with “organs” (organelles) and “cells” (defined structures and molecular arrangements) within these organs. It should therefore stand to reason that controlling drug disposition within the cell might also be necessary for optimal drug action [18–27]. Consequently, the next logical step in the development of targeted nanocarriers would be to extend our control over nanocarrier distribution to the subcellular level as well. At least two major schemes can be imagined to be useful in the design of nanocarriers with the potential for subcellular targeting: the first based on the inherent predisposition of the nanocarrier for a particular compartment and the second based on attaching subcellular targeting ligands to the surface of nanocarriers to redirect their accumulation to the desired compartment. Essential to the latter of these approaches is the use of a subcellular targeting ligand. Such ligands could be, as in the case of leader sequences, derived from normal cellular trafficking processes or, as in the case of triphenyl phosphonium, based on observations of a predisposition of an organic compound for subcellular compartments. The availability of a wide range of subcellular stains is proof enough that there are several molecules with an inherent ability to accumulate in a particular subcellular compartment. Based on the intracellular distribution of a large variety of fluorophores, a quantitative structure–activity relationship (QSAR) model for predicting cellular uptake and intracellular distribution of low molecular weight compounds

c01.indd 4

7/30/2010 2:17:25 PM

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

5

has been proposed [28]. This QSAR approach was recently applied to identify potential common chemical features of molecules that are known to selectively accumulate at or inside mammalian mitochondria within living cells [29]. The QSAR approach has additionally proved useful for the modeling of cationic transfection lipids [30] and could therefore be applicable to predicting the subcellular disposition of a potential therapeutic molecule and even to design molecules with a desired subcellular affinity for the development of subcellular targeting approaches. Approaches to nanocarrier-mediated subcellular delivery are based on the principle that the subcellular destination of a drug is the same as that of the nanocarrier. Nanocarriers by virtue of their particulate nature are believed to be subject to endocytic cell entry mechanisms and subsequent endolysosomal processing. As such, nanocarriers could be considered to be ideally suited for delivering bioactive molecules to the endolysosomal system. Indeed, directing nanomedicine complexes to the endolysosomal system has increasingly gained attention, as pathological conditions associated with endosomes and lysosomes could potentially benefit from therapies targeting these pathways [31– 34]. Although endocytosis is a common mechanism that almost all cells possess for the internalization of macromolecules, a wide array of such vesicular internalization mechanisms exist [31]. For example, nanoscale drug carrier systems taken up by clathrin-dependent receptor-mediated endocytosis (RME) are most likely to undergo lysosomal degradation, while clathrin-independent RME may lead to endosomal accumulation [31]. Consequently, the type of targeting moiety displayed by the nanocarrier system will determine whether the carrier delivers its cargo to either endosomes or lysosomes. Well-characterized endocytic targeting moieties potentially useful for nanocarrier-mediated drug delivery are folic acid, low-density lipoprotein, cholera toxin B, mannose-6-phosphate, transferrin, riboflavin, the tripeptide RGD, ICAM-1 antibody, and nicotinic acid, as recently reviewed by Bareford and Swaan [31]. The cellular internalization mechanisms utilized by these ligands involve clathrin-dependent RME, caveolin-assisted endocytosis, lipid raft associated endocytosis, and cell adhesion molecule (CAM) directed cellular uptake [31]. In addition to several approaches to exploiting the inherent tendency of nanoparticles to accumulate in the endolysosomal compartment for possible therapeutic purposes, there is a growing body of work that suggests the feasibility of modifying nanocarriers to redirect delivery of their cargo to other subcellular compartments as well. Liposomes modified with mitochondriotropic ligands have been shown to improve the efficacy of an anticancer drug both in vitro and in vivo [35]. AuNPs have already been targeted to the nucleus using the adenoviral nuclear localization signal (NLS) and integrin binding domain [36]. Such an approach has been reported to be useful in the development of probes for cell tracking by surface-enhanced Raman scattering [37]. Modification with a leader sequence peptide has also been applied to creating delivery systems for mitochondria. A mitochondrial leader peptide

c01.indd 5

7/30/2010 2:17:25 PM

6

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

(MLP), derived from the nucleocytosol expressed but mitochondria localized ornithine transcarbamylase, has been reported to render polyethylene imine (PEI) mitochondriotropic and represents a potential approach for mitochondrial DNA delivery [38]. It is interesting to note that the examples discussed above share a common assumption. Nanocarriers are assumed to have a predisposition for the endolysosomal pathway by virtue of their nanometer size and, without a subcellular targeting ligand, all nanocarriers would remain in the endolysosomal compartment. However, it is interesting to also consider the disposition of a nanocarrier made exclusively of a molecule with a predisposition for a subcellular compartment. A good example is the mitochondriotropic amphiphile dequalinium chloride. A serendipitous discovery while screening mitochondriotropic drugs potentially able to interfere with the mitochondrial DNA metabolism in Plasmodium falciparum [39] revealed this self-associating tendency of dequalinium chloride and its ability to form vesicles. At the time of their discovery, these unusual vesicles were termed DQAsomes (pronounced dequasomes), that is, dequalinium (DQA) based liposome-like vesicles [40]. Based on the fact that these carriers were composed exclusively of mitochondriotropic molecules and that they were able to bind and protect DNA, DQAsomes were explored as potential mitochondria-specific DNA delivery vehicles for direct mitochondrial gene therapy [41–44]. DQAsomes have also been explored as a mitochondria-targeted nanocarrier system for small drug molecules, in particular, for anticancer drugs known to trigger apoptosis via direct action on mitochondria [45, 46]. It would therefore appear that, for now, a basic proof of concept for an alternative strategy toward the design of subcellular targeting nanocarriers seems to have been established. It is also obvious that in order to design similar carriers for other subcellular compartments it would be necessary to first find self-assembling molecules with an affinity for the intended subcellular compartment. To this end, recent work on the subcellular distribution of micelle-forming agents offers some interesting insights [47–51]. Based on the examples discussed so far, it would seem that there is indeed hope that nanocarrier systems could be designed to achieve true molecular level targeting inside cells. However, to say that these systems will be available soon is perhaps premature given what little we know about the subcellular dynamics associated with nanoparticle trafficking. There are in our opinion several unanswered questions. For example, do all nanocarriers remain intact upon cell entry and subsequent disposition? Are there differences in the disposition of vesicles in comparison to particles? What is the true influence of size on the intracellular disposition of various nanocarriers? Most important, however, is the question of the mechanism by which the nanocarrier is able to achieve selective uptake and delivery into the subcellular compartment. All the strategies described so far report observations of altered or improved subcellular accumulation that appears to result in improved activity, but how exactly this happens is still unclear. Do the nanocarriers remain intact upon

c01.indd 6

7/30/2010 2:17:25 PM

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

7

internalization and then get trafficked as intact structures? If so, how is the therapeutic cargo released to the correct subcellular compartment? Alternatively, it could be imagined that once taken up into the early endosomal vesicle, the nanocarrier components undergo a redistribution to become part of the endosomal vesicle. There is some evidence to suggest that in fact cells actively traffic nanocarriers in cell membrane-derived vesicles [52]. Assuming the targeting ligand was able to redistribute to the surface of the endosomal vesicle, it might be possible then that the vesicle would have an altered subcellular fate that could involve transport to and association with a target compartment other than the lysosome. While this may seem to be farfetched speculation, there has already been some work along similar lines toward the development of nanocarrier systems for delivery of molecules to the nucleus and even the mitochondria. A strategy that involved stepwise membrane fusion was devised based on the premise that, to efficiently deliver DNA to the nucleus, a delivery system must penetrate through the plasma membrane, nuclear envelope, prior to DNA release in the nucleus. Using a multilayered nanoparticle called a Tetralamellar Multifunctional Envelopetype Nano Device (T-MEND) and consisting of a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion, transgene expression in nondividing cells was reported to be dramatically increased [53]. A similar approach in designing a mitochondria-specific delivery sytem has been reported as well. Liposomal carriers called MITO-Porters, which carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis) were prepared with lipid compositions that were identified in various experiments to promote both fusion with the mitochondrial membrane and the release of liposomal cargo to the intramitochondrial compartment in living cells. Using GFP protein as a model cargo, it was shown that MITO-Porter liposomes are able to selectively deliver their cargo to mitochondria [54, 55]. It is also interesting to note that changes in nanoparticle architecture result in changes in subcellular disposition [56]. Fluorescein isothiocyanate labeled layered double hydroxide (LDH) nanoparticles were prepared from Mg2Al under conditions that yielded either hexagonal sheets (50–150 nm wide and 10–20 nm thick) or nanorods (30–60 nm wide and 100–200 nm long). A comparison of the subcellular distribution of these two types of preparations revealed that the nanorods trafficked to the nucleus but the hexagonal sheets remained in the cytoplasm [56]. Not surprisingly, an active microtubule mediated transport process is hypothesized to be responsible for the observed rapid nuclear accumulation of the nanorods [56]. As discussed so far, various nanocarrier platforms have already undergone preliminary investigation for their ability to control the subcellular disposition of drugs and a potential improvement in therapy through the use of nanocarriers for subcellular targeting of bioactive molecules. A common paradigm that currently seems to apply to most of these approaches is the use of

c01.indd 7

7/30/2010 2:17:25 PM

8

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

subcellular targeting ligands to control subcellular distribution. Given the relative ease of modifying nanocarriers with various surface functionalities and the fact that such approaches are already in use to achieve targeting at the cellular and organ levels, the ligand-based approach does seem to be a logical extension of current technology. It helps that new tools are concurrently being investigated to understand some of the physicochemical aspects of how small molecules [28, 29] as well as proteins [57] are able to selectively accumulate in certain subcellular compartments to afford the rational design of a wide repertoire of subcellular targeting ligands. However, one must be cautious in the knowledge that the approaches described so far are based only on current understanding of subcellular trafficking. Current knowledge of subcellular trafficking processes is based largely on studies with solid nanoparticles and on quantum dots [58–64]. Whether these observations can be extended to vesicular carriers like liposomes and micelles remains in question and in our opinion is due in large part to current limitations in imaging technology. We are however hopeful that technological advances in real-time fluorescence confocal imaging of live cells [65–68], as well as the emergence of new imaging techniques like total internal reflection microscopy [69] and label-free approaches like Raman microscopy [70–72], will allow some of the questions raised to be more satisfactorily answered. This book is our attempt to bring the best of current knowledge together to provide a comprehensive resource for anyone interested in this emerging area of drug delivery. The subsequent chapters discuss in full detail the current state of the art in the various approaches to nanocarrier-mediated bioactives to cell organelles as well as emerging research methods for the identification of subcellular targeting ligands and the study of subcellular transport processes.

REFERENCES 1. Ehrlich, P. Experimental Researches on Specific Therapy. On Immunity with Special Reference to the Relationship Between Distribution and Action of Antigens. London: Royal Institute of Public Health, 1908, p. 107. 2. Ganta, S., Devalapally, H., Shahiwala, A., and Amiji, M. A review of stimuliresponsive nanocarriers for drug and gene delivery. J. Control. Release 126: 187– 204 (2008). 3. Jabr-Milane, L., Van Vlerken, L., Devalapally, H., Shenoy, D., Komareddy, S., Bhavsar, M., and Amiji, M. Multi-functional nanocarriers for targeted delivery of drugs and genes. J. Control. Release 130: 121–128 (2008). 4. Mishra, B., Patel, B. B., and Tiwari, S. Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery. Nanomedicine 6(1): 9–24 (2010). 5. Torchilin, V. P. Targeted pharmaceutical nanocarriers for cancer therapy and imaging. AAPS J. 9: E128–147 (2007).

c01.indd 8

7/30/2010 2:17:25 PM

REFERENCES

9

6. Ma, L., Liu, Y. L., Ma, Z. Z., Dou, H. L., Xu, J. H., Wang, J. C., Zhang, X., and Zhang, Q. Targeted treatment of choroidal neovascularization using integrinmediated sterically stabilized liposomes loaded with combretastatin A4. J. Ocul. Pharmacol. Ther. 25: 195–200 (2009). 7. Hatakeyama, H., Akita, H., Ishida, E., Hashimoto, K., Kobayashi, H., Aoki, T., Yasuda, J., Obata, K., Kikuchi, H., Ishida, T., Kiwada, H., and Harashima, H. Tumor targeting of doxorubicin by anti-MT1-MMP antibody-modified PEG liposomes. Int. J. Pharm. 342: 194–200 (2007). 8. Nallamothu, R., Wood, G. C., Kiani, M. F., Moore, B. M., Horton, F. P., and Thoma, L. A. A targeted liposome delivery system for combretastatin A4: formulation optimization through drug loading and in vitro release studies. PDA J. Pharm. Sci. Technol. 60: 144–155 (2006). 9. Frisch, B., Carriere, M., Largeau, C., Mathey, F., Masson, C., Schuber, F., Scherman, D., and Escriou, V. A new triantennary galactose-targeted PEGylated gene carrier, characterization of its complex with DNA, and transfection of hepatoma cells. Bioconjug. Chem. 15: 754–764 (2004). 10. Torchilin, V. P., Levchenko, T. S., Lukyanov, A. N., Khaw, B. A., Klibanov, A. L., Rammohan, R., Samokhin, G. P., and Whiteman, K. R. p-NitrophenylcarbonylPEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups. Biochim. Biophys. Acta. 1511: 397–411 (2001). 11. Minton, A. P. How can biochemical reactions within cells differ from those in test tubes? J. Cell Sci. 119: 2863–2869 (2006). 12. Ellis, R. J. and Minton, A. P. Cell biology: join the crowd. Nature 425: 27–28 (2003). 13. Sanabria, H., Kubota, Y., and Waxham, M. N. Multiple diffusion mechanisms due to nanostructuring in crowded environments. Biophys. J. 92: 313–322 (2007). 14. Goins, A. B., Sanabria, H., and Waxham, M. N. Macromolecular crowding and size effects on probe microviscosity. Biophys. J. 95: 5362–5373 (2008). 15. Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275: 1625–1629 (2000). 16. Seksek, O., Biwersi, J., and Verkman, A. S. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138: 131–142 (1997). 17. Weiss, M. Probing the interior of living cells with fluorescence correlation spectroscopy. Ann. N. Y. Acad. Sci. 1130: 21–27 (2008). 18. Duvvuri, M. and Krise, J. P. Intracellular drug sequestration events associated with the emergence of multidrug resistance: a mechanistic review. Front. Biosci. 10: 1499–1509 (2005). 19. Breunig, M., Bauer, S., and Goepferich, A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm. 68: 112–128 (2008). 20. Torchilin, V. P. Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu. Rev. Biomed. Eng. 8: 343–375 (2006). 21. Li, S. D. and Huang, L. Pharmacokinetics and biodistribution of nanoparticles. Mol. Pharm. 5: 496–504 (2008). 22. Kaufmann, A. M. and Krise, J. P. Lysosomal sequestration of amine-containing drugs: analysis and therapeutic implications. J. Pharm. Sci. 96: 729–746 (2007).

c01.indd 9

7/30/2010 2:17:25 PM

10

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

23. Panyam, J. and Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55: 329–347 (2003). 24. Weissig, V. Mitochondrial-targeted drug and DNA delivery. Crit. Rev. Ther. Drug Carrier Syst. 20: 1–62 (2003). 25. Weissig, V. Targeted drug delivery to mammalian mitochondria in living cells. Expert Opin. Drug Deliv. 2: 89–102 (2005). 26. Weissig, V., Boddapati, S. V., Jabr, L., and D’Souza, G. G. Mitochondria-specific nanotechnology. Nanomedicine 2: 275–285 (2007). 27. Weissig, V., Cheng, S. M., and D’Souza, G. G. Mitochondrial pharmaceutics. Mitochondrion 3: 229–244 (2004). 28. Horobin, R. W. Uptake, distribution and accumulation of dyes and fluorescent probes within living cells: a structure-activity modelling approach. Adv. Colour. Sci. Technol. 4: 101–107 (2001). 29. Horobin, R. W., Trapp, S., and Weissig, V. Mitochondriotropics: a review of their mode of action, and their applications for drug and DNA delivery to mammalian mitochondria. J. Control. Release 121: 125–136 (2007). 30. Horobin, R. W. and Weissig, V. A QSAR-modeling perspective on cationic transfection lipids. 1. Predicting efficiency and understanding mechanisms. J. Gene Med. 7: 1023–1034 (2005). 31. Bareford, L. M. and Swaan, P. W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Deliv. Rev. 59: 748–758 (2007). 32. Gregoriadis, G. and Ryman, B. E. Liposomes as carriers of enzymes or drugs: a new approach to the treatment of storage diseases. Biochem. J. 124: 58P (1971). 33. Castino, R., Demoz, M., and Isidoro, C. Destination “lysosome”: a target organelle for tumour cell killing? J. Mol. Recognit. 16: 337–348 (2003). 34. Tate, B. A. and Mathews, P. M. Targeting the role of the endosome in the pathophysiology of Alzheimer’s disease: a strategy for treatment. Sci. Aging Knowledge Environ. 2006(10): 2 (2006). 35. Boddapati, S. V., D’Souza, G. G., Erdogan, S., Torchilin, V. P., and Weissig, V. Organelle-targeted nanocarriers: specific delivery of liposomal ceramide to mitochondria enhances its cytotoxicity in vitro and in vivo. Nano. Lett. 8: 2559–2563 (2008). 36. Tkachenko, A. G., Xie, H., Liu, Y., Coleman, D., Ryan, J., Glomm, W. R., Shipton, M. K., Franzen, S., and Feldheim, D. L. Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjug. Chem. 15: 482–490 (2004). 37. Xie, W., Wang, L., Zhang, Y., Su, L., Shen, A., Tan, J., and Hu, J. Nuclear targeted nanoprobe for single living cell detection by surface-enhanced Raman scattering. Bioconjug. Chem. 20: 768–773 (2009). 38. Lee, M., Choi, J. S., Choi, M. J., Pak, Y. K., Rhee, B. D., and Ko, K. S. DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine. J. Drug Target. 15: 115–122 (2007). 39. Rowe, T. C., Weissig, V., and Lawrence, J. W. Mitochondrial DNA metabolism targeting drugs. Adv. Drug Deliv. Rev. 49: 175–187 (2001).

c01.indd 10

7/30/2010 2:17:25 PM

REFERENCES

11

40. Weissig, V., Lasch, J., Erdos, G., Meyer, H. W., Rowe, T. C., and Hughes, J. DQAsomes: a novel potential drug and gene delivery system made from Dequalinium. Pharm. Res. 15: 334–337 (1998). 41. D’Souza, G. G., Boddapati, S. V., and Weissig, V. Mitochondrial leader sequence— plasmid DNA conjugates delivered into mammalian cells by DQAsomes colocalize with mitochondria. Mitochondrion 5: 352–358 (2005). 42. D’Souza, G. G., Rammohan, R., Cheng, S. M., Torchilin, V. P., and Weissig, V. DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells. J. Control. Release 92: 189–197 (2003). 43. Weissig, V., D’Souza, G. G., and Torchilin, V. P. DQAsome/DNA complexes release DNA upon contact with isolated mouse liver mitochondria. J. Control. Release 75: 401–408 (2001). 44. Weissig, V., Lizano, C., and Torchilin, V. P. Selective DNA release from DQAsome/ DNA complexes at mitochondria-like membranes. Drug Deliv. 7: 1–5 (2000). 45. Cheng, S. M., Pabba, S., Torchilin, V. P., Fowle, W., Kimpfler, A., Schubert, R., and Weissig, V. Towards mitochondria-specific delivery of apoptosis-inducing agents: DQAsomal incorporated paclitaxel. J. Drug Deliv. Sci. Tech. 15: 81–86 (2005). 46. D’Souza, G. G., Cheng, S. M., Boddapati, S. V., Horobin, R. W., and Weissig, V. Nanocarrier-assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel. J. Drug Target. 16: 578–585 (2008). 47. Bae, Y., Nishiyama, N., Fukushima, S., Koyama, H., Yasuhiro, M., and Kataoka, K. Preparation and biological characterization of polymeric micelle drug carriers with intracellular pH-triggered drug release property: tumor permeability, controlled subcellular drug distribution, and enhanced in vivo antitumor efficacy. Bioconjug. Chem. 16: 122–130 (2005). 48. Maysinger, D., Lovric, J., Eisenberg, A., and Savic, R. Fate of micelles and quantum dots in cells. Eur. J. Pharm. Biopharm. 65: 270–281 (2007). 49. Savic, R., Azzam, T., Eisenberg, A., and Maysinger, D. Assessment of the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a fluorogenic-based approach. Langmuir 22: 3570–3578 (2006). 50. Savic, R., Azzam, T., Eisenberg, A., Nedev, H., Rosenberg, L., and Maysinger, D. Block-copolymer micelles as carriers of cell signaling modulators for the inhibition of JNK in human islets of Langerhans. Biomaterials 30(21): 3597–3604 (2009). 51. Xiong, X. B., Mahmud, A., Uludag, H., and Lavasanifar, A. Multifunctional polymeric micelles for enhanced intracellular delivery of doxorubicin to metastatic cancer cells. Pharm. Res. 25: 2555–2566 (2008). 52. Ruan, G., Agrawal, A., Marcus, A. I., and Nie, S. Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding. J. Am. Chem. Soc. 129: 14759– 14766 (2007). 53. Akita, H., Kudo, A., Minoura, A., Yamaguti, M., Khalil, I. A., Moriguchi, R., Masuda, T., Danev, R., Nagayama, K., Kogure, K., and Harashima, H. Multilayered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process. Biomaterials 30: 2940–2949 (2009).

c01.indd 11

7/30/2010 2:17:25 PM

12

AN INTRODUCTION TO SUBCELLULAR NANOMEDICINE

54. Yamada, Y. and Harashima, H. Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases. Adv. Drug Deliv. Rev. 60: 1439–1462 (2008). 55. Yamada, Y., Akita, H., Kamiya, H., Kogure, K., Yamamoto, T., Shinohara, Y., Yamashita, K., Kobayashi, H., Kikuchi, H., and Harashima, H. MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim. Biophys. Acta. 1778: 423–432 (2008). 56. Xu, Z. P., Niebert, M., Porazik, K., Walker, T. L., Cooper, H. M., Middelberg, A. P., Gray, P. P., Bartlett, P. F., and Lu, G. Q. Subcellular compartment targeting of layered double hydroxide nanoparticles. J. Control. Release 130: 86–94 (2008). 57. Nakai, K. and Horton, P. Computational prediction of subcellular localization. Methods Mol. Biol. 390: 429–466 (2007). 58. Yacobi, N. R., Malmstadt, N., Fazlollahi, F., Demaio, L., Marchelletta, R., HammAlvarez, S. F., Borok, Z., Kim, K. J., and Crandall, E. D. Mechanisms of alveolar epithelial translocation of a defined population of nanoparticles. Am. J. Respir. Cell. Mol. Biol. 42(5): 604–614 (2009). 59. Hillaireau, H. and Couvreur, P. Nanocarriers entry into the cell: relevance to drug delivery. Cell. Mol. Life Sci. 66(17): 2873–2896 (2009). 60. Smirnov, P. Cellular magnetic resonance imaging using superparamagnetic anionic iron oxide nanoparticles: applications to in vivo trafficking of lymphocytes and cell-based anticancer therapy. Methods Mol. Biol. 512: 333–353 (2009). 61. Harush-Frenkel, O., Altschuler, Y., and Benita, S. Nanoparticle-cell interactions: drug delivery implications. Crit. Rev. Ther. Drug Carrier Syst. 25: 485–544 (2008). 62. Huser, T. Nano-biophotonics: new tools for chemical nano-analytics. Curr. Opin. Chem. Biol. 12: 497–504 (2008). 63. Vasir, J. K. and Labhasetwar, V. Quantification of the force of nanoparticle–cell membrane interactions and its influence on intracellular trafficking of nanoparticles. Biomaterials. 29: 4244–4252 (2008). 64. Rajan, S. S., Liu, H. Y., and Vu, T. Q. Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells. ACS Nano. 2: 1153–1166 (2008). 65. Perrine, K. A., Lamarche, B. L., Hopkins, D. F., Budge, S. E., Opresko, L. K., Wiley, H. S., and Sowa, M. B. High speed method for in situ multispectral image registration. Microsc. Res. Tech. 70: 382–389 (2007). 66. Rabut, G. and Ellenberg, J. Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J. Microsc. 216: 131–137 (2004). 67. Sunaguchi, M., Nishi, M., Mizobe, T., and Kawata, M. Real-time imaging of green fluorescent protein-tagged beta 2-adrenergic receptor distribution in living cells. Brain Res. 984: 21–32 (2003). 68. Jester, J. V., Andrews, P. M., Petroll, W. M., Lemp, M. A., and Cavanagh, H. D. In vivo, real-time confocal imaging. J. Electron Microsc. Tech. 18: 50–60 (1991). 69. Byrne, G. D., Pitter, M. C., Zhang, J., Falcone, F. H., Stolnik, S., and Somekh, M. G. Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles. J. Microsc. 231: 168–179 (2008).

c01.indd 12

7/30/2010 2:17:25 PM

REFERENCES

13

70. Matthaus, C., Kale, A., Chernenko, T., Torchilin, V., and Diem, M. New ways of imaging uptake and intracellular fate of liposomal drug carrier systems inside individual cells, based on Raman microscopy. Mol. Pharm. 5: 287–293 (2008). 71. Fujita, K. and Smith, N. I. Label-free molecular imaging of living cells. Mol. Cells 26: 530–535 (2008). 72. Freudiger, C. W., Min, W., Saar, B. G., Lu, S., Holtom, G. R., He, C., Tsai, J. C., Kang, J. X., and Xie, X. S. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322: 1857–1861 (2008).

c01.indd 13

7/30/2010 2:17:25 PM

c01.indd 14

7/30/2010 2:17:25 PM

CHAPTER 2

Delivery of Nanosensors to Measure the Intracellular Environment PAUL G. COUPLAND and JONATHAN W. AYLOTT School of Pharmacy, University of Nottingham, Nottingham, United Kingdom

2.1

INTRODUCTION

Nanosensors can be internalized in viable cells, providing an optical signal to report on the intracellular environment. This allows an understanding of intracellular function to be gained in real time. Nanosized sensors are especially desirable as they provide an output with minimal cellular disruption, which enhances the quality of data generated from each cell. Chemical perturbation is also minimized by the structural design of a nanosensor; it is these characteristics of nanosized sensing particles that give the potential for observation that is minimally invasive [1–5]. In recent years, new nanosized sensing particles have been demonstrated and reported widely in the literature. Borisov and Klimant [6] provide a good summary for the state of the art of optical nanosensor technology and its increasing use in scientific fields, including biology, biotechnology, and clinical medicine. In this chapter we will focus our discussion on nanosensors of a single polymeric matrix, for example, polyacrylamide or silica sol-gel, and the delivery methods used for intracellular translocation of these nanoparticles. The delivery principles discussed here are transferable to many types of nanoparticle systems currently being researched. The most common technique for intracellular research utilizes fluorescent dyes in combination with confocal microscopy [7]. The small size of the free sensing molecule is a key advantage, providing high spatial resolution and allowing information throughout the cell to be collected en masse. A problem faced when using free dye in direct contact with the intracellular environment of the cell is chemical perturbation due to any cytoxic action of the dye molecules [5]. Equally, the intracellular environment can impact on the efficacy Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

15

c02.indd 15

7/30/2010 2:17:26 PM

16

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

of the sensing molecules [8, 9], where random protein binding and intracellular sequestration are major complications [10] as well as simply retaining the dye within the cell during the lifetime of an experiment [11]. To overcome the analytical difficulties of using free sensing dyes, an alternative approach evolved—optochemical sensors. These are typically a pulled optical fiber with a modified polymeric tip, to entrap sensing dye molecules, inserted directly into the cell [12, 13]. Pulled fiber sensors provide a biocompatible platform, protecting the dye and the cell from one another, and the polymer matrix of the modified tip facilitates complex sensing schemes to be devised by closely packing elements that interact complementarily [14, 15]. However, the large size of the sensor relative to a single cell causes damaging physical perturbation to all but the largest cells. This is due to the physical pathway required to transmit the signal generated at the tip of the sensor to the detector. Additionally, only low spatial resolution is achievable as few optodes can be inserted into a cell before it is damaged irreversibly [10, 16]. Fiber-optic sensors and free-dye molecules have advantages over each other, yet both have the limitations described that prevents them from being ideally suited to single cell intracellular measurement. A new technique that retained the benefits of free dyes (small size, good spatial resolution) and optochemical sensors (biocompatible dye entrapment, complex sensing schemes) that prevented chemical and physical perturbation of the cell was required. The development of PEBBLE (probes encapsulated by biologically localized embedding) nanosensors by Kopelman [1] was the next step in miniaturization of pulled fiber optochemical sensors and removed the fiber-optic component used for signal transmission. Understanding that the tip of the fiber-optic sensor was all that was needed for sensing, and recognizing that fluorescence microscopy was ideally suited to record the fluorescence output, Kopelman undertook to fabricate the same polymer tip as a single unit. Typically sub 200 nm in diameter, these sensors occupy approximately 1 ppb of a typical mammalian cell and cause negligible physical perturbation [4]. Nanosensors resemble the tip of an optode, in that they consist of a polymer matrix surrounding chemical sensing elements; there is no longer a physical signal path—thus being more akin to free dyes—but the protective capacity of a biocompatible polymer matrix is retained. Chemical perturbation to the cell is minimized, as is the effect of the intracellular environment upon the efficiency of the reporter molecule; in essence, the polymer matrix protects the cell from the dye and the dye from the cell [1]. Fluorophores usually affected through random protein binding respond characteristically when entrapped within the polymer matrix of a nanosensor [17, 18]. The matrix also allows for enhanced longevity of the sensing components (by protecting the dye from protein binding) enabling fully reversible sensors to be produced [3]. The nanosensor matrix is a discrete domain in which components making up a more complex sensing regime can be located. This allows for highly selective sensors incorporating ratiometric sensing regimes that use more than one dye [4], or electron transfer regimes using optically silent ionophores, in conjunc-

c02.indd 16

7/30/2010 2:17:26 PM

INTRODUCTION

17

tion with fluorophore partners [15]; significantly, the use of a sensor matrix increases the scope of analysis. Nanosensors retain the spatial resolution advantage of free dyes rather than being limited to the immediate locality of a single optode tip, because many nanosensors can be loaded into a single cell. In brief, a nanosensor is a nanometer-size polymeric spherical matrix in which chemical sensing elements/fluorophores are entrapped. The polymer matrix is porous, allowing diffusion of analytes to interact with the entrapped fluorophores, giving a fluorescent response that is captured with an optical system [19, 20]. By minimizing the chemical effects of free dyes and the physical effects of bulky optodes, a more natural state of the intracellular environment can be investigated [21]. Nanosensors are most commonly fabricated from polyacrylamide [2], silicate sol-gels [5], or liquid polymer matrices [22], each being porous, photostable, optically transparent, and chemically inert. Functionalization of the nanosensor matrix is possible when required, to provide chemical functional groups on the nanosensor surface for subsequent modification and/or conjugation. The variety of matrices available for nanosensor fabrication and their chemical diversity allow for a flexible and modular approach to nanosensor design (Figure 2.1). O NH

2

O

O

O

N

N H

O N H

O

R functional matrix, chemical groups

lon+ polymer matrix, (silica sol-gel, polyacrylamide, liquid polymer)

CPP attachment

NH

O

O

O

N O NO H

O O

O N

N HO

S

NH 2

O

ionic matrix, comonomer additives +/–

SP* Lipid Multi-layer multifunction surface chemistry

O 2 NH

S

Fluorophore(s) (incl. FRET pairing), ionophores, enzymes hv

O

S

hv

O 2

S

O

N

Nanosensor function

Encapsulation in liposomes

PEG CPP *Signal Peptide

Figure 2.1. Nanosensor schematic. (See color insert.)

c02.indd 17

7/30/2010 2:17:26 PM

18

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

The surface of the nanosensor is important due to its involvement in a number of biophysical interactions. Modification of the nanosensor surface with chemical moieties to provide a specific function allows for application to different cell types, delivery methods, and intracellular tasks. Modification of the nanosensor matrix can be achieved, for example, by the inclusion of an amine-modified acrylamide into a polyacrylamide matrix, providing primary amines throughout the nanosensor matrix [17]. Various molecules, from heterobifunctional linkers to phospholipids, can then be conjugated to the nanosensor, providing additional functionality and capability. As with all emerging technologies, continued development and improvements to the processes and protocols are ongoing. For nanosensors, one approach to improve their reporting of intracellular function and condition is by increasing the brightness of the signal emitted from each individual nanosensor. This is important in the realm of delivery and analytical performance, as brighter nanosensors will generate the same amount of signal from a cell with a lower nanosensor loading. This of course reduces the potential for physical disruption to the cell during and after delivery. Alternative strategies for attachment of fluorophores to the nanosensor surface, or entrapping them in the polymer matrix, to yield much brighter nanosensors are being developed. Historically, the most common approach is to physically entrap fluorophores into the polymer matrix. This method typically requires that fluorophores are attached to a large, inert molecule such as dextran, which will prevent subsequent leaching of the entrapped fluorophore from the polymer matrix. An alternative methodology exists for incorporating fluorophores in nanosensors, whereby the fluorophores are covalently attached directly to the backbone of the polymer matrix [23]. This eliminates the requirement of dextran altogether and allows for the potential incorporation of greater numbers of fluorophores by several orders of magnitude. However, indiscriminate attachment to the functional groups in the polymer matrix includes those on the nanosensor surface and as the nanosensor matrix provides a protective role, attachment of fluorophores on the nanosensor surface will contradict the idea of the protective capacity of a nanosensor. To prevent unwanted interaction between the fluorophore and its environment, additional steps are needed to remove or mask these surface-located fluorophores. Steps such as enzyme-mediated fluorophore removal would be required, where the fluorophores are attached via a cleavable linker allowing surface bound fluorophores to be easily removed [24]. Approaches to enhance the brightness of nanosensing particles have been investigated by other groups including the Wiesner group at Cornell University, who introduced superbright C-dots with brightness levels and enhanced photostability approaching those reported for semiconductor quantum dots [25]. C-dots are core-shell particles based on a dye-rich silica core surrounded by a pure silica shell. The shell acts as a protective layer similar to the polyacrylamide or sol-gel matrix of the nanosensors described here. Also, there are investigations into using quantum dots as intracellular probes and sensors [26–28]. The enhancement

c02.indd 18

7/30/2010 2:17:27 PM

INTRACELLULAR DELIVERY

19

in brightness and functionality of nanosensors and other types of nanosensing particles is varied and promising but for each type of nano(sensor)particle the challenges of delivery remain.

2.2

INTRACELLULAR DELIVERY

The nanosensor is a tool that has been designed to observe and analyze intracellular domains with minimal cellular perturbation. However, one of the major challenges to the widespread uptake of nanosensor technology has been the lack of a generic delivery system to translocate the nanosensor across the cell membrane and to the intracellular domain of choice. There are several techniques by which nanoparticles can be delivered into cells in culture; historically, many have been developed for the delivery of plasmids and oligonucleotides in molecular biology, or for the uptake of molecules packaged within organic polymer structures developed for drug delivery. The various delivery techniques investigated can be designated into two main categories: mechanical and membranal. In the mechanical category each delivery technique uses a mechanical and physical force to insert the nanosensors into the cells, for example, using a needle to inject them directly or a pulse of electricity to disrupt the cellular membrane. Membranal techniques are based on an interaction at the biological level between the cellular membrane and a chemical group on the nanosensor surface that aids the nanosensor in its translocation to the intracellular environment. 2.2.1

Mechanical Methods

2.2.1.1 Gene Gun Bombardment One of the most commonly used nanosensor delivery methods to date is gene gun delivery. Originally developed in molecular biology research for the delivery of DNA into living cells, it was easily adapted for nanosensor delivery [2]. The gene gun method can be described as a shotgun method in which nanosensors are literally fired into cells. Nanosensors are layered onto a disk or the inner surface of a small length of tube (to create a bullet) depending on the model of gene gun, by applying a thin layer of suspended nanosensors in solution and allowing it to dry. Pressurized helium is then used to propel the nanosensors off the disk or from the prepared bullet into the cell culture. It is reported that by controlling the original concentration of nanosensors in the suspension when layered onto the carrier it is possible to deliver a single sensor or thousands of sensors into the cell culture sitting a short distance away. It is also argued [2, 5] that certain levels of spatial delivery are possible by altering the helium pressure and the distance from carrier disk to cell culture. As the nanosensors are intrinsically fired in a random pattern, this only serves to selectively choose the distance the nanosensors will travel through the cell—with a certain amount of control

c02.indd 19

7/30/2010 2:17:27 PM

20

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

and practice (and luck!), this will allow one to deliver sensors to the nucleus or have them remain in the cytosol. Great care is needed to achieve the fine window of conditions that accomplishes cellular penetration without dislodging the cells from the culture plate. 2.2.1.2 Picoinjection Picoliter injection is a technique that has been applied to nanosensor internalization; physical damage and cellular distress are the main problems encountered during delivery. Needles are formed using a standard glass pipette puller and are used to inject a nanosensor solution directly into the cell. Generally, cell viability is good although normal cellular activity is likely affected and there is always the chance the cell becomes irreparably damaged. The deliverable range of nanosensor concentration is broad as this method dopes the cell with increasing numbers of nanosensorsolution aliquots until the desired level of concentration is reached. This method requires a high level of operator skill and is a batch process, meaning only one cell at a time can be loaded [9]. These limitations have led to this technique being used mostly in specialized applications where cell-by-cell analysis of a small number of cells is required, for example, embryology [21]. 2.2.1.3 Electroporation and Sonication These two methods have been used successfully for the delivery of various molecules into cells, such as plasmid DNA, for many years [29, 30]. One study suggests specifically that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell walls, for example, in algae and plants, because it nonspecifically disrupts cell-surface barriers [31]. In electroporation an electrical pulse temporarily permeabilizes the phospholipid bilayer of the plasma membrane. As this technique bypasses the endocytic pathway, nanosensors can be delivered directly to the cytoplasm with no need for subsequent endosomal escape, which is a clear benefit when, for example, considering CPP mediated delivery, described later in this chapter. However, as the cells are both permeabilized and subjected to very strong electrical pulses, there is usually a high rate of cellular mortality, which is often not reported [28]. 2.2.1.4 Patch Clamp and Scanning Ion Conductance Microscopy (SICM) Both these methods require a pulled micro/nanopipette to be held in contact, or at least in very close proximity, to the cell membrane. The notion of using these techniques as methods for nanosensor delivery is simple; with slight modification to each technique, a nanosensor-loaded solution is contained within the pipette as it is brought to the cell membrane. During patch clamping transient cell membrane disruption caused by creating an electrical current allows a temporary route for nanosensors to internalize. Similarly, in SICM the scanning tip can be used to provide a disruptive electrical field, which again disrupts the phospholipid bilayer, allowing the surrounding

c02.indd 20

7/30/2010 2:17:27 PM

INTRACELLULAR DELIVERY

21

nanosensors intracellular access. A major drawback to these techniques is the batch one-cell-at-a time nature of the technique. 2.2.1.5 Scrape Loading This procedure is used to transiently disrupt cell membranes and allow direct cell loading. This is achieved by drawing a tool (e.g., a glass rod with rubber end known as a rubber policeman) across an adherent population of cells, which scrapes them off the dish; the disrupted membranes temporarily allow various molecules [32], macromolecules [33], and nanoparticles [34] to cross this usually highly selective barrier. The method has not found great favor, however, due to the impracticality of severely reduced cell viability, approximately 50%, and because the method results in loaded cells that are in suspension. They must be replated and allowed to recover and spread, which is a serious disadvantage, as during this stage the labile probes/particles may be subjected to cytoplasmic redistribution and degradation. 2.2.2

Membranal Techniques

The dynamic nature of the endomembranal system is a consistent theme in different modes of intracellular delivery: specifically, sequestration through phagocytosis and pinocytosis in macrophages, liposomal delivery, and cell penetrating peptide mediated delivery. 2.2.2.1 Phagocytosis (Macrophages) and Pinocytosis Phagocytosis or “cell-eating” is the uptake of solid material by a cell. Only a few kinds of cells display this behavior, including protists (e.g., amebas and ciliates) and the phagocytic white blood cells of animals. The protists use it for feeding while the white blood cells use it as means of capturing bacteria and cleaning cell debris from infection sites. In phagocytosis the cellular membrane actively surrounds the solid object to be taken up, eventually pinching off internally to form a phagocytic vesicle, or phagosome. In general, this fuses with a lysosome whereupon the contents are digested or recycled. Macrophages have been shown to take up nanosensors from a surrounding solution, providing an easy delivery method for these types of cells [10]. The number of nanosensors internalized is a factor of the original concentration of nanosensors in the solution and how long the macrophages remain in solution. Cell viability is excellent. The disadvantages are that this technique is highly selective for macrophages, the sensors end up in certain cellular regions, as directed by the cell, and these specialized immune system cells can be difficult to culture. Pinocytosis or “cell-drinking” is the uptake of fluids from the outside of the cell. Here the plasma membrane tends to fold inwards, forming small vesicles containing the material. As in phagocytosis, the vesicle generally fuses with a lysosome and digestion of the contents takes place. Unlike receptor-mediated endocytosis, however, pinocytosis is nonspecific with regard to the materials taken up. Cell biologists first demonstrated pinocytosis by exposing amebas

c02.indd 21

7/30/2010 2:17:27 PM

22

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

to concentrated protein solutions and observing protein uptake. Effective uptake of nanoparticles via pinocytosis has been demonstrated in mouse macrophages [35] and for nanosensors with mouse embryos [21]. 2.2.2.2 Liposomal Delivery Commercially available liposomes, such as Lipofectamine 2000, Fugene, and Escort, can be used to produce nanosensor– liposome complexes. The technique relies on the natural fusion of liposomes with certain cells, which occurs due to the structural similarities between liposome or lipid micelle and the phosholipid bilayer of the cell membrane. The nanosensor–liposome complex fuses with the cell membrane and nanosensors, internalized or bound to the wall of the liposomes, can become internalized within the cell. With cell-specific tailoring of cell to nanosensor to liposome concentration a wide range of nanosensor concentrations can be delivered in a similar fashion to the macrophage system, specifically the concentration of the original nanosensor solution and of nanosensor–liposome complexes incubated with the cells is important for achieving defined final concentrations [9]. Delivery to the cytosol is possible, which is a great benefit of this technique; this can be demonstrated with pH measurements of the cytoplasmic environment of approximately pH 7 (cytoplasmic pH is similar to cell culture media pH [36, 37]), indicating that sensors are not sequestered into acidified vesicles (endosomes/lysosomes). 2.2.2.3 Cell-Penetrating Peptides Cell-penetrating peptides (CPPs) are individual short peptides able to translocate across cell membranes. They are being studied widely because of their ability to aid translocation and drive the uptake of varied cargos into mammalian cells. There are several reviews on the potential and advantages of using CPPs as delivery vectors, in particular, therapeutic agents for human treatment [38–41]. One example illustrates how the functional efficacy of therapeutic agents would be greatly enhanced through the controlled import and intracellular targeting to specific organelles [41]. The value of using CPPs as delivery agents became more appealing as it emerged that CPPs could offer targeted delivery and that the short peptide structure of a CPP could be tailored for improved delivery strategies. CPP endomembranal delivery may prove to be an ideal method that is near silent— for both chemical and physical disruption. The effective cell translocation of the CPP nature was observed prior to the isolation of an individual peptide; this is because CPP sequences in nature form part of a larger protein. It was the observation that some proteins were able to translocate across cellular membranes by a process known as protein transduction that initiated the search for the element of each protein that was responsible. Several proteins are well known for this behavior, for example, human immunodeficiency virus (HIV-1) Tat protein [42], herpes simplex virus (HSV-1) VP22 protein [43], and Drosophila Antennapedia homeoprotein [44]. A portion of each protein associated with intracellular translocation was found through cleavage or site-directed mutagenesis, identifying the amino

c02.indd 22

7/30/2010 2:17:27 PM

INTRACELLULAR DELIVERY

23

acids required for membrane translocation. This part of the protein was referred to as the “protein-transduction domain” (PTD) of each protein. The approach revealed PTDs that translocated the entire protein but also retained their activity in isolation, when either removed from the native protein or synthesized in the laboratory. In isolation, therefore, the PTD of a protein becomes a CPP [45]. Derossi et al. [46] working on the Drosophila Antennapedia homeoprotein revealed the PTD through site-directed mutagenesis, discovering the driving force behind the internalization of this protein; the third helix was essential for membrane translocation. Importantly, however, it was observed that the third helix was also sufficient for membrane translocation alone, and from this work it was possible to develop a 16 amino acid long CPP, referred to as penetratin (pAntp) [44, 47]. In a similar approach, Green and Loewenstein had identified an 86 amino acid section of the HIV-1 Tat protein that was internalized readily by viable cells [42]. Currently, CPPs are defined as peptides with a maximum of 30 amino acids, which are able to enter cells in a seemingly energy-independent manner, thus being able to translocate across membranes in a nonendocytotic fashion [48], although the exact biochemical and biophysical mechanisms involved in membrane translocation are yet to be established and debate is ongoing in this area of CPP research. It was thought that endocytosis did not play a part due to the evidence that peptide internalization occurred at 4 °C—because all active transport mechanisms involving endocytosis do not function at this low temperature. Also, CPPs being taken up by different cells and tissue types suggests a common internalization method, which suggests binding to conserved cell membrane determinants [49]. However, data from several groups argues for energydependent processes [50]. One regular feature of all CPPs and PTDs is the higher than average constitute of cationic amino acids [51]. This suggests that in some way at least the initiation of cellular uptake comes from ionic interactions between the cationic CPP and the anionic lipid membrane, although it is known that charge alone is not a sufficient driving force to induce translocation [40]. Recent literature describes a principal role for arginine in the CPP [52] and studies into the various permutations of Tat and poly-R-PTD (arginine-rich protein transduction domains) suggested an essential role of the guanidinium head-groups of the arginine residues for cellular uptake [52]. A good review of the delivery of quantam dots to cells, which describes the use of CPP in some detail, is provided by Delehanty et al. [28]. In our research we conjugated Tat peptide to the surface of aminefunctionalized polyacrylamide nanosensors for delivery to a variety of different cell lines, including CHO-K1, GH4 pituitary lactotrope, A172 human glioblastoma, and human embryonic stem cells [53] (Figure 2.2). It was demonstrated that the hydrodynamic radius of the cargo played a significant role in affecting the route of internalization. The function of the nanosensors was to determine pH and we showed that the nanosensors resided in vesicles with a measured pH of 5, postdelivery. These were presumed to be lysosomes, as they could be co-stained with LysoTracker Red® as shown in Figure 2.3. In

c02.indd 23

7/30/2010 2:17:27 PM

24

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

Figure 2.2. Confocal fluorescence microscopy images of Tat-conjugated nanosensors delivered to (from left): CHO cells incubated with Ca2+-sensitive nanosensors (green) and DRAQ5 nuclear stain (blue); GH4 pituitary lactotrope cells loaded with Tat conjugated Ca2+-sensitive nanosensors; A172 human glioblastoma cell loaded with fluorescein nanosensors; human mesenchymal stem cells (hMSCs) loaded with rhodamine B nanosensors (red) and co-stained with CD105:FITC membrane stain (green) and DRAQ5 nuclear stain (blue). (See color insert.)

Figure 2.3. Confocal fluorescence microscopy images of Chinese hamster ovary K1 (CHO-K1) cells containing: (left) Tat-FITC conjugate (green) which do not show colocalized fluorescence with endosomes/lysosomes stained with LysoTracker Red® when loaded into CHO-K1 cells; (middle) Tat-conjugated nanosensors and LysoTracker Red® showing colocalized fluorescence (orange color) indicating that the nanosensors are residing in acidified endosomes: (right) Tat-FITC conjugate (green) and Tat-TRITC-nanosensors (red) loaded into a single cell. The overlay of these two channels shows no colocalization of fluorescence of the two different cargo sizes mediated by Tat peptide delivery. (See color insert.)

contrast to this, fluorescently labeled Tat, which had a much smaller hydrodynamic radius, localized to different vesicles in the cytoplasm and did not colocalize with the LysoTracker Red® dye. It was apparent that the difference in size of cargo that the Tat peptide was conjugated to seemed to directly affect the mechanism of translocation into the cell. The precise mechanisms involved following the first ionic interaction will continue to be debated; they may consist of currently unknown mechanisms or, simply, endocytosis. It is

c02.indd 24

7/30/2010 2:17:27 PM

INTRACELLULAR TRAFFICKING, NANOSENSOR FATE, AND STRATEGIES

25

feasible, considering the physicochemical diversity of CPPs, that diverse mechanisms of uptake account for cellular uptake of different peptides. Even so, CPPs continue to be a useful research tool, having been demonstrated as delivery vectors for a variety of molecules and nanosized particles [54–57].

2.3 INTRACELLULAR TRAFFICKING, NANOSENSOR FATE, AND STRATEGIES FOR TARGETED DELIVERY After delivery, indicating the crossing of the cell membrane, the final location of nanosensors is often directed by cellular pathways that deal with the movement and trafficking of internalized particles and vesicles. As shown in Figure 2.3, CPP delivered nanosensors were found to locate to lysosomes within a few hours after incubation. This therefore means we have to carefully consider mechanisms by which the nanosensors can be directed to specific locations within the cell after internalization. Frequently, the required location of the delivered nanosensors is to reside freely in the cytoplasm without being sequestered into endosomal vesicles, for example, when wishing to monitor ion flux throughout the cell. Alternatively, the ultimate target may be a specific organelle. Either way, controlling the fate of the nanosensor is a detail that needs attention when developing a delivery system. 2.3.1

Cytochalasin-D

Cytochalasin-D is a cell-permeable fungal toxin from Zygosporium mansonii and an inhibitor of actin microfilament function. It binds to the barbed end of actin filaments, inhibiting the association and dissociation of subunits and causing disruption to the actin filaments and inhibition of actin polymerization. This action of cytochalasin-D is known to disrupt the fusion of the endosomes with lysosomes in late stage endocytosis [58], which is interesting with regard to nanoparticle delivery as internalization into lysosomes after translocating the membrane is limiting. Preincubation with cytochalasin-D has been shown to prevent nanoparticles being ultimately digested in lysosomes by blocking the fusion of endosomes with lysosomes [59]. For some delivery methods (e.g., CPP), this approach will only halt the endocytic pathway at the endosome stage; but this at least provides a point at which efforts can be focused on working to release the nanosensors from the endosome, before they are recycled out of the cell. 2.3.2

Targeted Delivery

For targeted delivery to specific intracellular organelles, like mitochondria, targeting molecules can be located on the nanosensor surface. These targeting molecules, often signaling peptides, are used ubiquitously in the cell to allow cell machinery to correctly transport proteins within the cellular environment

c02.indd 25

7/30/2010 2:17:27 PM

26

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

[60]. Many examples have been found in cytology for protein translocation, including the sorting of proteins to peroxisomes, the endoplasmic reticulum, mitochondria, and chloroplasts. If these signal peptides were conjugated to the nanosensor surface, then there is potential for directing the nanosensor to an organelle of choice. Other surface-bound chemistries that may be useful in nanosensor surface chemistry are those that would enable endosomal escape. A route of investigation may be to accept the inevitability of endocytosis and concentrate on devising mechanisms for breaking the delivered payload (nanosensors) out of the endosome once inside the cell. Utilizing the fusion of nanosensor-bound lipids with the endosomal membrane, which can then be cleaved by cellular enzymes, may provide a means for endosomal escape and cytoplasmic localization. 2.3.3

Multilevel, Multifunctional Surface Chemistry

The nanosensor surface is a valuable locale that should be used to provide biophysical properties for enhanced delivery methodology. A theoretical nanosensor surface could carry out multiple functions in a specific sequence that chaperones the nanosensor through the various membranes and on to a specific intracellular microdomain. Consider a CPP-mediated delivery method that triggers endocytosis. In the outer level of the molecular coat the CPP is presented to the cell membrane; directly beneath this is a layer of PEG that protects other functional molecules closer to the nanosensor surface. The CPP triggers translocation across the outer cell membrane via receptor-mediated endocytosis that ultimately leads to the nanosensor being located in a lysosome. Importantly, the CPP and PEG layer are attached to the nanosensor by a linker that is cleaved by the lysosomal environment—be that a lower pH or lysosome-specific enzymes. Removal of the CPP and PEG layers then reveals a lipid layer on the nanosensor that fuses with, and breaks through, the lysosomal lipid membrane, gaining the nanosensor access to the cytoplasmic environment. Here the nanosensor may be available to take measurement or perhaps, when required, a photocleavable linker is illuminated that removes the lipids and reveals signal peptides that direct the nanosensor to a cellular organelle. Alternatively, the linker could simply be susceptible to cytoplasmic enzymes such as esterases.

2.4

CELL BEHAVIOR DIAGNOSTICS

What is vital in all delivery techniques is the minimal level of cellular disruption. It is worthy of reiteration here that developing a delivery method that is successful, in terms of the numbers of internalized sensors, is of little value if cellular perturbation (physical or chemical) is such that normal cellular processes are disrupted. Methods for assessing cellular normality/viability must

c02.indd 26

7/30/2010 2:17:27 PM

CELL BEHAVIOR DIAGNOSTICS

27

therefore be carried out to appraise each delivery technique. This feature of nanosensor delivery can be referred to as “postdelivery cell behavior diagnostics,” which is the investigation that confirms and ensures a delivery method is as least disruptive as possible. A cell is considered viable if it has the ability to grow and develop [61, 62]; we extend our definition to include any morphogenic or phenotypic change that has occurred because of nanosensor delivery. For in vitro cultures of cells, assays to test viability can be based on the physical characteristics of the cells such as cell membrane integrity, cytoplasmic streaming in plant cells, deformability as monitored with optical tweezers [63], or granulation measured with flow cytometry. Several dyes are used for the assessment of cell membrane integrity, including trypan blue, methylene blue, and neutral red. These dyes are able to cross the disrupted membranes of dead cells and stain the intracellular contents. This is commonly referred to as a live : dead assay. The dead cells can be accounted for microscopically or with spectrometric estimation. Additionally, assays monitoring the metabolic activity, for example, the hydrolysis of fluorogenic compounds, are commonly used as indicators of cellular health. These assays often rely on the function of cellular enzymes (e.g., reductases or esterases) capable of altering the structure of an applied substrate to give an observable result. One such example is the administration of fluorescein diacetate, which is able to transfer across intact membranes. Inside the cell esterases cleave off the diacetate group yielding fluorescein; therefore the intracellular accumulation of this fluorescent compound correlates directly to the health of the cell. Again, fluorescent cells can be monitored with fluorescent microscopy, spectroscopy, or flow cytometry. The kinetic response to a chemoattractant could be monitored as another demonstration of normal cell function. Other methods based on the assessment of structural, morphological, and phenotypic characteristics can be used as additional or more convenient features of natural cell behavior including adherence and cell proliferation. For example, dielectrophoresis could be used to investigate changes in cell permittivity, and patterned electrode pair structures, used to measure impedance, can provide inferential measurement of proliferation. A method we employed to assay the well-being of a cell after delivery was to assess the degree of cellular identity. By this we refer to the unique identity of a cell, in terms of it being able to carry out a particular function or the type of receptors on the cell surface. Other proteinomic or genomic assessments may be used to identify the expressed biomolecular landscape of the cell and compare this with control populations. Human embryonic mesenchymal stem cells (hMSCs) were loaded with nanosensors prior to assessment with flow cytometry. A benefit of flow cytometry is the ability to investigate far greater numbers of cells than microscopy-based techniques; here each plot of data represents 50,000 cells. Flow cytometry can provide information on cellular size and granulation, as well as cell surface markers when the cells are previously incubated with relevant CD markers. In Figure 2.4 the dual use of flow cytometry data and confocal microscopy images show hMSC, containing

c02.indd 27

7/30/2010 2:17:27 PM

28

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

104

104

104

A

B

C PE

103

PE

103

PE

103

102

102

102

101

24.0 73.4

0 1.77

100 0 10

101

nic

102

103

104

101

20.3 60.5

0.01 1.43

100 0 10

101

2 nic 10

103

104

101

94.4 0.50

4.01 0.17

100 0 10

101

2 nic 10

103

104

Figure 2.4. Use of cell surface markers to confirm cellular identity. (Top) Simultaneous detection of CD29 expression on hMSC containing Tat-functionalized FITCnanosensors. (A) Isotype control showing only FITC fluorescence coming from internalized nanosensors. (B) CD29 second antibody control showing only FITC fluorescence coming from internalized nanosensors. (C) hMSC containing Tat-functionalized FITC nanosensors and stained with CD29-PE. Both signals are detected, as evidenced by the shift along the y-axis of the cell population. (Bottom) hMSC cells loaded with rhodamine B nanosensors (red) and counterstained with mouse anti-human CD105:FITC antibody (green). (See color insert.)

internalized Tat nanosensors, that retain the expression of CD29 and CD105, respectively. The hMSCs were positive for cell surface markers CD29 and CD105, and negative for CD34 and CD45 (control experiments; data not shown), demonstrating a population of undifferentiated hMSCs of nonhemopoietic lineage as defined by Pittenger et al. [64]. The combination of flow cytometry and fluorescence microscopy can be a powerful tool for cell behavior diagnostics.

2.5

CHALLENGES AND FUTURE PERSPECTIVES

The ultimate goal of nanosensor research is to have the ability to measure the analyte of choice at a cellular location of choice in real time. Further advantages accrue if the measurement system can be multiplexed to measure multiple analytes simultaneously. There is currently a rich stream of investigation to develop new measurement technologies at the nanoscale level to provide

c02.indd 28

7/30/2010 2:17:27 PM

REFERENCES

29

devices capable of probing the intracellular environment, concisely summarized by Borisov and Klimant [6]. New nanostructures, including “smart” sensors, and theranostics can be envisaged with increasing complexity and functionality being built into the devices. However, without improved delivery methods and, importantly, better understanding of how nanoparticles enter the cell and cross cell membranes the potential of these devices for intracellular analysis will not be realized. Each of the existing delivery methods described in this chapter has disadvantages when looked at in isolation. Most notable are the distress and physical perturbation to the cell from the “mechanical” loading techniques; gene gun delivery, picoliter injection, and scrape loading. Patch clamp and SICM methods have not yet found wide usage probably due to the cell-by-cell nature of loading, similar to picoinjection. Electroporation and sonication are thought to cause considerable disruption of the cellular membranes, which presents difficulties if trying to make measurements of “normal” cells. Of the membranal techniques phagocytosis and pinocytosis are limited to specific cell types, but liposomal delivery is used routinely for delivery of oligonucleotides and has been shown to work for nanoparticles too. It remains, however, that specific conditions need to be developed for each cell line and general applicability is notably limited. The attachment of cell-penetrating peptides has been investigated and shows promise as a technique for enabling nanosensor internalization without disruption to the cell wall or cellular biochemical activity of the cell. With nanosensors particularly, and most other nanosized particles destined for intracellular translocation, the surface of the particle provides the potential to be utilized for delivery strategies capable of traversing complicated routes through the cellular environment. Some of the exciting possibilities, in terms of intracellular targeting and fabricating multifunctional nanodevices were outlined. A final thought concerns the quality of fluorescence imaging as demand leads to the requirement of nanoscale resolution. A limitation at present is that the fluorescent output from nanosensors has to be assumed to arise from an ensemble of nanosensors as the standard optical microscopy resolution limit of λ/2 does not allow individual nanosensors to be imaged. The rapidly expanding field of superresolution microscopy, including the techniques of STED, PALM, STORM, and RESOLFT (see Hell [65] for a comprehensive review) will present opportunities to overcome the resolution limit; this may prove to be as transformative to intracellular imaging and analysis as the development and application of confocal microscopy has been to cellular analysis in the past 20 years.

REFERENCES 1. Buck, S. M., et al. Optochemical nanosensor PEBBLEs: photonic explorers for bioanalysis with biologically localized embedding. Curr. Opin. Chem. Biol. 8(5): 540–546 (2004).

c02.indd 29

7/30/2010 2:17:28 PM

30

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

2. Clark, H. A., et al. Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors. Anal. Chem. 71(21): 4831–4836 (1999). 3. Clark, H. A., et al. Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors. Anal. Chem. 71(21): 4837–4843 (1999). 4. Park, E. J., et al. Ratiometric optical PEBBLE nanosensors for real-time magnesium ion concentrations inside viable cells. Anal. Chem. 75(15): 3784–3791 (2003). 5. Xu, H., et al. A real-time ratiometric method for the determination of molecular oxygen inside living cells using sol-gel-based spherical optical nanosensors with applications to rat C6 glioma. Anal. Chem. 73(17): 4124–4133 (2001). 6. Borisov, S. M. and Klimant, I. Optical nanosensors—smart tools in bioanalytics. Analyst 133(10): 1302–1307 (2008). 7. Srivastava, A. and Krishnamoorthy, G. Time-resolved fluorescence microscopy could correct for probe binding while estimating intracellular pH. Anal. Biochem. 249(2): 140–146 (1997). 8. Clark, H. A., et al. Subcellular optochemical nanobiosensors: probes encapsulated by biologically localised embedding (PEBBLEs). Sensors and Actuators B—Chemical 51(1–3): 12–16 (1998). 9. Clark, H. A., et al. Optochemical nanosensors and subcellular applications in living cells. Mikrochim. Acta. 131(1–2): 121–128 (1999). 10. Buck, S. M., et al. Nanoscale probes encapsulated by biologically localized embedding (PEBBLEs) for ion sensing and imaging in live cells. Talanta 63(1): 41–59 (2004). 11. Sumner, J. P. and Kopelman, R. Alexa Fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors. Analyst 130(4): 528–533 (2005). 12. Tan, W. H., Shi, Z. Y., and Kopelman, R. Development of submicron chemical fiber optic sensors. Anal. Chem. 64(23): 2985–2990 (1992). 13. Vo-Dinh, T. and Kasili, P. Fiber-optic nanosensors for single-cell monitoring. Anal. Bioanal. Chem. 382(4): 918–925 (2005). 14. Vo-Dinh, T., et al. Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell. Nature Biotechnol. 18(7): 764–767 (2000). 15. Brasuel, M., et al. Production, characteristics and applications of fluorescent PEBBLE nanosensors: Potassium, oxygen, calcium and pH imaging inside live cells. Sensors and Materials 14(6): 309–338 (2002). 16. Tan, W. H., et al. Ultrasmall for cellular. Anal. Chem. 71(17): 606A–612A (1999). 17. Webster, A., Compton, S. J., and Aylott, J. W. Optical calcium sensors: development of a generic method for their introduction to the cell using conjugated cell penetrating peptides. Analyst 130(2): 163–170 (2005). 18. Sumner, J. P., et al. A fluorescent PEBBLE nanosensor for intracellular free zinc. Analyst 127(1): 11–16 (2002). 19. Koo, Y. E. L., et al. Photonic explorers based on multifunctional nanoplatforms for biosensing and photodynamic therapy. Appl. Optics 46(10): 1924–1930 (2007). 20. Peterson, J. I., et al. Fiber optic pH probe for physiological use. Anal. Chem. 52(6): 864–869 (1980).

c02.indd 30

7/30/2010 2:17:28 PM

REFERENCES

31

21. Webster, A., et al. The delivery of PEBBLE nanosensors to measure the intracellular environment. Biochem. Soc. Trans. 035(3): 538–543 (2007). 22. Brasuel, M., et al. Fluorescent nanosensors for intracellular chemical analysis: decyl methacrylate liquid polymer matrix and ion exchange-based potassium PEBBLE sensors with real-time application to viable rat C6 glioma cells. Anal. Chem. 73(10): 2221–2228 (2001). 23. Sun, H., et al. Synthesis and characterization of ratiometric, pH sensing nanoparticles with covalently attached fluorescent dyes. Chem. Materials 18(15): 3381–3384 (2006). 24. Ulijn, R. V. Enzyme-responsive materials: a new class of smart biomaterials. J. Materials Chem. 16(23): 2217–2225 (2006). 25. Fuller, J. E., et al. Intracellular delivery of core-shell fluorescent silica nanoparticles. Biomaterials 29(10): 1526–1532 (2008). 26. Hild, W. A., Breunig, M., and Goepferich, A. Quantum dots—nano-sized probes for the exploration of cellular and intracellular targeting. Eur. J. Pharm. Biopharm. 68(2): 153–168 (2008). 27. Zorov, D. B., et al. Examining intracellular organelle function using fluorescent probes—from animalcules to quantum dots. Circ. Res. 95(3): 239–252 (2004). 28. Delehanty, J. B., Mattoussi, H., and Medintz, I. L. Delivering quantum dots into cells: strategies, progress and remaining issues. Anal. Bioanal. Chem. 393(4): 1091– 1105 (2009). 29. Hartikka, J., et al. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188. Mol. Ther. 4(5): 407–415 (2001). 30. Wyber, J. A., Andrews, J., and Demanuele, A. The use of sonication for the efficient delivery of plasmid DNA into cells. Pharm. Res. 14(6): 750–756 (1997). 31. Azencott, H. R., Peter, G. F., and Prausnitz, M. R. Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication. Ultrasound Med. Biol. 33: 1805–1817 (2007). 32. McNeil, P. L., et al. A method for incorporating macromolecules into adherent cells. J. Cell Biol. 98(4): 1556–1564 (1984). 33. McKeon, J. and Khaledi, M. G. Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 22(17): 3765–3770 (2001). 34. Hosseinkhani, H. DNA nanoparticles for gene delivery to cells and tissue. Int. J. Nanotechnol. 3(4): 416–461 (2006). 35. Buono, C., et al. Fluorescent pegylated nanoparticles demonstrate fluid-phase pinocytosis by macrophages in mouse atherosclerotic lesions. J. Clin. Invest. 119(5): 1371–1381 (2009). 36. Chu, G. L., et al. The role of intracellular pH and its variance in low pH sensitization of killing by hyperthermia. Radiat. Res. 122(3): 288–293 (1990). 37. Kneen, M., et al. Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys. J. 74(3): 1591–1599 (1998). 38. Foged, C. and Nielsen, H. M. Cell-penetrating peptides for drug delivery across membrane barriers. Expert Opin. Drug Deliv. 5(1): 105–117 (2008).

c02.indd 31

7/30/2010 2:17:28 PM

32

DELIVERY OF NANOSENSORS TO MEASURE THE INTRACELLULAR ENVIRONMENT

39. Vives, E., Schmidt, J., and Pelegrin, A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochim. Biophys. Acta Rev. Cancer 1786(2): 126–138 (2008). 40. Futaki, S., et al. Arginine-rich peptides—an abundant source of membranepermeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276(8): 5836–5840 (2001). 41. Gariepy, J. and Kawamura, K. Vectorial delivery of macromolecules into cells using peptide-based vehicles. Trends Biotechnol. 19(1): 21–28 (2001). 42. Green, M. and Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55(6): 1179–1188 (1988). 43. Elliott, G. and Ohare, P. Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88(2): 223–233 (1997). 44. Derossi, D., Chassaing, G., and Prochiantz, A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8(2): 84–87 (1998). 45. Richard, J. P., et al. Cell-penetrating peptides—a reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278(1): 585–590 (2003). 46. Derossi, D., et al. The 3rd helix of the Antennapedia homeodomain translocates through biological-membranes. J. Biol. Chem. 269(14): 10444–10450 (1994). 47. Derossi, D., et al. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271(30): 18188–18193 (1996). 48. Hansen, M., Kilk, K., and Langel, U. Predicting cell-penetrating peptides. Adv. Drug Deliv. Rev. 60(4–5): 572–579 (2008). 49. Silhol, M., et al. Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat. Eur. J. Biochem. 269(2): 494–501 (2002). 50. Vives, E. Cellular uptake of the Tat peptide: an endocytosis mechanism following ionic interactions. J. Mol. Recognition 16(5): 265–271 (2003). 51. Futaki, S., Goto, S., and Sugiura, Y. Membrane permeability commonly shared among arginine-rich peptides. J. Mol. Recognition 16(5): 260–264 (2003). 52. Lundberg, P. and Langel, U. A brief introduction to cell-penetrating peptides. J. Mol. Recognition 16(5): 227–233 (2003). 53. Coupland, P. G., et al. Internalisation of polymeric nanosensors in mesenchymal stem cells: analysis by flow cytometry and confocal microscopy. J. Control. Release 130(2): 115–120 (2008). 54. Mae, M., et al. Design of a tumor homing cell-penetrating peptide for drug delivery. Int. J. Pept. Res. Ther. 15(1): 11–15 (2009). 55. Chen, B., et al. Transmembrane delivery of the cell-penetrating peptide conjugated semiconductor quantum dots. Langmuir 24(20): 11866–11871 (2008). 56. Torchilin, V. P. Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery. Biopolymers 90(5): 604–610 (2008). 57. Palm-Apergi, C. and Hallrink, M. A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery. J. Control. Release 132(1): 49–54 (2008). 58. Qualmann, B., Kessels, M. M., and Kelly, R. B. Molecular links between endocytosis and the actin cytoskeleton. J. Cell Biol. 150(5): F111–F116 (2000).

c02.indd 32

7/30/2010 2:17:28 PM

REFERENCES

33

59. Morgan, T. T., et al. Encapsulation of organic molecules in calcium phosphate nanocomposite particles for intracellular imaging and drug delivery. Nano Lett. 8(12): 4108–4115 (2008). 60. Emanuelsson, O., et al. Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols 2(4): 953–971 (2007). 61. Pegg, D. E. Viability assays for preserved cells, tissues, and organs. Cryobiology 26(3): 212–231 (1989). 62. Palta, J. P., Levitt, J., and Stadelmann, E. J. Plant viability assay. Cryobiology 15(2): 249–255 (1978). 63. Lincoln, B., et al. High-throughput rheological measurements with an optical stretcher. In: Cell Mechanics. Elsevier Academic Press, San Diego, CA, 2007, pp. 397–423. 64. Pittenger, M. F., et al. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411): 143–147 (1999). 65. Hell, S. W. Microscopy and its focal switch. Nature Methods 6(1): 24–32 (2009).

c02.indd 33

7/30/2010 2:17:28 PM

c02.indd 34

7/30/2010 2:17:28 PM

CHAPTER 3

Cytoplasmic Diffusion of Dendrimers and Dendriplexes ALEXANDER T. FLORENCE School of Pharmacy, University of London, London, United Kingdom

PAKATIP RUENRAROENGSAK Lung Cell Biology, Respiratory Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom

3.1

INTRODUCTION

Diffusion of nanoparticulate carriers within the cytoplasm is one of the controlling factors in the delivery of active therapeutic agents not only to the cell nucleus but also to a large extent to adjacent cells. Delivery to the nucleus is the goal of gene therapy but the overall flux of particles in tissues is determined cell by cell. The cytoplasm may be an aqueous environment but it is a complex one, crowded molecularly and far from isotropic. Hence the diffusion process of particles that have gained access to the individual cell in a free state, or escape from organelles in which they have been snared, and then diffuse, is complex. The flux of particles is determined by several factors: (1) by the properties of the medium in which the particles move; (2) by the properties of the particle, such as its radius, surface properties, shape, and flexibility; (3) by the concentration gradients that drive diffusion; and (4) by a range of other influences such as physical obstructions, which increase the effective path length that particles must traverse, and binding of nanoparticles to components within the cytoplasm. Convection currents may enhance or retard diffusion. The problem in both modeling and exact mathematical description is that the cytoplasm is a dynamic medium.

Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

35

c03.indd 35

7/30/2010 2:18:35 PM

36

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

In this chapter we describe some of the work conducted in our laboratories on the diffusion of dendrimers and dendriplexes, and discuss it in the light of findings in the literature. The bulk of the work discussed here centers on a novel synthetic 6.5-nm dendrimer based on branched lysine residues, which we found serendipitously to be autofluorescent. This feature of the dendrimer allowed us to investigate nanoparticle movement within the cytoplasm using confocal microscopy and other techniques. We also studied the diffusion of the dendrimer in aqueous media (water and glycerol solutions) and in aqueous gels for comparison with the much more complex cytoplasmic transport. One complication, which may not be unique to our dendrimer, is the finding that, at least in vitro, it has a biphasic effect on actin polymerization. Dendrimers are three-dimensional hyperbranched macromolecules [1]. They have been employed mainly in experimental applications encompassing diagnostic tools and as drug carriers; some have intrinsic biological activity. Despite this, elucidation of the uptake mechanisms and the cellular pathways of dendrimer carrier systems and dendrimers per se has been slow. Before diffusion can occur in the cytoplasm, the particles have to be taken up; the mode of uptake may influence subsequent events, depending on whether the particle emerges in the cytoplasm as a free entity or encapsulated within vesicles. The uptake of nanoparticles and dendrimers has perforce been studied by attachment of fluorescent probes either by conjugation [2] or by physical interactions [3]. Scission or loss of the fluorescent moiety will result in difficulty with interpretation of resulting data [4]. The use of an intrinsic fluorescent particle can overcome these problems as there are no chemical changes required and surface properties are the original. In our work, for example, with dendrimer–DNA complexes (dendriplexes), their diameter can increase by up to 20–25 nm on fluorescently labeling the DNA; this is accompanied by a twofold increase in the zeta potential of the complex. Artifacts are also created by fixing cells; thus experiments in live cells are preferable for estimating cytoplasmic transport [5, 6]. The synthetic self-fluorescent sixth generation amino-terminated polyamide polylysine dendrimer (Figure 3.1A), namely [(Gly)(Lys63)(NH2)64] with a molecular weight of 8149 Da, has been reported previously by us [7]. The use of this compound as a nanoscopic probe is discussed in this chapter, using confocal laser scanning microscopy (CLSM). The results relate both to the native dendrimer and to a dendrimer–DNA complex and are discussed in terms of diffusion, obstruction, and binding effects. We can only speculate on its effects on actin polymerization, which we found in vitro. From a naïve physicochemical point of view, if such effects occur in vivo, depolymerization would ease the passage of the dendrimer and polymerization would complicate it. However, actin polymerization actually provides the propulsive force for intracellular transport of endosomes and some microorganisms, so this is a complex subject [8].

c03.indd 36

7/30/2010 2:18:35 PM

DIFFUSION WITHIN CELLS

A

Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

Lys Lys

Lys

Lys Lys

Lys Lys

Lys

Lys Lys

Lys

Lys

Lys

Lys

Lys

Lys Lys

Lys Lys

Lys Lys

Lys

Lys

Gly

Lys

NH2

Lys

Lys Lys

Lys

Lys Lys Lys Lys

Lys

Core Lys Lys Branch Surface

Lys

Lys Lys

Lys Lys

Lys Lys

Lys Lys

C

B

Lys

Lys

Lys

Lys Lys

Lys Lys Lys

Lys

Control D

10 μm

10 μm

G

t=1h

10 μm

10 μm

t=2h

10 μm

t=1h

10 μm

I

H

t=2h

t=15min F

E

t=30min

37

10 μm

t=4h

10 μm

Figure 3.1. The structure (A) of the sixth generation polylysine dendrimer used in this study and a time-dependent study of the interaction between the dendrimer (green fluorescence) and Caco-2 cells (B–I): the cells were incubated with complete media containing 0.1% (w/v) of dendrimers for 15 min (C), 30 min (D), 1 h (E, F), 2 h (G, H), and 4 h (I). The cells were observed under confocal microscopy. In the control (A), the cells were exposed to the complete medium without dendrimer. The dendrimer is found to attach to the cell membrane periphery (red arrows) as a result of electrostatic attraction (C, D). At 1 h the dendrimer distributes toward the cell nuclei in all regions of the cells (E, F). At longer incubation times (G–I), the dendrimer appears to be concentrated on the surface of the cells. This technique cannot prove internalization. (See color insert.)

3.2

DIFFUSION WITHIN CELLS

Diffusion of spherical nanoparticles in a liquid medium obeys the Stokes– Einstein equation relating diffusion coefficient (D) to the radius of the particle (r) and the viscosity (η) of the medium in which the particle moves as

c03.indd 37

7/30/2010 2:18:35 PM

38

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

D = kT 6πηr One can demonstrate in binary aqueous mixtures (e.g., glycerol–water) that the diffusion coefficient reduces with increase in bulk viscosity, as these are simple fluids composed of small molecules. In macromolecular systems, the viscosity parameter in the Stokes–Einstein equation is not the bulk viscosity but in fact the intrinsic or “microscopic” viscosity. Movement of particles through some gels is rapid because the “pore” sizes are large and the medium in which the particles move is akin to bulk water. We have calculated the intrinsic viscosity of the interior of HPMC gels to be very close to that of water, using a variety of nanoparticle probes. In nonionic viscous vehicles such as glycerol solutions and HPMC gels, there is no or little influence of particle charge [9]. As other chapters in this book discuss, the cell cytoplasm is concentrated and crowded, occupied as it is by a variety of macromolecules between 5% and 40% of the total cell volume or 400 mg/L [10]. The cellular architecture provides a complex non-Newtonian fluid comprising an aqueous phase filling the spaces between an entangled mesh of filamentous cytoskeleton and other macromolecular structures, resembling a gel-like structure [11–14]. This phenomenon is well known as “macromolecular crowding”, which plays a decisive role on several levels of cellular organization, particularly in the diffusion and binding of matter inside cells [13]. Translocation of nanosystems occurs in this three-dimensional forest. The Stokes–Einstein equation may not be entirely applicable to diffusion phenomena within the constantly changing interior of cells. Diffusion of matter in cells relies on fluid-phase viscosity (F1), solute binding to macromolecules (F2), and collisions between solutes and macromolecules (F3) [15, 16]. Diffusion theory of solutes in the cell cytoplasm has been described by Kao et al. [16], who assumed that the cytoplasm was composed of an aqueous fluid-phase compartment bathing a matrix of mobile and static macromolecules/particles much larger than water molecules and small solutes. They identify three main factors involved in the reduction in diffusion coefficient of a small solute in cytoplasm (Dcyto) relative to that in water (D0): Dcyto = F1 ( η) × F2 ( Du , {Db,i , fb,i }) × F3 ({ni , Vi }) D0

(3.1)

The function F1(η) represents the deceleration of net solute translational diffusion because of an increase in true fluid-phase cytoplasmic viscosity, an increase due to solute-induced perturbation in solvent structure, which can be written F1 ( η) =

c03.indd 38

η0 ηcyto

(3.2)

7/30/2010 2:18:36 PM

DIFFUSION WITHIN CELLS

39

where η0 represents the viscosity of water and ηcyto is the true fluid-phase microviscosity of the cytoplasm. The function F2(Du,{Db,i, fb,i}) indicates the total reduction of solute translational diffusion due to the transient binding of solute molecules to cytoplasmic structures as addressed in Equation 3.3 below [16]. F2 represents the ratio of the weighted diffusion coefficient of bound and unbound solute to the diffusion coefficient of the unbound solute. In Equation 3.3 Du and Db,i are, respectively, the diffusion coefficients of unbound and ith bound solute, and fb,i is the fraction of net solute bound to component i. F2 ( Du , {Db,i , fb,i }) = fu + ∑ ( Db,i Du ) fb,i

(3.3)

fu + ∑ fb,i = 1

(3.4)

i

where i

The function F3({ni,Vi}) illustrates the deceleration of the total reduction of solute translational diffusion owing to collisional interaction with cytoplasmic structures assuming there are ni structures of type i, each of volume Vi. The “volume exclusion” by mobile obstacles has been used as a model (a stretchedexponential [SE] model) to explain this function [17–19]. This SE model is applicable to the diffusion of Brownian particles (or solutes), which are relatively large compared to the solvent molecules. v F3,SE ( ni , Vi ) = exp ⎡⎣ −α ( ni , Vi ) ⎤⎦

(3.5)

Here niVi represents the volume fraction occupied by the occluding molecule and the prefactor α and ν represent scaling parameters obtained by fitting Equation 3.5 to data, but can be calculated independently from theory [16]. Viscosities of the cytoplasm of various cell types have been calculated. Values ranged from 2 to 20 cP [16, 20–22]. By using time-resolved fluorescence anisotropy, Verkman and colleagues found that the cytoplasmic solvent measured in different types of cell lines may be significantly different from that of bulk water [23]. This has also been demonstrated [24] using a different approach in two mammalian tissue culture cell lines. Luby-Phelps et al. [25] characterized the nature of the diffusion barrier by comparing the diffusion of 3.2–25.8 nm Ficoll probe molecules. The results showed that relative to water the diffusion rate in the cytoplasm, which is already low with the smallest (3.2-nm) probe, reduces with an increase of the probe size. To describe this relationship, diffusion of various sizes of FITCFicoll was measured in model solutions [19]. The relative diffusion coefficient of FITC-Ficoll in concentrated solutions of Ficoll is low, but it is independent of the size of the FITC-Ficoll probe. In a tangle of F-actin filaments, on the

c03.indd 39

7/30/2010 2:18:39 PM

40

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

other hand, relative diffusion of the 3.2-nm probe is not impeded, but its diffusion decreases with the larger FITC-Ficoll probes. Taken together, it was noticed that the effects of Ficoll (10%) and F-actin filaments (5 mg/mL) resemble the pattern seen in the cytoplasm. The solution conditions calculated to most closely mimic the size dependency of FITC-Ficoll diffusion in the cells are 120 mg/mL of bovine serum albumin dissolved in a fluid matrix containing 37 mg/mL F-actin fibers. Based on these results, a model of cytoplasm as a densely entangled filament network interpenetrated by a fluid phase crowded with globular macromolecules was developed. This is in good agreement with cell structures proposed by Medalia et al. [14]. Uncoated GFP-M6tail-associate vesicles within cells [26], found during the process of clathrin-dependent endocytosis, are trapped in an actin-rich region of the cytoplasm of ARPE-19 cells. These vesicles exhibited Brownian-like motion. They could exit from the actin-rich region by a slow diffusion-based mechanism. The diffusion coefficient of the vesicles (n = 250) was found to be 1.42 × 10−12 cm2/s (SD = 1.24). Small molecules traverse cell membranes by passive transport, whereas macromolecules cross membranes by endocytosis, the process of membranebound vesicles originated from the invagination and pinching off of the membrane [27]. Phagocytosis occurs in mammalian cells, while pinocytosis may occur in all cell types. It has been reported that some dendrimers may create holes in cell membranes ranging between 15 and 40 nm, allowing passive diffusion of dendriplexes [28–32]. In addition, polylysine polymer was found to initiate holes on the cell membrane with a diameter ranging from 1 to 10 nm [29]. This suggests that, as we believe, the polylysine dendrimers used in our work are taken up intact and are not sequestered. The use of cell-penetrating peptides to increase the bioavailability of drug/gene/siRNA delivery has been seen to overcome both extracellular and intracellular barriers [33]. Peptide carried MPG and Pep was found to form a stable nanoparticle complex with the active macromolecules. The particles were found to enter the cells independently of endosomal pathways. Both lipoplexes and dendriplexes possessing positive charges first adhere to the cell membrane as a result of electrostatic interactions. Endocytosis has been invoked depending on types of carrier systems and cell lines [34, 35]. The nature of the carrier surface, in fact, regulates specific or nonspecific binding of the complexes onto the cell membrane and triggers different endocytic pathways such as clathrin- and caveolin-dependent endocytosis, and clathrinand caveolin-independent endocytosis. Clathrin- and caveolin-dependent endocytosis has been proposed as the major pathway of internalization of lipoplexes and dendriplexes [36–38]. Interestingly, a difference in uptake pathway was also found between surface-engineered dendrimers with amine and carboxyl functional groups. The carboxyl-modified dendrimers were taken up by caveolin-dependent endocytosis, whereas the amine-modified dendrimers were taken up by the clathrin- and caveolin-independent pathway [2].

c03.indd 40

7/30/2010 2:18:39 PM

DIFFUSION AND UPTAKE OF DENDRIMER AND DENDRIPLEXES

41

3.3 METHODS USED TO ASSESS THE UPTAKE AND CELLULAR DIFFUSION OF NANOPARTICLES The self-fluorescent sixth generation amino-terminated polyamide polylysine dendrimer (Figure 3.1A), with a molecular weight of 8149 Da, was used in this study and the details of the synthesis method can be found elsewhere [7]. For the dendriplexes, the same dendrimer was complexed with plasmid DNA (pDNA), pDsRed2-N1 (4.689 kb, Clonetech, USA), at 10 : 1 molar charge ratio (+/−). As the fluorescence of the dendrimer decreases after the complexation, the pDNA was fluorescently labeled using Label IT® Tracker™ Fluorescein kit (Mirus Bio Coporation, USA) before forming the complex. Dynamic uptake of the dendrimer and dendriplexes in all types of cells was followed every 5 min before and after adding the dendrimer and dendriplexes using confocal microscopy and data analyzed using custom procedures written in IgorPro software (Wavemetric Inc). The z-stack image at time t = 5 min before the dendrimer or the dendriplexes was combined and used as the control image. The fluorescent intensity detected in the green channel in this stack represents the background fluorescent intensity. The regions of interest (ROIs) were drawn and saved as the reference file. The fluorescent intensity values of the dendrimer/dendriplexes inside each z-stack image were averaged and standardized by subtraction of the background fluorescent intensity. The diffusion coefficients (D) and relative diffusion coefficients (D/D0) of the dendrimer/dendriplexes were calculated assuming that the cytoplasm is homogeneous. The focus was on the transport of the dendrimer and dendriplexes from the plasma membrane to the nucleus [39]. The lag time (tL) for the dendrimer/dendriplexes to develop a uniform concentration gradient within cytoplasm allows calculation of a diffusion coefficient where the thickness (h) of the diffusion layer is known, as D = h2/6tL. The thickness is the mean distance between the plasma membrane and nuclear membrane, termed the “cytoplasmic radius.” The cytoplasmic radius was measured using TEM and confocal microscopy. The value of tL was obtained by measuring the fluorescence intensity of the dendrimer over time from uniformly sized regions of interest placed equidistantly across the cell using custom procedures written in IgorPro software. The diffusion coefficient (D) of the dendrimer in the cell cytoplasm was then calculated, using the experimental tL value. The set fluorescent intensity of ROIs in each time point (F) was then plotted against time (minutes).

3.4

DIFFUSION AND UPTAKE OF DENDRIMER AND DENDRIPLEXES

This topic is discussed here as uptake may be a rate-limiting step in subsequent events in the cytoplasm. There is of course a concentration dependency of both flux and diffusion coefficient [40]; hence rates and extents of uptake are important.

c03.indd 41

7/30/2010 2:18:39 PM

42

3.4.1

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

Adhesion of Dendrimer to Different Cell Lines

The adhesion of the dendrimer to Caco-2 cells is time dependent as illustrated in Figure 3.1B–I. At 15 min, the dendrimer (seen by the green fluorescence) concentrates at the periphery of cell membrane (Figure 3.1C). Within 30 min, the dendrimer is found to accumulate in the cytoplasm (Figure 3.1D). The relatively rapid uptake into the Caco-2 cells and specifically the nucleus can be attributed first to the electrostatic attraction between the amino surface groups of the dendrimer and the negative charge of membrane proteins, allowing attachment prior to endocytosis. Within an hour, the dendrimer covers all parts of the cell (Figure 3.1E–F). Distribution of the dendrimer toward the cell nucleus is observed in dividing (D) and nondividing cells (E). The dendrimer was found to cover the cells and apparently concentrate in the cytoplasm region (Figure 3.1G–H). The cells after exposure to the dendrimer for 4 h are seen in Figure 3.1I; the higher number of areas of dendrimer coverage are found on the cells. Different results were found in SKMES-1 cells. The dendrimer appeared to randomly adhere to the cell membrane. At longer incubation periods a more typical trend was found: the dendrimer was found to cover the cell surface. The reasons for this variation remain unclear but may result from differences in uptake mechanisms. Xia et al. [41] found such diferences in uptake mechanisms for 60-nm diameter amine-modified polystyrene nanoparticles when studying macrophage (RAW 264.7) and endothelial cells (BEAS2B). The uptake of the particles in the latter was found to be caveolin-dependent endocytosis whereas that in the former was independent of caveolin. We have demonstrated differences in the rate of binding of our dendrimer to three different cell types, and also the reduction of cellular adhesion when the medium bathing the cells is flowing (results not shown here). The extent of reduction is dependent on the force of adhesion (unpublished data).

3.4.2 Diffusion and Uptake of Dendrimer and Dendriplexes in Living Cells At low concentrations of the dendrimer, the colocalization of the dendrimer may not be very obvious, but increased levels of the fluorescent signal of the dendrimer are found inside the cell nucleus after background subtraction. Interestingly, the same pattern was noticed in the nucleus after the dendrimer was added into the system (Figure 3.2A–C). Hoechst33342 was used for locating the nuclear compartment. The dynamic uptake of the dendrimer into the nucleus was examined by overlaying images of the dendrimers (in green channel (Ch2)) over images of the nucleus (in blue channel (Ch1)) as can be seen in Figure 3.2. The fluorescent signal in the green channel, representing the dendrimers in each ROI, was normalized by background fluorescence subtraction (white boxes no. 14

c03.indd 42

7/30/2010 2:18:39 PM

DIFFUSION AND UPTAKE OF DENDRIMER AND DENDRIPLEXES

43

Fluorescent Intensity

60 50 40 † Boxes in both channels are regions of interest (ROIs)

30

ROI 5 ROI 6 ROI 7 ROI 8 ROI 9 ROI 10

20 10 0 0

20

40 60 Time (min)

80

100

Figure 3.2. An overlay of the compressed z-stack images of the dendrimer uptake process in living Caco-2 cells. The cells were incubated with medium containing 0.1% dendrimer (represented in green) and images collected every 5 min before (A) and after addition of dendrimer (B–F). Images presented are at time t = 5 (B), 15 (C), 30 (D), 40 (E), and 90 (F) min of incubation. Data were analyzed over the time and plotted in (G); the inserts show the positions of regions of interest (ROIs, white boxes) for data analysis. The mean fluorescence signal in each ROI was measured from the projected z-stack images at different time points with background subtraction. The dendrimer was found in the cytoplasmic compartment 5–35 min after exposure to the dendrimer (ROI 9 and 10). The concentration of the dendrimer declined after 30 min, whereas the relative fluorescent intensity of the dendrimer inside the nucleus was detected (ROI 5–8). The cell nuclei were stained with Hoechst33342 represented in blue. The scale bar is 20 μm. (See color insert.)

c03.indd 43

7/30/2010 2:18:39 PM

44

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

in the insert pictures of Figure 3.2G). The relative fluorescent signal of the dendrimer was then plotted (F) against time (t, min) as in Figure 3.2G. The results (n = 5) demonstrated that the dendrimer reached the nucleus of Caco-2 cells within 35–45 min (tL) of incubation (Figure 3.2G), compared to 25–30 min in SKMES-1 cells (data not shown). This might be due to the differences of the intracellular architecture within each cell line, but not cell size as the cell radius is taken into account in the calculation of D. Average cell radii were 12.09 (± 6.02 (SD)) and 8.01 (± 3.21 (SD)) μm for the Caco-2 cells and SKMES cells, respectively. Dcyto values of the dendrimer in Caco-2 cells and SKMES-1 cells were found to be 9.82 (± 0.98) × 10−11 cm2/s and 5.99 (± 0.16) × 10−11 cm2/s, respectively. Cellular components such as secretory granules have diffusion coefficients of 1.9 × 10−11 cm2/s, lower than that of the dendrimers [42] as might be expected from their larger dimensions. The diffusion coefficients relative to those in water (Dcyto/D0) are, respectively, 1.24 (± 0.12) × 10−4 and 0.76 (± 0.05) × 10−4 (D0 = 79.54 × 10−8cm2/s). These values imply that macromolecular crowding and obstacles in the cytoplasm provide a formidable barrier to dendrimer transport. They also emphasize that differences between cell lines can be significant. In work on transfection with dendriplexes, we have found that there can be a 1000-fold difference in efficiencies when using the same DNA construct with a range of cell lines [43]. The cytoplasm hinders the mobility of the dendrimer by some 1000-fold compared to that in water. This may be explained in loose terms by exclusion and obstruction effects, the former due to the loss of free water by hydration of macromolecules and the latter due to the tortuous pathway that the dendrimer must travel in the cytoplasm, avoiding organelles and other structural features, such as actin fibers. The diffusion of globular proteins in muscle cells approximates to zero when the hydrodynamic radius of the proteins increased from 1 nm (Dcyto = 16.3 ± 3 μm2/s) to 7 nm (Dcyto = 4.0 (± 0.7) × 10−3 μm2/s) [44]. This slower-than-predicted diffusion of the proteins or nanoparticles may be explained by an elasticity of F-actin/α-actinin networks in the cell cytoplasm [45]. 3.4.3

Dendrimer–Actin Interactions

We have evidence of an interaction due to the electrostatic attraction between the dendrimer and actin cytoskeleton in vitro, and we have determined the diffusion coefficient of the dendrimer in the presence of actin gel using the fluorescent recovery after photobleaching (FRAP) technique. The ratio Dactin/D0 of the dendrimer was found to be 0.26, at a concentration of actin gel of 1 mg/mL, much lower than the concentration inside cells (∼10 mg/mL). Although the medium in which particles diffuse may approximate to that of pure water [16, 46], the auxiliary reduction of the translation diffusion of the dendrimers in cytoplasm can be attributed to microscopic barriers, enhanced tortuosity, and binding. Further studies on this are needed.

c03.indd 44

7/30/2010 2:18:40 PM

DIFFUSION AND UPTAKE OF DENDRIMER AND DENDRIPLEXES

A

B

C

D

45

Relative fluorescent intensity (F)

E

90 80 70 60 50 40 30 20 10 0 0

20

40

60

Time (min)

80

100

ROI 2 ROI 3 ROI 4 ROI 5 ROI 6 ROI 7 ROI 8 ROI 9 ROI 10 ROI 11 ROI 12 ROI 13

White boxes are regions of interest (ROIs)

Figure 3.3. An overlay of the compressed z-stack images of the dendriplex uptake process in living Caco-2 cells. The cells were incubated with complete medium containing dendriplexes (represented in green) and images were collected every 5 min before (A) and after adding the dendrimer (B–D). Images presented here are at time t = 5 (B), 30 (C), and 60 (D) min of incubation period. Data were analyzed and plotted in (E) and the insert pictures show the positions of regions of interest (ROIs, white boxes) for data analysis in both channels. The mean fluorescent signal in each ROI was measured from projected z-stack images taken at different time points, with background subtraction. The dendriplexes were found to be taken up into the cytoplasmic compartment (ROI 2–3, 7, 9, 12–13) but there is no uptake of the dendriplexes into the nuclear compartment within 2 h of incubation (ROI 4, 8, 10). (See color insert.)

c03.indd 45

7/30/2010 2:18:40 PM

46

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

3.4.4

Dendriplex Diffusion

The diffusion coefficients of the dendriplexes were expected to be lower than that of the parent dendrimer as they consist of many dendrimer molecules complexed with the DNA. In the dynamic uptake study (Figure 3.3) they were found after 2 h only in the cytoplasmic compartment throughout the stack images (A–D). Although dendriplexes were first found attached to the cell membrane after 5 min incubation, they remained in the cytosol until the end of the experiment (A–D). This was confirmed by the quantification of the fluorescent signal of the dendriplexes as plotted in Figure 3.3E (ROI 2–3, 7, 9, 12–13). There is no fluorescence in the nuclear compartment after 2 h. Changes in cell morphology were found in the cells after 2 h under the conditions of the experiment. Further work was carried out using living cells incubated with the dendrimer at longer time points; cells were removed to view at the end of each selected time point. The dendriplexes were found to be taken up into the nuclear compartment after 2.5 h of incubation and uptake was seen to be widespread in nearly every cell after 3 h. As this lag time is long, the calculated diffusion coefficient of the dendriplexes is lower than that of the parent dendrimer, as shown in Table 3.1. The lag time (tL) here was found to be between 2.5 and 3 h (n = 5), the diffusion coefficient of the dendriplexes in cytoplasm (Dcyto) was calculated to be 2.36 (± 0.34 (SD)) × 10−11 cm2/s (2.36 × 10−3 μm2/s) and the diffusion coefficients (Dcyto) relative to that in water (4.53 × 10−8 cm2/s (D0)) was 5.21 (± 0.75) × 10−4. In Caco-2 cells the cytoplasmic diffusion coefficient, Dcyto, of the dendriplexes relative to the dendrimer was found to be 0.24. The cytoplasm hinders the mobility of the dendriplexes up to 2000 times that in water. This might be explained by the interaction between the cationic dendriplexes and the cell organelles, which impede the mobility of the dendriplexes apart from the effect of particle size, sieving effects, and obstruction of the crowded condition of the cells. The larger unit should be more affected by the obstruction effect. Whether or not binding of the dendriplex, say, to actin filaments occurs is not known; it may be that dissociation of the complex occurs and the diffusion coefficients are mixed values. TABLE 3.1 Diffusion Coefficients and Relative Diffusion Coefficients of the Parent Dendrimers and the Dendriplexes in Cell Cytoplasm Compared to Other Media Diffusion Coefficients in Various Media (cm2/s)

Relative Diffusion Coefficients (cm2/s)

SKMES-1 Cells

Dactin D0

Dcyto D0

Ddendriplexes Ddendrimer

Dendrimer (6.5 nm)

79.54 × 10−8 23.75 × 10−8 9.82 × 10−11 5.99 × 10−11

0.26

—

Dendriplexes (107.3 nm)

4.53 × 10−8

1.24 × 10−4 (Caco-2) 0.76 × 10−4 (SKMES-1) 5.21 × 10−4

Nanoparticles

c03.indd 46

Water

Actin Gel

—

Caco-2 Cells

2.36 × 10−11

—

—

0.24

7/30/2010 2:18:40 PM

MECHANISMS OF PARTICLE UPTAKE INTO THE CELL NUCLEUS

47

The differences are caused by two main factors: the physicochemical properties (size, charge, and mass) of the species and the uptake processes. The hydrodynamic diameter and zeta potential of the dendriplexes were, respectively, 107.3 nm and 39.80 mV compared to values for the parent dendrimer of 6.5 nm and 47.27 mV. The positive surface charge of both compounds facilitates electrostatic attraction with the cell membrane as a prelude to being taken up by endocytosis. Concomitantly, the dendrimer may be taken up by transient nanopores, which can be created after adherence to the cell membrane, allowing also passive diffusion and resulting in the shorter uptake process [28–32]. The larger size of the dendriplexes would attenuate their diffusion inside the cytoplasm. In comparison to the native dendrimer the diffusion coefficient of the dendriplex is reduced by a factor of 4. One might have expected a larger decrease simply because the radius has increased by ∼15-fold from 6.5 to 107 nm (D0 of dendrimer and dendriplexes are 79.54 × 10−8 and 4.53 × 10−8 cm2/s, respectively) in accord with the Stokes–Einstein equation. The diffusion coefficient in water of the dendriplexes is 20 times lower than that of the dendrimer. Although particle size precisely controls the diffusion of the particle in bulk or aqueous solution, it was found to also play a more complex role in the diffusion of the two particle types discussed here. Dcyto values of dendriplexes and dendrimer were reduced from 9.82 × 10−11 to 2.36 (± 0.34 (SD)) × 10−11 cm2/s (fourfold reduction). This may be related somehow to uptake pathways. In the cells, diffusion coefficients generally can be explained by the obstruction effect and sieving effects, which are a function of particle size (Figure 3.4A) [13, 25, 47–49]. Janson and Luby-Phelps proposed the pore slit model having an average cutoff radius between 15 and 50 nm. Following this model the diameter of the dendriplexes is beyond the maximum cutoff size and thus cannot pass through the cellular meshwork, which presents as the slow mobility of the dendriplexes at the rim of the plasma membrane, and their ability to traverse the cytoplasm. 3.5 MECHANISMS OF PARTICLE UPTAKE INTO THE CELL NUCLEUS The cell nucleus is the ultimate target to achieve efficient gene transfection. The exact process by which lipoplexes deliver their plasmid DNA to nuclei after escaping from endosomes has remained elusive. The nuclear pore complex (NPC) responsible for nucleocytoplasmic transport [51, 52] plays a fundamental role in human and other eukaryotic cells. The NPC possesses remarkable transport competencies with two distinct modes: passive and facilitated (or active) transport [53, 54]. Passive transport is a nonspecific process that involves ordinary diffusion of matter through the nuclear pore with a cutoff at about 10 nm in diameter [50] and/or molecular mass less than 25 kDa [53]. Facilitated transport is the highly specific process acting against concentration gradients [55] with cutoff diameters of approximately 50 nm [50] and a molecular mass (m) range of 25 < m < 75 kDa [53]. This limit easily includes

c03.indd 47

7/30/2010 2:18:40 PM

48

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

the size of most protein transport substrates and macromolecules, for which the molecular weight of the cargo–receptor complex should be in the low hundreds of kilodaltons. In addition, Thachenko et al. [56] have suggested a cutoff size for particles for targeted nuclear delivery; particles should have diameters less than 100 nm to enter the cell membrane and less than 30 nm to be imported through the NPC, respectively. The passive diffusion of the DNA through nuclei in nonmitotic cells has been reported [57, 58]. Salman et al. [57] found that the uptake of DNA, 2 nm in linear dimensions, is independent of ATP or GTP hydrolysis, indicating linear diffusion without the need for conformation change or specific biochemical interaction with nuclear pore complexes (NPCs). The kinetic results, however, show diffusion to be much slower than would be estimated from purely hydrodynamic considerations. This perhaps suggested that the DNA may possible disassemble from its carrier before it passively diffuses through the NPC into the nucleus. Once through the NPC the journey of the DNA is not over. The nucleus is crowded. Lukacs et al. [59] have found that DNA diffusion in the nucleus is negligible—and, unlike diffusion in the cytoplasm, independent of DNA size, in agreement with our data shown in Figure 3.2. Active transport entails nuclear localization signal (NLS) peptides that can be linked to the plasmid DNA; this peptide triggers the transport of the DNA through the NPC by forming a complex with nuclear membrane importers, but such a strategy does not always produce higher transfection levels and can indeed reduce the effect, possibly because the added peptide changes the physical properties of the complex, not least its size or susceptibility to aggregation [60, 61]. The dendrimers and the dendriplexes traverse the cell cytoplasm to the nucleus as can be proposed in the model in Figure 3.4. The 1000- and 2000 fold reduction in diffusion coefficient of the dendrimer and the dendriplexes in the cell cytoplasm can be dissected into two main steps: diffusion within cell cytoplasm and passage through the nuclear pore complex (NPC). The factors involved in the first step are the obstruction and exclusion effects regarding the molecular crowding and the sieving effect mentioned earlier. The cell cytoplasm is depicted here acting as a meshwork, providing a sieving effect with maximum cutoff size at 50 nm in radius (Figure 3.4A). Likewise for a gel, the meshwork structure and crowded state appear as an insoluble compartment through which the dendrimer and the dendriplexes cannot diffuse (Figure 3.4B). Thus the dendrimer and the dendriplexes have to detour in the nonexcluded aqueous volume, until they reach their destination. After endocytosis or passive diffusion through transient nanopores ( – ), the dendrimer (red arrow) and the dendriplexes (green arrow) diffuse toward the nuclear compartment. Some of the dendrimer may interact with cell organelles ( ) and the interaction can be found after endosomal escape of the dendriplexes ( ). Particle size and type of carrier system seem to play a decisive role in the diffusion of the particles in this first step. After endosomal escape, the debate is still whether or not DNA disassembles from the dendrimer and how the DNA reaches the nucleus. Knowledge

c03.indd 48

7/30/2010 2:18:40 PM

49

MECHANISMS OF PARTICLE UPTAKE INTO THE CELL NUCLEUS

A

Cell radius (h) B 4 1

Dendriplexes Dendrimer

2 3

Cell membrane

Nuclear membrane

FG surface

C 1

Cytoplasmic filaments Threading area

2

Passive transport

Selectivity filter Nuclear filaments

3

Active transport

Figure 3.4. An uptake model for dendrimer and dendriplexes: (A) demonstrates the sieving effect of the cytoplasmic meshwork with a cutoff size of 100 nm in diameter. The crowded condition and the obstruction effect cause the slow diffusion of the dendrimer and dendriplexes after endocytosis ( – ) or passive diffusion ( ) through transient nanoholes, 1–10 nm in diameter, illustrated in (B). The dendrimer and dendriplexes have to detour in the aqueous compartment excluding crowded volumes. Some of the dendrimer and dendriplexes may interact with cell organelles or cytoplasmic proteins ( ). Passing through the NPC, the DNA released from the endosome ( ) may cross the NPC by passive diffusion ( ). As small as 6.5 nm, the dendrimer can diffuse through the central pore having a cutoff size of 8–10 nm ( ), whereas the larger of the dendriplexes, 107 nm, may employ active transport ( ). (Part C – is modified from Peters [50]; other parts are original.) (See color insert.)

c03.indd 49

7/30/2010 2:18:40 PM

50

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

of transport mechanisms through the nuclear pore complexes (NPCs) might help to make this clear. A vertebrate somatic cell usually contains between 1000 and 10,000 NPCs [62]. Transport of the DNA through the NPC is assumed to be the rate-limiting step for gene therapy, a process divided into two strategies: passive and active pathways (Figure 3.4C). Particles with diameters between 8 and 10 nm can be transported by passive diffusion, whereas particles of larger size and particles with molecular masses between 25 and 75 kDa [53] utilize active transport processes. Recent models of the NPC have been proposed as a hydrogel [60, 63, 64], as seen in Figure 3.4C. The selectivity is supplied by the meshwork construction of the phenylalanine glycine (FG) motif and the nonselectivity or passive transport is acquired from the aqueous tube of the channel center, 8–10 nm in diameter. DNA and dendrimers may passively diffuse though this channel (Figure 3.4C – ), while the dendriplexes may have to interact as transport complexes in the perinuclear membrane. These transport complexes are searching for the FG motif in the “threading area,” seen in Figure 3.4C , and they will only selectively passage via FG active transport pathways through the NPC (Figure 3.4C ).

3.6

CONCLUSION

The intrinsically fluorescent lysine-based dendrimer used in our work has proved to be useful as a nanofluorescent probe for tracking uptake and translocation within the cells, as it possesses a fluorescent signal detected by both spectrofluorimetry and confocal microscopy. The uptake of the dendrimer and dendriplexes formed from it depend on the cell type and naturally incubation periods. The dendrimer is taken up into the nuclear compartment. Dendriplexes, because of their size, do so also at a slower rate. One uptake mechanism investigated here was found to be due to endocytic-mediated endocytosis. Other uptake mechanisms have also been proposed. A model of the uptake pathway of the dendrimer and the dendriplexes from the cytoplasm throughout the nuclear pore complexes (NPCs) has been proposed here. Macromolecular crowding in the cytoplasm presents a significant barrier to diffusion of dendrimer and dendriplexes and this has been discussed here in terms of exclusion and obstruction effects. Finally, the uptake of the dendrimer was found to be impeded by the influence of fluid medium flow. The diffusion of the dendrimer and the dendriplexes within the cell nucleus may be expected to be dependent on similar key factors obtained inside the cell cytoplasm, but the nucleus is even more crowded than the cytoplasm. Whether or not the dendriplexes release their DNA before or after passing through the NPC is not known. The transport of the parent dendrimer through the NPC into the cell nucleus is expected to be dependent on passive diffusion. Further investigation to clarify this issue is indispensable so that more successful gene delivery systems can be developed. The main challenge is to determine why there are differences in transfection and diffusion in different cell

c03.indd 50

7/30/2010 2:18:41 PM

REFERENCES

51

types, and more work needs to be done on tissues in vivo, with the challenges of getting sufficiently high concentration gradients of nanoparticles such as dendimers and dendriplexes. The physical nature of the cytoplasm in so far as it allows translocation of particles requires further definition and investigation. In conclusion, an understanding of movement of delivery vectors within cells and in the nucleus is essential for the development of effective systems. Good models of the physical structure of cell interiors are required along with probes such as the one described here to advance the design of new delivery vectors. REFERENCES 1. Tomalia, D. A. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog. Polym. Sci. 30: 294–324 (2005). 2. Perumal, O. P., Inapagolla, R., Kannan S., and Kannan, R. M. The effect of surface functionality on cellular trafficking of dendrimers. Biomaterial 29: 3469–3476 (2008). 3. El-Sayed, M., Kiani, M. F., Naimark, M. D., Hikal, A. H., and Ghandehari, H. Extravasation of poly(amidoamine) (PAMAM) dendrimers across microvascular network endothelium. Pharm. Res. 18: 23–28 (2001). 4. Preston, R. A., Murphy, R. F., and Jones, E. W. Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran. J. Cell Biol. 105: 1981–1987 (1987). 5. Lundberg, M. and Johansson, M. Positively charged DNA-binding proteins cause apparent cell membrane translocation. Biochem. Biophys. Res. Commun. 291: 367–371 (2002). 6. Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. Cell-penetrating peptides: a reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278: 585–590 (2003). 7. Al-Jamal, K. T., Ruenraroengsak, P., Hartell, N., and Florence, A. T. An intrinsically fluorescent dendrimer as a nanoprobe of cell transport. J. Drug Target. 14: 405–412 (2006). 8. Dickinson, R. B. and Purich, D. I. Diffusion rate limitations in actin-based propulsion of hard and deformable particles. Biophys. J. 91: 1548–1563 (2006). 9. Ruenraroengsak, P. and Florence, A. T. The diffusion of latex nanospheres and the effective (microscopic) viscosity of HPMC gels. Int. J. Pharm. 298: 361–366 (2005). 10. Ellis, R. J. and Minton, A. P. Cell biology: join the crowd. Nature 425: 27–28 (2003). 11. Fulton, A. B. How crowded is the cytoplasm? Cell 30: 345–347 (1982). 12. Goodsell, D. S. Inside a living cell. TIBS 16: 203–206 (1991). 13. Luby-Phelps, K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. 192: 189–221 (2000).

c03.indd 51

7/30/2010 2:18:41 PM

52

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

14. Medalia, O., Weber, I., Frangakis, A. S., Nicastro, D., Gerisch, G., and Baumeister, W. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science 298: 1209–1213 (2002). 15. Zimmerman, S. B. and Minton, A. P. Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu. Rev. Biophys. Biomol. Struct. 22: 27–65 (1993). 16. Kao, H. P., Abney, J. R., and Verkman, A. S. Determinants of the translational mobility of a small solute in cell cytoplasm. J. Cell Biol. 120: 175–184 (1993). 17. Furukawa, R., Arauz-Lara, J. L., and Ware, B. R. Self-diffusion and probe diffusion in dilute and semidilute aqueous solutions of dextran. Macromolecules 24: 599–605 (1991). 18. Phillies, G. D. J. The hydrodynamic scaling model for polymer self-diffusion. J. Phys. Chem. 93: 5029–5039 (1989). 19. Hou, L., Lanni, F., and Luby-Phelps, K. Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm. Biophys. J. 58: 31–43 (1990). 20. Clegg, J. S. Properties and metabolism of the aqueous cytoplasm and its boundaries. Am. J. Physiol. 246: R133–R151 (1984). 21. Mastro, A. M., Babich, M. A., Taylor, W. D., and Keith, A. D. Diffusion of a small molecule in the cytoplasm of mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 81: 3414–3418 (1984). 22. Nicolay, K., van der, T. A., and Dijkhuizen, R. M. In vivo diffusion spectroscopy: an overview. NMR Biomed. 8: 365–374 (1995). 23. Verkman, A. S., Armijo, M., and Fushimi, K. Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains. Biophys. Chem. 40: 117–125 (1991). 24. Luby-Phelps, K., Mujumdar, S., Mujumdar, R. B., Ernst, L. A., Galbraith, W., and Waggoner, A. S. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65: 236–242 (1993). 25. Luby-Phelps, K., Castle, P. E., Taylor, D. L., and Lanni, F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 84: 4910–4913 (1987). 26. Aschenbrenner, L., Naccache, S. N., and Hasson, T. Uncoated endocytic vesicles require the unconventional myosin, myo6, for rapid transport through actin barriers. Mol. Biol. Cell 15: 2253–2263 (2004). 27. Conner, S. D. and Schmid, S. L. Regulated portals of entry into the cell. Nature 422: 37–44 (2003). 28. Hong, S., Bielinska, A. U., Mecke, A., Keszler, B., Beals, J. L., Shi, X., Balogh, L., Orr, B. G., Baker, J. R., Jr., and Banaszak Holl, M. M. Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport. Bioconjug. Chem. 15: 774–782 (2004). 29. Hong, S., Leroueil, P. R., Janus, E. K., Peters, J. L., Kober, M. M., Islam, M. T., Orr, B. G., Baker, J. R., Jr., and Banaszak Holl, M. M. Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. Bioconjug. Chem. 17: 728–734 (2006). 30. Mecke, A., Lee, D. K., Ramamoorthy, A., Orr, B. G., and Banaszak Holl, M. M. Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers. Biophys. J. 89: 4043–4050 (2005).

c03.indd 52

7/30/2010 2:18:41 PM

REFERENCES

53

31. Mecke, A., Lee, D. K., Ramamoorthy, A., Orr, B. G., and Holl, M. M. Synthetic and natural polycationic polymer nanoparticles interact selectively with fluid-phase domains of DMPC lipid bilayers. Langmuir 21: 8588–8590 (2005). 32. Mecke, A., Majoros, I. J., Patri, A. K., Baker, J. R., Jr., Holl, M. M., and Orr, B. G. Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group. Langmuir 21: 10348–10354 (2005). 33. Morris, M. C., Deshayes, S., Heitz, F., and Divita, G. Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol. Cell 100: 201–217 (2008). 34. Mislick, K. A. and Baldeschwieler, J. D. Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93: 12349–12354 (1996). 35. Manunta, M., Izzo, L., Duncan, R., and Jones, A. T. Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging. J. Drug Target. 15: 37–50 (2007). 36. Woodle, M. C. and Scaria, P. Cationic liposomes and nucleic acids. Curr. Opin. Colloid Interface Sci. 6: 78–84 (2001). 37. Manunta, M., Nichols, B. J., Tan, P. H., Sagoo, P., Harper, J., and George, A. J. Gene delivery by dendrimers operates via different pathways in different cells, but is enhanced by the presence of caveolin. J. Immunol. Methods 314: 134–146 (2006). 38. Elouahabi, A. and Ruysschaert, J. M. Formation and intracellular trafficking of lipoplexes and polyplexes. Mol. Ther. 11: 336–347 (2005). 39. Ruenraroengsak, P., Al-Jamal, K. T., Hartell, N., Braeckmans, K., De Smedt, S. C., and Florence, A. T. Cell uptake, cytoplasmic diffusion and nuclear access of a 6.5 nm diameter dendrimer. Int. J. Pharm. 331: 215–219 (2007). 40. Sagidullin, A. I., Muzafarov, A. M., Krykin, M., Ozerin, A. N., Skirda, V. D., and Ignat’eva, G. M. Generalized concentration dependence of self-diffusion coefficients in poly(allylcarbosilane) dendrimer solution. Macromolecules 35: 9472–9479 (2002). 41. Xia, T., Kovochich, M., Liong, M., Zink, J. I., and Nel, A. E. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS Nano 2: 85–96 (2008). 42. Kaether, C. and Gerdes, H. H. Monitoring of protein secretion with green fluorescent protein. Methods Enzymol. 302: 11–19 (1999). 43. Bayele, H. K., Sakthivel, T., O’Donell, M., Pasi, K. J., Wilderspin, A. F., Lee, C. A., Toth, I., and Florence, A. T. Versatile peptide dendrimers for nucleic acid delivery. J. Pharm. Sci. 94: 446–457 (2005). 44. Arrio-Dupont, M., Foucault, G., Vacher, M., Devaux, P. F., and Cribier, S. Translational diffusion of globular proteins in the cytoplasm of cultured muscle cells. Biophys. J. 78: 901–907 (2000). 45. Tseng, Y. and Wirtz, D. Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks. Biophys. J. 81: 1643– 1656 (2001). 46. Fushimi, K. and Verkman, A. S. Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. J. Cell Biol. 112: 719–725 (1991).

c03.indd 53

7/30/2010 2:18:41 PM

54

CYTOPLASMIC DIFFUSION OF DENDRIMERS AND DENDRIPLEXES

47. Janson, L. W., Ragsdale, K., and Luby-Phelps, K. Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains. Biophys. J. 71: 1228–1234 (1996). 48. Luby-Phelps, K. Effect of cytoarchitecture on the transport and localization of protein synthetic machinery. J. Cell Biochem. 52: 140–147 (1993). 49. Provance, D. W. Jr., McDowall, A., Marko, M., and Luby-Phelps, K. Cytoarchitecture of size-excluding compartments in living cells. J. Cell Sci. 106(2): 565–577 (1993). 50. Peters, R. Introduction to nucleocytoplasmic transport: molecules and mechanisms. Methods Mol. Biol. 322: 235–258 (2006). 51. Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E., and Goldberg, M. W. The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. J. Cell Sci. 113(10): 1651–1659 (2000). 52. Rout, M. P., Aitchison, J. D., Suprapto, A., Hjertaas, K., Zhao, Y., and Chait, B. T. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148: 635–651 (2000). 53. Lusk, C. P., Blobel, G., and King, M. C. Highway to the inner nuclear membrane: rules for the road. Nat. Rev. Mol. Cell Biol. 8: 414–420 (2007). 54. Bickel, T. and Bruinsma, R. The nuclear pore complex mystery and anomalous diffusion in reversible gels. Biophys. J. 83: 3079–3087 (2002). 55. Dingwall, C., Sharnick, S. V., and Laskey, R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30: 449–458 (1982). 56. Tkachenko, A. G., Xie, H., Liu, Y., Coleman, D., Ryan, J., Glomm, W. R., Shipton, M. K., Franzen, S., and Feldheim, D. L. Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjug. Chem. 15: 482–490 (2004). 57. Salman, H., Zbaida, D., Rabin, Y., Chatenay, D., and Elbaum, M. Kinetics and mechanism of DNA uptake into the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 98: 7247–7252 (2001). 58. de Gennes, P. G. Passive entry of a DNA molecule into a small pore. Proc. Natl. Acad. Sci., U.S.A. 96: 7262–7264 (1999). 59. Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275: 1625–1629 (2000). 60. Sakthivel, T., Toth, I., and Florence, A. T. Synthesis and physicochemical properties of lipophilic polyamide dendrimers. Pharm. Res. 15: 776–782 (1998). 61. Toth, I., Sakthivel, T., Wilderspin, A. F., Toth, I., Bayele, H. K., O’Donnell, M., Perry, D. J., Pasi, K. J., Lee, C. A., and Florence, A. T. Novel cationic lipidic peptide dendrimer vectors—in vitro gene delivery. STP Pharm. Sci. 9: 93–99 (1999). 62. Burke, B. Cell biology. Nuclear pore complex models gel. Science 314: 766–767 (2006). 63. Frey, S., Richter, R. P., and Gorlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314: 815–817 (2006). 64. Elbaum, M. Materials science. Polymers in the pore. Science 314: 766–767 (2006).

c03.indd 54

7/30/2010 2:18:41 PM

CHAPTER 4

Endocytosis and Intracellular Trafficking of Quantum Dot–Ligand Bioconjugates TORE-GEIR IVERSEN, NADINE FRERKER, and KIRSTEN SANDVIG Centre for Cancer Biomedicine, Faculty Division, Norwegian Radium Hospital, University of Oslo, and Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway

4.1

INTRODUCTION

Nanoparticles (NPs) are gaining increasing relevance for use in biological and biomedical applications both within diagnostic imaging and therapeutic drug delivery systems. These applications require the targeting of specific tissues and cells, and important for several of the actions is the uptake of NPs into cells. In vivo and in vitro labeling with NPs allow detection and tracing of molecules into cells as well as cell fractionation. The entry of NPs into a targeted cell is determined and restricted by the endocytic pathways operating. Not all endocytic mechanisms have been identified and characterized so far. The following paragraphs give a short overview of the most studied pathways and introduce important factors and observations relevant in endocytosis and trafficking of NPs.

4.2

DIVERSITY OF ENDOCYTIC MECHANISMS

Endocytosis is a process in which molecules are internalized into the cell interior without passing through the membrane. There are different types of Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

55

c04.indd 55

7/30/2010 2:18:43 PM

56

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

pinocytosis

phagocytosis clathrin-mediated endocytosis

clathrin-independent endocytosis

dynamin-dependent endocytosis

dynamin-independent endocytosis

RhoA = dynamin = clathrin

caveolae

Cdc42

Arf6

Rac ruffling

Figure 4.1. Endocytic pathways. Phagocytosis (left: enclosure of large particle) and different forms of endocytosis—clathrin-mediated endocytosis and clathrinindependent forms such as macropinocytosis, caveolae, RhoA-, Cdc42-, and Arf6mediated endocytosis.

endocytosis—each involving the formation of intracellular vesicles by means of invagination of the plasma membrane and membrane fission. In general, a rough division can be made between phagocytosis (“cell eating”) and pinocytosis (“cell drinking”). Large particles such as bacteria are taken up via phagocytosis. The particle is engulfed by extrusions from the cell membrane (a “phagocytic cup”), which then mediates the formation of a phagosome. Phagocytosis occurs mainly in specialized mammalian cells such as macrophages, monocytes, and neutrophils but also to a lesser extent in nonprofessional phagocytes (i.e. fibroblasts, endothelial and epithelial cells). Phagocytosis constitutes the initial step for the degradation of particles larger than 0.5 μm. Pinocytosis is used to internalize fluid simultaneously with whatever substance is found within the area of invagination. There are multiple types of endocytotic pathways, which can be classified in part based on their requirement for the coat protein clathrin, the GTPase dynamin, or the formation of caveolae. Thus a general division into clathrindependent/independent and dynamin-dependent/independent endocytic pathways can be made (Figure 4.1). 4.2.1

Clathrin-Dependent Endocytosis

The clathrin-dependent endocytosis is one of the best characterized endocytic pathways and constitutes a major route for selective receptor internalization

c04.indd 56

7/30/2010 2:18:43 PM

DIVERSITY OF ENDOCYTIC MECHANISMS

57

in eukaryotic cells [1]. The formation of a clathrin-coated pit starts with adaptor protein-mediated recruitment of clathrin to the plasma membrane, where the adaptor proteins bind transmembrane receptor molecules with cargo. Finally, the coated pit invaginates and, regulated by the GTPase dynamin and GTP, pinches off to form a clathrin-coated vesicle. In general, clathrin-mediated endocytosis (CME) is a rapid uptake process (<2 min). 4.2.2

Clathrin-Independent Endocytosis

A number of clathrin-independent pathways that can be classified upon involvement of dynamin and various other GTPases have been detected so far. A classification scheme based on dependence on the small Ras-superfamily GTPases RhoA, Cdc42, and Arf6 and the formation of caveolae has been suggested [2]. The GTP-binding protein dynamin is required in caveolar as well as in RhoA-regulated endocytosis. Caveolae are flask-shaped plasma membrane invaginations of 50–100 nm in diameter that are enriched in sphingolipids, cholesterol, and the transmembrane protein caveolin [3]. Although caveolin-1 has been shown to be important for the formation of caveolae, morphologically similar structures to caveolae have been observed in some cells that lack caveolin proteins [4, 5]. Interestingly, overexpression of flotillin1 and flotillin2 in HeLa cells was recently reported to generate caveolae-like structures [6]. Caveolae are mostly static structures but they can be induced to pinch off by certain ligands such as SV40 [7, 8]. Moreover, less characterized endocytic pathways exist sharing some of the above mentioned characteristics or none of them [9, 10]. In this context, the existence of polymorphous structures with an elongated and tubular shape that endocytose glycosphingolipids and other plasma membrane components have been described [5]. Similar structures can be induced by the Shiga toxin B subunit [11]. An endocytic mechanism involving tubular structures (CLICs, clathrin-independent carriers) has been proposed [12, 13], and recently GRAF1 was identified as specific marker for CLICs [14]. However, the picture is not clear since it has been reported that CLICs can form without dynamin whereas GRAF1 is able to bind dynamin. Macropinocytosis, a special form of pinocytosis, is one of the best characterized types of clathrin-independent endocytosis, and it is often preceded by the actin-driven process of membrane ruffling, which can be stimulated by growth factors (for recent reviews see Mercer and Helenius [15] and Kerr and Teasdale [16]). In macropinocytosis, the cell engulfs mass quantities of fluid after formation of protrusions that can be lamellipodia-like, bleb-shaped, or appear in the form of circular ruffles. These protrusions may collapse onto and fuse with the plasma membrane. After membrane fission, large noncoated vesicles of heterogeneous sizes (0.5–10 μm in diameter) and irregular in

c04.indd 57

7/30/2010 2:18:43 PM

58

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

shape—the macropinosomes—are formed. Macropinocytosis depends on several kinases such as p21-activated kinase 1 or protein kinase C [17], but also Rho family GTPases such as Rac1 [18], which plays an important role in remodeling of the actin cytoskeleton, can be involved.

4.3 UPTAKE OF NANOPARTICLES OF VARIOUS SIZE AND SURFACE CHARGE Endocytosis mechanisms involved in uptake of nanoparticles of varying sizes and compositions have been investigated in a number of studies relying mainly on inhibitors that are supposed to perturb a specific endocytic pathway. It has been shown that anionic polymeric particles of 24 nm were endocytosed in HeLa and primary HUVEC cells via a cholesterol-independent, non-clathrinand non-caveolae-dependent pathway, which did not route them to the degradative pathway, whereas particles with a size of 43 nm were mainly internalized via clathrin-dependent endocytosis and further directed to the endolysosomal pathway, suggesting that cells can precisely distinguish and accordingly sort these particles via different uptake mechanisms [19]. In another study, Hoekstra and co-workers examined the uptake mechanisms of latex beads of various sizes (50, 100, 200, 500, and 1000 nm) in the nonphagocytic mouse cell line B16 by using inhibitors of either clathrin-dependent or caveolae-dependent endocytosis to discriminate between these two pathways. They found that beads with a diameter below 200 nm were internalized via clathrin-mediated endocytosis (CME), although at slower rates for the larger particles. Surprisingly, considering the restricted size of caveolae, they reported that a caveolae-dependent mechanism was the predominant endocytic mechanism for particles of 500 nm, whereas no uptake could be observed with particles of 1000 nm size [20]. The glycosphingolipid (GSL) lactocyl-ceramide (LacCer) was used as a fluorescent marker for caveolae and was found to colocalize with the 500-nm nanoparticles. However, one should be cautious when using Bodipy-LacCer as a marker for caveolae since it has been shown that exogenously added GSL can cause formation of GSL-enriched lipid domains at the plasma membrane and induce internalization of integrins via caveolae-like structures [21]. DNA lipoplexes containing the cationic lipid DOTAP were exclusively internalized by CME, while polyplexes prepared from the cationic polymer polyethylene imine (PEI) were internalized equally by both clathrin-dependent and clathrin-independent pathways. However, luciferase transfection by the PEI/DNA-polyplexes was blocked by inhibitors such as genistein and filipin, and the result was interpreted as involvement of caveolae. However, it should be noted that filipin can be expected to inhibit any cholesterol-dependent internalization, and genistein is an inhibitor of tyrosine phosphorylations that will affect uptake via other pathways as well, for instance, uptake through clathrin-coated pits when

c04.indd 58

7/30/2010 2:18:43 PM

UPTAKE OF NANOPARTICLES OF VARIOUS SIZE AND SURFACE CHARGE

59

receptor phosphorylations on tyrosine are involved. Interestingly, the fraction internalized via the caveolae-mediated pathway was not directed into lysosomes [22, 23]. In general, particles with positive surface charge will bind to the negatively charged membrane proteins and lipids at the cell surface and are therefore likely to be easily captured and taken up. Thus the surface charge of the NPs could be decisive for endocytosis and trafficking. A study of endocytosis of cationic and anionic polymeric NPs with a similar size (80 nm) in HeLa cells showed that the cationic NPs displayed a twofold higher endocytosis rate compared to the anionic NPs [24]. In addition, cationic beads were internalized via CME, whereas internalization of anionic beads was shown to be independent of clathrin and caveolin. Suprisingly, upon inhibition of dynamin-dependent endocytosis by expression of dominant negative mutant dynamin, there was an increase in endocytosis especially of the cationic NPs, which was attributed to the induction of macropinocytosis previously reported in these cells [25]. In another study, Harush-Frenkel et al. [26] found that, in polarized MDCK cells, anionic as well as cationic polymeric nanoparticles mainly entered the apical pole of these cells via CME. Furthermore, a significant fraction of the NPs was also transcytosed to the basolateral pole. Recently, it was suggested that uptake of charged polystyrene NPs (120 nm) in HeLa cells occurred via a dynamin-dependent and lipid-raft-dependent pathway independently of their surface charge [27]. Macropinocytosis seemed to be involved in uptake of the cationic NPs due to the inhibiting effect of 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Surprisingly, cholesterol depletion and filipin treatment did not inhibit uptake of the NPs, which contradicts previous studies demonstrating that cholesterol is involved both in macropinocytosis and lipid-raft-dependent uptake. Chlorpromazine was used as an inhibitor of CME and resulted in a minor inhibition in the uptake of positively charged NPs, but had no effect in uptake of the negatively charged NPs. The effect of surface charge on the uptake of mesoporous silica NPs (MSNs) has been studied in 3T3-L1 cells and mesenchymal stem cells (hMSCs) [28]: In both cell types, uptake of the anionic NP was inhibited by phenyl arsene oxide and cytochalasin-D, suggesting involvement of clathrinand actin-dependent endocytosis. With the strongly cationic MSNs, the inhibitory effects on uptake were only observed in the 3T3-L1 cells but not in the hMSCs, implying that the effect of NP surface charge is dependent on cell type. The endocytic pathway followed by a nanoparticle without bound ligands seems to depend on its size and surface charge. Macropinocytosis seems to be a preferred pathway for positively charged nanoparticles. However, endocytosis mechanisms followed by a certain nanoparticle apparently vary between cell lines and types depending on the endocytic pathways operating (Table 4.1). Thus most cell lines seem to have different endocytic mechanisms available for internalizing nanoparticles (<200 nm).

c04.indd 59

7/30/2010 2:18:43 PM

c04.indd 60

60 TABLE 4.1 NP Type

Endocytosis Studies on Different Nanoparticles Including QDs Charge (+/−)

Size (Diameter, nm)

Ligand

Cell Line

Reported Endocytosis Mechanisms or Intracellular Localization

mPeg-PLA

+32 mV −26 mV

Polystyrene

+ −

120 120

HeLa HeLa

Silica

+ −

110 110

Polystyrene

− − − −

24 45 and 200 11 2

3T3-L1 hMSC 3T3-L1, hMSC HeLa, HUVEC HeLa, HUVEC HeLa Macrophages

CME: inhibited by dynamin and clathrin mutants, then macropinocytosis; clathrin/ dynamin-independent Dynamin/actin-dependent, clathrinindependent, macropinocytosis Dyn/actin-dependent, clathrin-independent Clathrin/actin-dependent Clathrin/actin-independent Clathrin/actin-dependent in both cell types Clathrin/caveolae-independent pathway Clathrin-dependent Significant lysosomal localization Transport into the nucleus

−

6 25–30 45

A431 HeLa, HEp-2, SW480

Accumulation in the cytoplasm CME CME, inhibited by dynamin, no recycling but endosomal/lysosomal accumulation

CdSe QDs CdTe QDs

CdSe QDs CdSe QDs

90 96

Stearyl-amine

–COO− COO− DHLA Thioglycolic acid (TGA) TGA EGF Tf

HeLa dyn K44A

Reference 24

27

28

19 Here 29

30 31, 32

7/30/2010 2:18:43 PM

USE OF PHARMACOLOGICAL INHIBITORS AND MICROSCOPY

61

4.4 USE OF PHARMACOLOGICAL INHIBITORS AND MICROSCOPY IN STUDIES OF ENDOCYTOSIS Most studies on the various endocytosis mechanisms responsible for membrane and ligand internalization have so far involved the use of pharmacological inhibitors. It is important to stress that many of the inhibitors used in these studies are not specific for one endocytic mechanism. Genistein is an inhibitor of several tyrosine kinases and thus has been shown to inhibit internalization of ligands via clathrin-dependent endocytosis, in addition to affecting changes required for pinching off of caveolae. Furthermore, perturbing the function of cholesterol either by using filipin or by cholesterol depletion with methylβ-cyclodextrin has been shown to affect other endocytic pathways beside the caveolae-mediated ones. Interestingly, it has been demonstrated that cholesterol is required not only for raft-dependent endocytosis, giving rise to small vesicles, but also for macropinocytosis [33]. More robust conclusions should be obtained by using a collection of inhibitors, mutant proteins, and siRNAs. Internalization of fluorescently labeled NPs into cells has mostly been studied by confocal microscopy, which has a limited resolution. Nanoparticles might be forming clusters at the cell’s surface, giving rise to a spotted and vesicular pattern, and upon imaging of flat adherent cells it might be difficult to judge whether a spot represents a true vesicle even from a z-stack of confocal images. Therefore counterstaining with various endosomal markers and measuring the degree of colocalization are generally helpful in resolving whether the NPs have been internalized. Moreover, an apparent colocalization can be obtained from vesicles in close proximity, especially in the thicker perinuclear area of the cell, and should not necessarily be interpreted as localizing to the same organelle.

4.4.1

Endocytosis of Fluorescent QDs Studied by Microscopy

The production of compact functionalized QDs that are stable in serum/ blood and display little unspecific binding to cells is a prerequisite for their use as probes to target specific cells in vivo. Using the intrinsic lipophilic QDs in biological applications requires further modification of their surface to make them soluble in aqueous solution. This can be achieved by absorption of various amphiphilic polymers to the QDs or by exchange of the native capping agent with thiolated molecules such as peptides [34], cysteine [35], or dihydrolipoic acid (DHLA) [36]. The capping molecules might contain functional groups, carboxyls, or amines, for further covalent attachment to the QD surface of biological molecules such as proteins, peptides, or nucleic acids required to achieve the desired targeting.

c04.indd 61

7/30/2010 2:18:43 PM

62

4.4.2

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

Endocytosis of QDs Without Ligands that Bind to Receptors

Once the QDs are made hydrophilic and stable in aqueous media, they can bind to cells through electrostatic interactions with molecules at the cell surface. The cell surface displays an anionic charge, and therefore cationic NPs in general bind more strongly to the cells. Despite an overall electrostatic repulsion to the cell surface, anionic dihydrolipoic acid (DHLA)-capped CdSe/ZnS QDs at a high concentration (300 nM) have been found to bind unspecifically to cells and be endocytosed [37]. Recent studies have shown that unspecific binding to cells could be greatly reduced by attaching a PEG-spacer to DHLA while keeping a compact size of the QDs (<15 nm) [38]. Recently, we also monitored significant endocytosis of DHLA-capped QDs (hydrodynamic diameter, diamh, 11 nm) into HeLa cells after 1 h at a ∼tenfold lower concentration (30 nM) (Figure 4.2). After incubation for 1 h at 37 °C the particles displayed partial colocalization with the early endosomal marker EEA1 (Figure 4.2, upper panel), and after 4 h incubation they displayed a

DHLA-QDs 1h:

EEA1

CD63 merged

EEA1

CD63 merged

10 μm

DHLA-QDs

10 μm

10 μm

4h:

10 μm

10 μm

10 μm

Figure 4.2. Intracellular location of dihydrolipoic acid (DHLA)-capped quantum dots (QDs, Em. 585 nm) in HeLa cells. The DHLA-QDs (30 nM) in complete growth medium were added to the HeLa cells and incubated at 37 °C for 1 and 4 h, respectively. Yellow and magenta colors in the merged images indicate colocalization with the early endosomal marker EEA1 and with the lysosomal marker CD63, respectively. Bars, 10 μm. (See color insert.)

c04.indd 62

7/30/2010 2:18:43 PM

USE OF PHARMACOLOGICAL INHIBITORS AND MICROSCOPY

63

significant increase in colocalization with the lysosomal marker CD63 (Figure 4.2, lower panel). Interestingly, it has recently been demonstrated that differences in cancer cell phenotypes can lead to significant differences in intracellular sorting, trafficking, and localization of nanoparticles. Unconjugated anionic QDs displayed dramatically different intracellular profiles in three closely related human prostate cancer cells [39]. Moreover, hypoxia conditions in tumor cells have been show to change intracellular trafficking and sorting of receptor tyrosine kinase (RTK) receptors, delaying their transport down the endolysosomal pathway and thus their deactivation [40]. Furthermore, in a study of cellular uptake of tiny thioglycolic acid-capped CdTe QDs, Nabiev et al. [29] observed endocytosis in macrophages only, and not in epithelial or endothelial cells. Moreover, they reported that the intracellular distribution of the QDs varied with particle size, in that the smallest QDs (2 nm) seemed to target histones in the nucleus, whereas the larger QDs (6 nm) remained in the cytoplasm [29].

4.4.3

Endocytosis of Ligand-Bound QDs

In order to achieve the desired targeting to specific cells, various peptides, proteins, or nucleic acids can be coupled to the QDs. Another key issue to address is whether the functionalized QDs can act as relevant intracellular probes to investigate the routing of ligands in live cells. Except for a few recent studies, little information has been published about the mechanisms of intracellular trafficking of different Qdot-bound ligands [41–43]. QDs bound to receptors such as glycine and epidermal growth factor (EGF) receptors have been used to measure and resolve the dynamics of single receptors at the cell surface. Biotinylated epidermal growth factor has been conjugated to streptavidin-coupled QDs for the binding to and activation of EGF receptors at the cell surface [30]. Importantly, a retrograde transport of EGF-QDs from filopodia to the cell body was observed. Furthermore, the EGF-QDs were rapidly endocytosed and the internalized receptor dynamics were followed over time. Dahan and co-workers used QDs to track individual glycine receptors (GlyRs) and analyzed their lateral dynamics in neuronal membrane cells [44]. Very recently, the rate of lateral diffusion of individual glycosyl-phosphatidyl-inositide anchored proteins coupled to avidin (Av-GPI) has been studied by labeling with QD and using dual-color total internal reflection fluorescence (TIRF) microscopy [45]. It was shown that the Av-GPI probe exhibited a fast and a slow diffusion regime in different membrane regions, and that cholesterol-/ sphingolipid-rich microdomains can compartmentalize the diffusion of GPIanchored proteins. Internalization of the transferrin (Tf) bound iron by the transferrin receptor is the major route of iron uptake. The complexes are constitutively

c04.indd 63

7/30/2010 2:18:43 PM

64

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

endocytosed by clathrin-dependent endocytosis, and after release of iron in an acidic endosome, the Tf bound to its receptor very efficiently recycles from the sorting endosomes to the plasma membrane. Tf has also been investigated as a ligand for targeted delivery of therapeutic agents that suffer from nonoptimal pharmacokinetic properties [46]. Tf has been used both by us and by others to facilitate uptake of QDs and other NPs into various cells [47, 48]. In a recent study [31], we coupled biotinylated Tf to commercially available streptavidin QDs resulting in bioconjugates with a hydrodynamic diameter (diamh) of 40 nm. The Tf : QD bioconjugates specifically bound to the transferrin receptors and were internalized via dynamin-dependent endocytosis. The Tf : QDs displayed good colocalization with the early endosomal marker (EEA1) already after 15 min of endocytosis. After chasing of their endocytosis for 3–4 h they accumulated in more perinuclear endosomes, displaying only partial colocalization with lysosomal markers such as LAMP-1 and CD63. Endocytosis of Tf : QDs was also studied in two more cancer cell lines, HEp-2 cells and SW480, where the Tf : QDs displayed a more significant (40%) colocalization with the lysosomal marker after 3 h of endocytosis [32]. Thus, contrary to transferrin itself, they did not recycle to the plasma membrane. This endosomal retention outside lysosomes can be considered advantageous in relation to the issue of drug delivery and avoiding lysosomal degradation.

4.4.3.1 Toxin : QD Conjugates Several protein toxins have cytosolic targets, and studies of their uptake and intracellular transport have provided knowledge about mechanisms used to gain access to the cytosol as well as details about endocytosis mechanisms. Some toxins such as diphtheria toxin escape from endosomes in response to low endosomal pH, whereas Shiga toxin and ricin are transported all the way back to the Golgi apparatus and the ER before they are translocated into the cytosol. In fact, retrograde transport to the Golgi and ER is necessary for the toxins to reach the cytosol, where they exert their toxic effect [49]. If toxin : QD bioconjugates can be transported to the Golgi apparatus and the ER, this would facilitate studies of retrograde transport. So far, one has not been able to visualize ricin in the ER. Thus if the toxin : QDs could mediate transport to such destinations and be monitored there by live-cell microscopy, it would be a step forward in clarifying further details about endocytic and retrograde transport. Shiga toxin B subunit binds to the globotriaosyl ceramide Gb3 receptor, which is expressed and binds toxin on the cell surface of the kidney endothelial cells of children [50]. Gb3 is also overexpressed at the cell surface of various metastatic cancer cells, making the Shiga B subunit an interesting ligand for specific targeting of cytostatika to these cells and for imaging [51, 52]. Ricin B subunit binds to glycoproteins and glycolipids containing a terminal galactose moiety present at the cell surface of nearly all mammalian cells. Thus it

c04.indd 64

7/30/2010 2:18:44 PM

USE OF PHARMACOLOGICAL INHIBITORS AND MICROSCOPY

65

has been used to facilitate intracellular delivery of other molecules, such as immunotoxins. In our previous studies, we reported that the ricinB : QDs (diamh, 30 nm) were internalized and partially routed into perinuclear vesicles, displaying a significant overlap with lysosomal structures in HeLa, HEp-2, and SW480 cells after 3 h of endocytosis [31, 32]. However, another more dispersed fraction of these ricinB : QD-positive structure did not stain positive for lysosomal markers. Furthermore, whereas a fraction of ricin can be visualized in the Golgi apparatus after 3 h of endocytosis, we did not observe any colocalization of the ricinB : QDs with the Golgi markers used. The ricinB : QDs remained accumulated in these endosomes for 24 h. Cholera toxin subunit B-QD conjugates (diamh, 25 nm) have been found to be internalized into dispersed vesicles of various cells, where they also were retained for days [53]. Here, we show internalization of ricinB : QDs (molar ratio, 5 : 1) in HeLa cells and HEp-2 cells, respectively (Figure 4.3). A fraction of the ricinB : QDs was routed into LAMP-1 positive endosomes after 90 min of endocytosis in both cell types, and the degree of colocalization between ricinB : QDs and LAMP-1 was similar after 180 min. However, after internalization for 24 h a decrease in overall ricinB : QD fluorescence was observed in both cell lines. Moreover, the ricinB : QDs displayed less colocalization with LAMP-1. This suggests that the lysosomal content with accumulated ricinB : QDs was slowly secreted [54, 55], or that there was a certain extent of lysosomal and oxidative degradation of the QD coats resulting in aggregation of the particles and thereby to a decrease in their photoluminescence [56]. 4.4.3.2 Antibodies QDs coupled to antibodies or a humanized antibody fragment against cell surface proteins that are endocytosed is another way to deliver the QDs into the cell. This approach has been especially appealing for detection of cancer cells with a cancer-associated overexpressed membrane marker [57, 58]. 4.4.3.3 Peptide-Mediated Endocytosis Instead of using entire proteins as ligands, peptides can be bound to the QDs, for example, constituting the receptor-binding motif of the ligand. The TAT peptide motif derived from the HIV-1 virus TAT protein has proved to be a popular targeting molecule to deliver cargo into cells. It is a highly positively charged ligand that interacts with negatively charged receptors at the cell surface. The TAT peptide has been self-assembled onto the surface of DHLA-QDs [59]. The TAT-peptide-coupled DHLA-QD conjugates were endocytosed efficiently within a 1-h period and accumulated within endosomes for 24 h without causing any cell death. Cellular uptake of TAT peptide coupled to the end groups of PEG-capped QDs has been used to label mesenchymal stem cells that subsequently were injected into nude mice and tracked to the liver, lung, and spleen [60].

c04.indd 65

7/30/2010 2:18:44 PM

66

c04.indd 66

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

7/30/2010 2:18:44 PM

DISTURBANCES IN CELLULAR PROCESSES INDUCED

67

Figure 4.3. Intracellular location of ricinB : QD655 bioconjugates in HeLa cells (left panel) and HEp-2 cells (right panel). The ricinB : QDs were constituted and bound to the cell surface at 4 °C, and then allowed to be internalized into the cells for various times at 37 °C. The cells were then fixed with 4% paraformaldehyde and prepared for IF microscopy imaging by labeling them with a rabbit antibody against LAMP-1 and with a corresponding secondary anti-rabbit-Cy2 conjugate. Images show ricinB : QD conjugates in red, LAMP-1 in green, and the merged z-stack. Yellow in the merged images indicates colocalization. As seen in the z-stack, the main fraction of QD conjugates is still located at the cell surface in HeLa as well as in HEp-2 cells after 30 min. RicinB : QDs show increasing uptake and partial colocalization with LAMP-1 after longer incubation (90–180 min). RicinB : QD staining is significantly reduced after 24 h of incubation in HeLa and even more in HEp-2 cells. (See color insert.)

4.5 DISTURBANCES IN CELLULAR PROCESSES INDUCED BY LIGAND–NANOPARTICLE BIOCONJUGATES Most of the ligand–nanoparticle bioconjugates studied contain a multivalent binding of ligands to the nanoparticle. In addition to the resulting increase in binding avidity, this can definitely also trigger adverse cellular effects. Multivalent ligand–QD conjugates might cause crosslinking of cell surface receptors and thereby trigger cell signaling pathways. In a recent study by Howarth et al. [61], they prepared compact QDs with monovalent binding of streptavidin. They observed that ephrin receptors EphA3 were evenly distributed at the cell surface upon binding of the monovalent-QDs without triggering of EphA3 kinase activation, whereas the multivalent-QDs lead to EphA3 receptor clustering and tyrosine kinase signaling resulting in their endocytosis [61]. In our study of Tf : QDs and ricinB : QDs we prepared a substoichiometric derivatization of the bioconjugates by varying the ligand : QD molar ratio from 1 : 1 up to 20 : 1 [31]. The ligand : QDs with a 1 : 1 ratio displayed weaker fluorescent intensities when bound to the cells compared with the ligand : QDs of a 5 : 1 ratio. However, we could not observe any differences in their degree of endocytosis or intracellular routing. It has been found that multivalent binding of ricin to HRP and gold nanoparticles directed ricin to lysosomes, whereas a monovalent binding also directed ricin transport into the Golgi apparatus [62]. Furthermore, endocytosis of polyvalent IgG to the Fc receptors of macrophages directed the IgG-Fc receptor complexes to lysosomes for degradation, whereas a monovalent antireceptor antibody caused the endocytosed Fc receptors to recycle [63, 64]. Importantly, we demonstrated that accumulation of the ricinB : QDs perturbed the intracellular transport of the unconjugated ligands ricin and Shiga toxin to the Golgi apparatus. Transport to the Golgi apparatus of ricin was severely inhibited, whereas transport of Shiga was increased. Thus in addition to measuring the typical cytotoxic effects (effects on mitochondria, protein

c04.indd 67

7/30/2010 2:18:44 PM

68

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

synthesis, apoptosis, etc.), it seems important to investigate their potential disturbances of cellular activities related to cell signaling and intracellular trafficking, for instance downregulation of growth factors.

4.6

PERSPECTIVES

Many questions remain open about the mechanisms of endocytosis and their involvement in QD/nanoparticle entry into cells. Precise answers are difficult to give as long as there are still a number of endocytic pathways that are incompletely characterized. Furthermore, the endocytic pathways involved in cellular trafficking of NPs will probably also depend on the cell types studied. Moreover, the possibility of tracking single QD–ligand molecules makes them valuable tools for identifying and resolving the specific endocytic pathways involved. A main objective is to develop QD-labeled ligands that can serve as relevant fluorescent probes for assessing the detailed itinerary of the ligands. However, the large sizes (>20 nm) of many QDs encapsulated within polymers may limit the access into the relevant intracellular sorting stations, and thereby alter the normal trafficking and activity of the ligand. Thus it is important to continue the development of stable QD–ligand conjugates with a small size.

4.7

CONCLUSION

The study of membrane protein dynamics at the molecular level in living cells has become a major issue in understanding important cellular processes such as endocytosis, intracellular transport, and signal transduction. The semiconductor quantum dots (QDs) with their remarkable fluorescent and colloidal properties have potential as probes for membrane proteins at the single molecule level. Furthermore, the rational development of drug delivery and contrast agents based on nanoparticles requires a deeper understanding of particle internalization mechanisms to target cellular pathways capable of providing the required response. Herein, we provided a brief overview of the various endocytosis mechanisms (clathrin-mediated, caveolar, dynamin-independent: noncaveolar, macropinocytosis, phagocytosis) operating in the cell, and discussed studies on endocytosis of different biologically active nanoparticles and QDs varying with respect to size, surface charge, and the ligands conjugated to them.

ACKNOWLEDGMENT This work was supported by Grant 172663/S10 to TGI from the FUGE and NANOMAT programmes of the Research Council of Norway.

c04.indd 68

7/30/2010 2:18:44 PM

REFERENCES

69

REFERENCES 1. Ungewickell, E. J. and Hinrichsen, L. Endocytosis: clathrin-mediated membrane budding. Curr. Opin. Cell Biol. 19: 417–425 (2007). 2. Mayor, S. and Pagano, R. E. Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol. 8: 603–612 (2007). 3. Rothberg, K. G., et al. Caveolin, a protein component of caveolae membrane coats. Cell 68: 673–682 (1992). 4. Anderson, R. G. The caveolae membrane system. Annu. Rev. Biochem. 67: 199– 225 (1998). 5. Kirkham, M. and Parton, R. G. Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim. Biophys. Acta. 1746: 349–363 (2005). 6. Frick, M., et al. Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding. Curr. Biol. 17: 1151–1156 (2007). 7. van Deurs, B., Roepstorff, K., Hommelgaard, A. M., and Sandvig, K. Caveolae: anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol. 13: 92–100 (2003). 8. Lajoie, P. and Nabi, I. R. Regulation of raft-dependent endocytosis. J. Cell Mol. Med. 11: 644–653 (2007). 9. Sandvig, K., et al. Pathways followed by protein toxins into cells. Int. J. Med. Microbiol. 293: 483–490 (2004). 10. Sandvig, K., Torgersen, M. L., Raa, H. A., and van Deurs, B. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem. Cell Biol. 129: 267–276 (2008). 11. Romer, W., et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450: 670–675 (2007). 12. Sabharanjak, S., Sharma, P., Parton, R. G., and Mayor, S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2: 411–423 (2002). 13. Kirkham, M., et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168: 465–476 (2005). 14. Lundmark, R., et al. The GTPase-activating protein GRAF1 regulates the CLIC/ GEEC endocytic pathway. Curr. Biol. 18: 1802–1808 (2008). 15. Mercer, J. and Helenius, A. Virus entry by macropinocytosis. Nat. Cell Biol. 11: 510–520 (2009). 16. Kerr, M. C. and Teasdale, R. D. Defining macropinocytosis. Traffic 10: 364–371 (2009). 17. Dharmawardhane, S., et al. Regulation of macropinocytosis by p21-activated kinase-1. Mol. Biol. Cell 11: 3341–3352 (2000). 18. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70: 401–410 (1992). 19. Lai, S. K., et al. Privileged delivery of polymer nanoparticles to the perinuclear region of live cells via a non-clathrin, non-degradative pathway. Biomaterials 28: 2876–2884 (2007).

c04.indd 69

7/30/2010 2:18:44 PM

70

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

20. Rejman, J., Oberle, V., Zuhorn, I. S., and Hoekstra, D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem. J. 377: 159–169 (2004). 21. Sharma, D. K., et al. The glycosphingolipid, lactosylceramide, regulates beta1integrin clustering and endocytosis. Cancer Res. 65: 8233–8241 (2005). 22. Rejman, J., Bragonzi, A., and Conese, M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol. Ther. 12: 468–474 (2005). 23. Rejman, J., Conese, M., and Hoekstra, D. Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis. J. Liposome Res. 16: 237–247 (2006). 24. Harush-Frenkel, O., Debotton, N., Benita, S., and Altschuler, Y. Targeting of nanoparticles to the clathrin-mediated endocytic pathway. Biochem. Biophys. Res. Commun. 353: 26–32 (2007). 25. Meier, O., et al. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J. Cell Biol. 158: 1119–1131 (2002). 26. Harush-Frenkel, O., Rozentur, E., Benita, S., and Altschuler, Y. Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells. Biomacromolecules 9: 435–443 (2008). 27. Dausend, J., et al. Uptake mechanism of oppositely charged fluorescent nanoparticles in HeLa cells. Macromol. Biosci. 8: 1135–1143 (2008). 28. Chung, T. H., et al. The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells. Biomaterials 28: 2959–2966 (2007). 29. Nabiev, I., et al. Nonfunctionalized nanocrystals can exploit a cell’s active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nano Lett. 7: 3452–3461 (2007). 30. Lidke, D. S., et al. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 22: 198–203 (2004). 31. Tekle, C., van Deurs, B., Sandvig, K., and Iversen, T. G. Cellular trafficking of quantum dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands. Nano Lett. 8: 1858–1865 (2008). 32. Iversen, T. G., Frerker, N., and Sandvig, K. Quantum dot bioconjugates: uptake into cells and induction of changes in normal cellular transport. In: M. Osinski, T. M. Jovin, and K. Yamamoto, eds. Colloidal Quantum Dots for Biomedical Applications IV. Progress in Biomedical Optics and Imaging. SPIE, San Jose, CA, 2009, 7189[1], 71890T-9. 33. Grimmer, S., van Deurs, B., and Sandvig, K. Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J. Cell Sci. 115: 2953–2962 (2002). 34. Pinaud, F., King, D., Moore, H. P., and Weiss, S. Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc. 126: 6115–6123 (2004). 35. Liu, W., et al. Compact cysteine-coated CdSe(ZnCdS) quantum dots for in vivo applications. J. Am. Chem. Soc. 129: 14530–14531 (2007).

c04.indd 70

7/30/2010 2:18:44 PM

REFERENCES

71

36. Clapp, A. R., Goldman, E. R., and Mattoussi, H. Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins. Nat. Protocols 1: 1258–1266 (2006). 37. Jaiswal, J. K., Mattoussi, H., Mauro, J. M., and Simon, S. M. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 21: 47–51 (2003). 38. Liu, W., et al. Compact biocompatible quantum dots functionalized for cellular imaging. J. Am. Chem. Soc. 130: 1274–1284 (2008). 39. Barua, S. and Rege, K. Cancer-cell-phenotype-dependent differential intracellular trafficking of unconjugated quantum dots. Small 5: 370–376 (2008). 40. Wang, Y., et al. Regulation of endocytosis via the oxygen-sensing pathway. Nat. Med. 15: 319–324 (2009). 41. Lidke, D. S., Lidke, K. A., Rieger, B., Jovin, T. M., and Arndt-Jovin, D. J. Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors. J. Cell Biol. 170: 619–626 (2005). 42. Yong, K. T., et al. Quantum rod bioconjugates as targeted probes for confocal and two-photon fluorescence imaging of cancer cells. Nano Lett. 7: 761–765 (2007). 43. Bharali, D. J., Lucey, D. W., Jayakumar, H., Pudavar, H. E., and Prasad, P. N. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. J. Am. Chem. Soc. 127: 11364–11371 (2005). 44. Dahan, M., et al. Diffusion dynamics of glycine receptors revealed by singlequantum dot tracking. Science 302: 442–445 (2003). 45. Pinaud, F., et al. Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking. Traffic 10: 691–712 (2009). 46. Qian, Z. M., Li, H., Sun, H., and Ho, K. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol. Rev. 54: 561–587 (2002). 47. Chan, W. C. and Nie, S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281: 2016–2018 (1998). 48. Sahoo, S. K. and Labhasetwar, V. Enhanced antiproliferative activity of transferrin-conjugated paclitaxel-loaded nanoparticles is mediated via sustained intracellular drug retention. Mol. Pharm. 2: 373–383 (2005). 49. Sandvig, K. and van Deurs, B. Delivery into cells: lessons learned from plant and bacterial toxins. Gene Ther. 12: 865–872 (2005). 50. Khan, F., Proulx, F., and Lingwood, C. A. Detergent-resistant globotriaosyl ceramide may define verotoxin/glomeruli-restricted hemolytic uremic syndrome pathology. Kidney Int. 75: 1209–1216 (2009). 51. Viel, T., et al. In vivo tumor targeting by the B-subunit of Shiga toxin. Mol. Imaging 7: 239–247 (2008). 52. Falguieres, T., et al. Human colorectal tumors and metastases express Gb3 and can be targeted by an intestinal pathogen-based delivery tool. Mol. Cancer Ther. 7: 2498–2508 (2008). 53. Chakraborty, S. K., et al. Cholera toxin B conjugated quantum dots for live cell labeling. Nano Lett. 7: 2618–2626 (2007).

c04.indd 71

7/30/2010 2:18:44 PM

72

ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING

54. Jaiswal, J. K., Andrews, N. W., and Simon, S. M. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol. 159: 625–635 (2002). 55. Andrews, N. W. Regulated secretion of conventional lysosomes. Trends Cell Biol. 10: 316–321 (2000). 56. Liu, Y. S., et al. pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells. J. Phys. Chem. C Nanomater. Interfaces 111: 2872– 2878 (2007). 57. Yong, K. T., et al. Imaging pancreatic cancer using bioconjugated InP quantum dots. ACS Nano 3: 502–510 (2009). 58. Zhang, H., et al. Detection and downregulation of type I IGF receptor expression by antibody-conjugated quantum dots in breast cancer cells. Breast Cancer Res. Treat. 114: 277–285 (2009). 59. Delehanty, J. B., et al. Self-assembled quantum dot–peptide bioconjugates for selective intracellular delivery. Bioconjug. Chem. 17: 920–927 (2006). 60. Lei, Y., et al. Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body. Bioconjug. Chem. 19: 421–427 (2008). 61. Howarth, M., et al. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat. Methods 5: 397–399 (2008). 62. van Deurs, B., et al. Estimation of the amount of internalized ricin that reaches the trans-Golgi network. J. Cell Biol. 106: 253–267 (1988). 63. Mellman, I. and Plutner, H. Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes. J. Cell Biol. 98: 1170–1177 (1984). 64. Mellman, I., Plutner, H., and Ukkonen, P. Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: possible role of a prelysosomal compartment. J. Cell Biol. 98: 1163–1169 (1984).

c04.indd 72

7/30/2010 2:18:44 PM

CHAPTER 5

Synthesis of Metal Nanoparticle-Based Intracellular Biosensors and Therapeutic Agents NEIL BRICKLEBANK Faculty of Health and Wellbeing, Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom

5.1

INTRODUCTION

The emergence of nanotechnology as an independent field of research in the late 1980s and early 1990s has led to a surge in interest in the synthesis of nanoscale (1–100 nm) particles of metals, metal oxides, and semiconductors (also known as quantum dots), due to their technological potential as electronic, magnetic, and photonic materials, as catalysts and as substrates for a host of biological applications including diagnostics and transport vectors. However, metal nanoparticles or colloids are not a new phenomenon; they have been known for many thousands of years, albeit unrecognized as such, and utilized primarily as pigments for decorative purposes. During the Middle Ages aqueous solutions of gold colloids gained a reputation as a remedy for a variety of illnesses, a use which, somewhat paradoxically, anticipated the aims of much current research in the area of nanobiotechnology [1]. Many biomolecules have size dimensions in the range 5–200 nm, which means that materials science and biotechnology converge at the nanoscale. The properties of nanoparticles that make them attractive for biotechnological applications result from a number of attributes including size, shape, and surface morphology. The physical properties of nanoparticles are very different from those of the parent bulk materials and are more complex than those of the individual constituent atoms. These differences arise through the small size and large surface area of the particles; for a 10-nm diameter particle about Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

73

c05.indd 73

7/30/2010 2:18:46 PM

74

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

15% of the atoms occupy “surface” positions, compared with 0.0015% for a 1-μm (1000-nm) particle. Such is the volume of published work that a review such as this cannot comprehensively cover the field of metal nanoparticle synthesis. In this chapter we will concentrate on providing an overview of recent studies, with the first part describing the primary routes for the synthesis of metal nanoparticles, functionalized with organic molecules. The latter sections of this chapter will overview the applications of functional metal nanoparticles as agents for labeling and detection and as therapeutics. The final section addresses the issue of toxicology of nanoparticles, an area of particular relevance to pharmacology.

5.2

SYNTHESIS OF METAL NANOPARTICLES

A wide variety of routes have been developed for the synthesis of metal nanoparticles. The main methods can generally be categorized as wet chemical reduction of metal salts, thermal degradation of metal complexes, or electrochemical synthesis. Less common routes include photochemical decomposition, laser ablation, and biological routes [2]. The latter are of interest because they rely on the fact that certain species of plants and microorganisms are able to uptake large quantities of metallic ions, which they reduce and precipitate as nanoparticles [3]. The shape, size, and particle size distribution of nanoparticles are dependent on many variables, including the reaction temperature and time, solvent, concentration, and nature of the precursors and reagents, particularly the capping ligand. Alteration of any of these factors can lead to a change in the size or shape of the resulting particles [4]. 5.2.1

Role of the Capping Ligand

The capping ligand is a crucial component in both the synthesis and subsequent use of metal nanoparticles. During the synthesis of nanoparticles the capping ligand helps to stabilize the particle, preventing uncontrolled growth and agglomeration. The choice of capping ligand influences the size and shape of the resulting particles. Many different species have been employed including organic bases, surfactants, and polymers. Other approaches for controlling the growth of nanoparticles and templating their morphology include the use of gels, micelles, zeolites, or layered minerals. However, among the most frequently used species are organic thiolates (RS−, where R = alkyl or aryl), usually derived from the corresponding thiols or disulfides. Other common ligands include tertiary phosphines or alkyl amines. Mixed ligand systems offer a further opportunity (and level of complexity) to control or template the formation of particles with a specific size or morphology. The resulting species are often referred to as monolayer protected clusters (MPCs) or, when two

c05.indd 74

7/30/2010 2:18:46 PM

SYNTHESIS OF METAL NANOPARTICLES

75

or more ligands are attached to the surface, mixed monolayer protected clusters (MMPCs). Polymers have also been used for the synthesis of biocompatible nanoparticles and inorganic silicon-based coatings have also been utilized, although molecular organic species predominate. In addition to their stabilizing role, the capping ligands can be used to functionalize the particles, enabling them to be used as sensors, for biomolecular recognition, or as transport vectors. Functionalization can be achieved by a number of ways. The organic groups that are attached to the particle during the initial synthesis can be modified by subjecting the particle to further chemical protocols. Alternatively, the particles can be used to form conjugates with biomolecules. The capping ligands that are utilized in the synthesis stage can be displaced by other species that exhibit the required functionality or biocompatibility through ligand exchange procedures. The latter is attractive for biological species that would not survive the frequently harsh conditions employed in traditional “wet chemistry” procedures. Organic thiolates have been widely used as capping ligands, particularly for gold nanoparticles, because of the high affinity of the sulfur for gold and the high stability of the resulting S–Au bonds. The nature of the bond formed between the donor atom of capping ligand and the surface of nanoparticle has been the subject of much investigation and speculation! However, a significant breakthrough came in 2007 with the reporting of the single crystal X-ray diffraction study of a sample of monodisperse gold particles functionalized with mercaptobenzoic acid (MBA) [5]. The structure revealed a core containing 102 gold atoms that is surrounded by 44 MBA ligands. The gold core comprises 89 Au atoms, 49 of which are arranged in a Marks decahedron with two 20-atom caps. The remaining 13 atoms form a band around the equator of the core. A significant finding was that the MBA ligands not only bind to the gold through Au–S bonds but also interact with each other through a series of face-to-face and face-to-edge interactions between adjacent phenyl rings and also phenyl–sulfur interactions. The interligand interactions observed in this system may provide an indication of how capping ligands are ordered on the particle surface. The significance of this discovery lies in the fact that it enables us to consider metal nanoparticles as “molecular” species, with a clearly defined chemical formula, rather than the more abstract images that have been hitherto accepted. Furthermore, it will aid our understanding of the formation and reactivity of functionalized metal nanoparticles and how these can be manipulated for the benefit of biotechnology. 5.2.2 Synthesis by Direct Chemical Reduction of Metal Salts Arguably the simplest route for the preparation of metal nanoparticles is the direct reduction of metal salts with a suitable reducing agent and/or a capping ligand; in some cases the capping ligand also acts as the reducing agent (Figure 5.1). The best example of this route is the citrate reduction of gold(III) chloride salts in water, the citrate forming a loosely bound shell around the

c05.indd 75

7/30/2010 2:18:46 PM

76

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

AuCI4

(C)

(A)

(i) p-HOC6H4SH,MeOH (ii) NaBH4,H2O

Sodium citrate, H2O, Δ

(B) (i) HAuCl4·H2O (ii) Oct4N+Br–,PhMe (iii) CH3(CH2)11SH, (iv) NaBH4,H2O

–

CO2

– –

OH

O2C

OH Au

–

O2C

O2C – O C 2 –

–

CO2

O2C

CO2

HO

S

S S S

S Au

S

OH

–

OH

CO2

S

S S

CO2 CO2

OH

HO –

HO

OH

HO

–

CO2

S

OH

HO

–

–

HO

OH

S SSS S S S Au S S S S SS S S

Figure 5.1. Common routes for the synthesis of gold nanoparticles: (A) citrate reduction, (B) Brust–Schiffrin two-phase system, and (C) Brust–Schiffrin one-phase system.

gold core. This method was first reported by Turkevitch in 1951 and leads to gold nanoparticles of approximately 20 nm in diameter [6]. The method was refined by Frens, who found that the size of the gold nanoparticles could be controlled by varying the ratio of gold salt and citrate [7]. Similar strategies can be used to prepare nanoparticles of other metals; for example, silver nitrate is readily reduced by sodium borohydride or citrate in water producing relatively monodisperse silver nanoparticles, about 27 nm in diameter [8, 9]. The silver particles produced using this route display excellent stability and their advantageous optical properties, which result from their surface plasmon resonance, have been used in analytical science in SERRS (surface enhanced resonance Raman spectroscopy) [9]. One point worth noting is that the nanoparticles produced using these routes generally display a comparatively broad particle size distribution. Production of particles with a single size and shape remains a challenge.

c05.indd 76

7/30/2010 2:18:46 PM

SYNTHESIS OF METAL NANOPARTICLES

77

A significant breakthrough came through the seminal work of Brust and Schiffrin who devised a method for producing gold nanoparticles with good polydispersity, which are very easily handled and can be isolated and redissolved in common organic solvents without aggregation or decomposition [10]. The initial method utilized an aqueous-toluene two-phase system with dodecanethiol as the capping ligand, sodium borohydride as the reducing agent, and tetraoctylammonium bromide as a phase transfer agent. The colorful reaction mixture changes from orange to dark brown upon addition of the reducing agent. The size of the particles can be varied by modifying the relative amounts of capping ligand and gold salt; the greater the amount of capping ligand the smaller the average particle size. Varying the temperature of the reaction also affects the particle size. Brust and co-workers refined the initial method by developing a single-phase method that obviated the need for the phase transfer agent, which can be difficult to remove from the metallic particles produced using the two-phase method [11]. Subsequently, many workers have adopted the Brust–Schiffrin method and have prepared gold nanoparticles functionalized with a huge variety of thiolate-derived capping ligands, and the methodology has been extended for the production of metallic particles of other metals [1]. The use of surfactants is another common route for the production of nanoparticles. Jana and Peng have reported a surfactant-mediated protocol for the gram-scale synthesis of monodisperse metal nanoparticles [12]. In their procedure, the metal salt (gold(III)chloride, silver acetate, copper acetate, or platinum chloride) was dissolved in toluene together with didodecyldimethylammonium bromide. Fatty acids, aliphatic amines, or thiols were added as stabilizing ligands and play a role in dictating the particle size. The reduction was effected by tetrabutylammonium bromide either alone or with hydrazine. Metal nanoparticles produced using this route cannot be used directly in biomedical applications. However, the particles can be precipitated from toluene solution and the ligands exchanged for water-soluble, biocompatible species. 5.2.3

Reduction of Organometallic Complexes

The reduction of low valent organometallic complexes has been widely used for the preparation of nanoparticles of semiconductor materials such as zinc sulfide and cadmium chalcogenides [13]. Reduction is achieved by chemical, thermal, or photochemical decomposition of the organometallic complex. The nanoparticles produced are often of high purity with very closely defined, and reproducible, sizes and shapes. However, limitations include the requirement for specialist equipment and facilities and the safety hazards associated with the precursors and reactants, many of which are highly toxic. Quantum dots have found significant use as biological labels and this has led a number of research groups to seek improvements in their synthesis that obviate the need for hazardous reagents. For example, Green and co-workers have devised an improved protocol for the production of water-soluble cadmium telluride

c05.indd 77

7/30/2010 2:18:46 PM

78

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

nanoparticles which uses ammonium telluride, (NH4)2Te, in place of H2Te. The CdTe nanoparticles, which were capped with cysteine or mercaptoundecanoic acid, are taken up by human breast cancer cells [14]. 5.2.4

Electrochemical Synthesis

Here the metal of interest is used as the sacrificial anode of an electrochemical cell [15]. As current is passed through the electrochemical cell, metal ions dissolve in the solvent, which contains a tetraalkylammonium salt, this having a dual role of stabilizing ligand and supporting electrolyte. The metal ions migrate to the cathode where nucleation and growth of the nanoparticles occurs as the metal ions are reduced to the zero-valent state. Electrochemical synthesis has been used to prepare particles of a host of transition metals including Fe, Co, Ni, and Pd [2]. A key advantage of this process is that the particles it yields tend to be purer than those obtained from chemical reduction methods, avoiding contamination with reducing agents and chemical by-products. 5.2.5

Other Routes for the Synthesis of Metal Nanoparticles

In addition to the more general approaches described above, a number of other protocols have been developed for the synthesis of functionalized nanoparticles. These have often been developed to produce materials for particular applications or with specific properties. For example, precipitation or seeding protocols are widely used to produce nanoparticles such as CdS quantum dots [13] and gold nanoparticles [1]. Size control is affected by varying the ratio of reagents. Laser ablation is a method that does not necessarily require the use of stabilizing ligands because the species produced using this method are charged and the Coulombic repulsions between particles prevents agglomeration [16, 17]. The reaction can be carried out in either aqueous or organic solvents with reasonable control of concentration, aggregation, and particle size. Irradiation of a gold plate suspended in water with a Nd:YAG laser yields gold nanoparticles with a diameter of about 18 nm [17]. These particles were coated with a thermoresponsive thiol-terminated poly-N-isopropylacrylamide-co-acrylamide co-polymer; above a transition temperature of 37 °C the polymer facilitates the reversible aggregation of the AuNPs. These polymer-functionalized particles are taken up by human breast adenocarcinoma MCF7 cells, with cells treated above 40 °C showing an 80-fold greater uptake compared to cells treated at 34 °C. This difference can be attributed to the aggregated particles having increased hydrophobicity [17]. Biological and biomimetic routes have used bacteria, plants, and plant extracts to synthesize noble metal nanoparticles [2]. Proteins such as ferritin, devoid of iron, have been utilized as templates and cages that can be filled with other nanomaterials, including cadmium sulfide, creating a quantum

c05.indd 78

7/30/2010 2:18:46 PM

SYNTHESIS OF FUNCTIONALIZED PARTICLES THROUGH LIGAND EXCHANGE

79

dot–ferritin nanocomposite [18]. Subsequently, this biomimetic approach has been applied to produce nanocomposites of other metals and proteins [19, 20]. An illustration of the level of complexity in the procedures that have been devised is provided by a recent report by Lin and co-workers, who have prepared fluorescent gold nanoparticles [21]. Their protocol started with the production of 5.5-nm surfactant-stabilized gold nanoparticles obtained using chemical reduction of AuCl3 in toluene. The particles were isolated and their size reduced by adding further quantities of HAuCl4 precursor, which etches the gold nanoparticles, producing smaller particles with a mean diameter of 3.7 nm. The particles were then transferred from toluene to aqueous media by exchanging the surfactant molecules with dihydrolipoic acid. Further purification by centrifugation and filtration yields stable AuNPs that are readily conjugated with biological molecules and taken up by cells.

5.3 SYNTHESIS OF FUNCTIONALIZED PARTICLES THROUGH LIGAND EXCHANGE The concept of ligand exchange was first described by Murray and co-workers [22]. The process is deceptively simple; mixing nanoparticles capped with a particular ligand with a second free ligand results in the replacement of a proportion of the original ligands (Figure 5.2). The method is particularly useful for preparing MMPCs and for introducing ligands onto the surface of the particle that may not be able to withstand the conditions employed during the original synthetic protocol. Biological molecules that possess a functional group that can bind to the surface of the particle, especially thiols, are readily attached to the surface of nanoparticles in this way. Examples include oligonucleotides, peptides, and biotin. An alternative strategy is to introduce thiol ligands bearing free carboxylate groups onto the surface of the nanoparticle. These can then be chemically coupled to biological molecules containing amino groups.

R R S S S

S S S Au

S SS

S S S

+R −

R S S S S

SH

S S

SH

Au S S S

R

S S S R

R

Figure 5.2. Schematic representation of ligand exchange.

c05.indd 79

7/30/2010 2:18:46 PM

80

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

The apparent simplicity of ligand exchange belies the fact that exchange reactions involve a series of complex equilibria and kinetics that are difficult to study using conventional techniques. Moreover, most samples are not polydisperse and contain a range of particle sizes and hence surface coverage of ligands, with different ligand-binding sites on the nanoparticle surface displaying different levels of reactivity. A number of mechanisms are possible including associative or dissociative pathways and the formation of intermediate species. In addition to exchange between the functionalized particles and free ligand, interparticle ligand exchange may also occur. The latter possibility has been elegantly demonstrated by Murray, who investigated interparticle ligand exchange between two different functionalized nanoparticles that were dissolved in different, immiscible solvents [23]. For example, when an aqueous solution of tiopronin (N-(2-mercaptopropionyl)glycine)-coated gold particles was mixed with a toluene solution of alkanethiolate-coated gold particles, tiopronin-coated gold nanoparticles could be detected in the toluene phase and alkanethiolate-coated gold nanoparticles in the aqueous layer. Similar observations were made when aqueous solutions of tiopronin-capped silver nanoparticles were mixed with toluene solutions of alkanethiolate-capped gold nanoparticles. Interestingly, the ligand and metal exchange is inhibited when reactions are completed under N2, suggesting that oxidation reactions play a role in the transfer. This latter point illustrates the complexity of exchange reactions beautifully. At present, there is no standard procedure for determining the surface coverage of nanoparticles or the absolute number of ligands in MMPCs and a key conclusion of a recent review of mechanistic studies of ligand exchange is that for thiol-protected gold particles, at least, it is never possible to displace or exchange all of the ligands attached to the nanoparticle surface [24].

5.4

NANOPARTICLE–DNA INTERACTIONS

Conjugates of metal nanoparticles and DNA are of much current interest because of their potential biomedical applications and have been the subject of several recent reviews [25, 26]. DNA is usually attached to the surface of nanoparticles covalently. This can be done directly through the formation of bonds between functional groups, such as cysteine residues or thiols, present in the nucleotide. Alternatively, the DNA can be bound to functional groups that are appended to the capping ligands on the nanoparticle surface using conventional chemical coupling strategies. Mirkin and co-workers pioneered the synthesis of metal nanoparticle– DNA assemblies. In their initial work two batches of 13-nm gold nanoparticles were each functionalized with a noncomplementary thiol-derivatized oligonucleotide. When a third oligonucleotide that contains end sequences that are complementary to those that are grafted onto the surface of the nanoparticles is added, aggregation occurs forming the nanoparticle–DNA assembly. The

c05.indd 80

7/30/2010 2:18:46 PM

NANOPARTICLE–DNA INTERACTIONS

81

assembly process is reversed by thermal denaturation [27]. Simultaneously, Alivisatos and co-workers reported a slightly different approach, using DNA to template two or three much smaller 1.4-nm gold nanoparticles, functionalized with oligonucleotides, into larger assemblies [28]. These seminal papers have catalyzed a phenomenal amount of research into nanoparticle–DNA assemblies with current emphasis focusing on their functionality [25, 26]. For example, it has recently been shown that DNA can be used to direct the assembly of gold nanoparticles into different micrometer-sized crystals, with either face-centered-cubic or body-centered-cubic lattices, dependent on the choice of DNA sequences attached to the surface of the gold nanoparticle and the DNA linkers [29]. An alternative approach to covalent linkage is the formation of bioconjugates through noncovalent interactions (Figure 5.3) [26, 30–32]; it is well known that noncovalent interactions play crucial structure-directing roles in biological molecules. The attachment of capping ligands bearing charged

–

O O P O O

–

Au

S

O –

O P O O

O

–

O

NH2

N

H2N

Base

Base

O

+

+

SH

N

SH 11

(B)

(A)

O +

–

S SO3

P

(C)

+

S

P

CH3 Br

–

(D)

Figure 5.3. Schematic representation of the interaction of cationically functionalized gold nanoparticles with oligonucleotides and the molecular structures of some cationic capping ligand precursors: (A) ethidium thiol, (B) trimethylammonium alkylthiol, (C) phosphonioalkylthiosulfate zwitterions, and (D) ω-thioacetylalkylphosphonium salts.

c05.indd 81

7/30/2010 2:18:46 PM

82

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

groups to the surface of nanoparticles is an attractive proposition for improving the aqueous solubility of the resulting MPCs. Noncovalent interactions between DNA and functionalized nanoparticles can be facilitated through intercalation, groove binding, or simple electrostatic attractions. Ethidium bromide is a well-known DNA intercalator and this species has been introduced onto the surface of cationic and anionic gold nanoparticles as a means of inducing binding to DNA. The ethidium groups are grafted onto the gold nanoparticles as thiolate ligands through a ligand exchange reaction; each particle contains only a few ethidium thiolate groups. The remaining capping ligands are TMA (trimethyl(mercaptoundecyl)ammonium) for the cationic species, or tiopronin (N-(2-mercaptopropionyl)glycine) for the anionic particles. In the absence of DNA, the normal fluorescence of the ethidium groups is quenched by the gold core. Introduction of DNA results in a partial restoration of the fluorescence. As one might anticipate, the cationic TMAfunctionalized gold nanoparticles bind rapidly and efficiently with the DNA, whereas the binding of the anionic tiopronin-functionalized is slow compared to ethidium that is not bound to the gold [30]. Rotello and co-workers have advanced the use of nanoparticles functionalized with alkylthiolate ligands bearing cationic ammonium head groups, which provide a complementary surface for binding DNA through electrostatic interactions with the negatively charged phosphate groups in the DNA backbone [26, 31, 32]. For example, the nanoparticles have been used to recognize a 37-mer DNA duplex inhibiting transcription by T7 RNA polymerase. This result illustrated the high affinity of the DNA for the functionalized nanoparticle and highlights their potential use in gene regulation [32]. Our own contribution to this area has been the synthesis of a series of phosphonium-capped gold nanoparticles (Figure 5.4) [33, 34]. The most widely

Ph

Ph

Ph Ph Ph + P Ph

Ph P+

Ph Ph Ph P+

Ph + Ph P

Ph + Ph P Ph

–

3S

SO3 – 4

or

+ AuCI O

Ph + Ph P Ph

–

3S

Ph

CH3 Br

NaBH4 H2O/CH2CI2

Ph

P

Ph Ph + P Ph

s

s s s s

s s

Ph + Ph P Ph

+ Ph

s

Ph Ph

Ph Ph

s

s

P

+

P

P +P Ph Ph Ph

Ph Ph

+ Ph

s

+

Ph

P

s

s

Ph + P

+ Ph

s

Au

+

P Ph Ph Ph

Ph

Ph Ph

Ph Ph

Figure 5.4. Schematic representation of protocol for the synthesis of phosphoniumcapped gold nanoparticles.

c05.indd 82

7/30/2010 2:18:46 PM

NANOPARTICLE–DNA INTERACTIONS

83

studied cationic capping ligands feature ammonium head groups as the chargecarrying species [26, 31]. Nonetheless, phosphonium groups offer a number of advantages including the availability of a wide range of organic substituents, which allows the possibility of creating a range of additional functionality, such as fluorescence or chirality and, most importantly, biocompatability; the ability of the triphenylphosphonium group to travel across cell membranes is well established. The latter property has led to the use of phosphonium compounds as transport vectors for targeting mitochondria [35, 36], anticancer agents [37], and as diagnostic agents [38]. Although there are many studies of the use of organophosphorus ligands, notably phosphines and phosphine oxides [39–41], for passivating the surface of nanoparticles, there have been far fewer studies of the use of phosphorus ligands to impart functionality to nanoparticles. Tris(hydroxymethyl) phosphine (THP)-capped gold nanoparticles, which have an average diameter of 1.5 nm, have been formed through the reduction of HAuCl4 with tetrakis (hydroxymethyl)phosphonium chloride. The resulting THP-gold nanoparticles, although negatively charged, readily form conjugates with DNA. It is believed that the interaction with DNA is facilitated by hydrogen bonds between the DNA bases and the hydroxy groups of the THP [41]. Biocompatible gold nanoparticles, stabilized by phosphorylcholine thiolate ligands, have been prepared and show enhanced stability compared to gold nanocrystals stabilized with polyethylene glycol [42]. Phosphonium-functionalized gold nanoparticles are synthesized using a variation of the Brust method. The phosphonium ligands are derived from either phosphonioalkylthiosulfate zwitterions or ωthioacetylalkylphosphonium salts, which behave as masked thiolate ligands [33, 34]. The term “masked thiolate” is used to describe alkylthiolate species that are “protected” as another group, in this case either thiosulfate or thioacetate, which, under reductive conditions or contact with metal surfaces, undergo cleavage of the S–SO3 or S–C(O)CH3 bonds, generating thiolate anions that attach to the surface of the growing nanoparticle. Masked thiolates are easier to handle and less prone to oxidation than conventional thiol ligands. The ability of the phosphonium gold nanoparticles to interact with RNA and DNA and has been investigated using a Biacore instrument that relies on changes in the surface plasmon resonance of sensor chips when exposed to analytes to study biomolecular interactions [43]. The phosphoniumfunctionalized gold nanoparticles interact strongly with negatively charged carboxylate groups and streptavidin, which are attached to the Biacore sensor chip. Neutral, citrate-stabilized, gold nanoparticles do not interact in the same way and are easily released by the sensor chip. Once bound to the sensor chip, the phosphonium-capped gold nanoparticles will bind other biomolecules such as DNA and RNA and this is currently being investigated for biomolecular recognition.The cellular uptake of phosphonioalkylthiosulfate zwitterions, ω-thioacetylalkylphosphonium salts, and phosphonium-functionalized gold

c05.indd 83

7/30/2010 2:18:47 PM

84

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

nanopartciles has also been investigated. The results indicate that the ligands and the functionalized gold nanoparticles are accumulated by the cells. The presence of the capping ligands and the gold within the cells was confirmed using MALDI-MS, which provides a relatively straightforward means of detecting these species in cells. The results confirm that phosphonium ligands offer a convenient way of transporting gold nanoparticles into cells.

5.5 FUNCTIONALIZED NANOPARTICLES FOR BIOLOGICAL DETECTION The use of nanoparticles in biological imaging and detection is one of the most widely explored applications of metal nanoparticles [14, 21, 26, 44–46]. Traditional methods for the labeling and detection of biological molecules utilize fluorescent chemical dyes or radiolabels. Both of these approaches have their limitations, including broad emission spectra of dyes, which makes use of multiple dyes difficult. Moreover, many organic dyes suffer from photobleaching, resulting in the deterioration of the emission signals after prolonged exposure to the excitation light. The challenges associated with radiolabels include the requirement for specialist facilities for handling and disposal of radioactive species. Many of these disadvantages are overcome by quantum dots, which have been widely explored as agents for biomedical imaging [43, 44]. Semiconductor quantum dots display tunable emission spectra, dependent on the size, shape, and functionalization of the particles. Furthermore, the emission spectra are narrow, which enables nanoparticles to be utilized in multiplexed assays. Quantum dots also show greater stability to photobleaching and a number of materials are now available commercially as reagents for biological imaging. One of the challenges associated with the synthesis of quantum dots for imaging applications is transfer from organic solvents to aqueous systems, although this can be overcome by growing a silica shell around the semiconductor nanoparticle core to which biocompatible ligands can be attached, or by swapping the capping ligands used in the synthetic procedure with water-soluble species such as mercaptoacetic acid by ligand exchange [44, 45]. A potential downside of nanoparticulate imaging agents is their production of cytotoxic side products upon degradation, an issue that needs to be considered when conducting biological imaging experiments [14]. The surface plasmon resonance of gold nanoparticles, which occurs when the particles absorb and emit light, has been exploited in biological contrast agents [46]. The nanoparticles are functionalized with antibodies that bind with antigens in the cells or regions of interest. In addition to visualization by light, gold nanoparticles can also be imaged by X-ray methods or by microscopic techniques such as TEM and AFM. Gold particles smaller than 2 nm do not display plasmon resonance but do exhibit size-dependent fluorescence.

c05.indd 84

7/30/2010 2:18:47 PM

METAL NANOPARTICLES AS THERAPEUTIC AGENTS

85

Recent studies have also shown that this fluorescence can be used for biological labeling, offering an alternative to quantum dots [21].

5.6

METAL NANOPARTICLES AS THERAPEUTIC AGENTS

Funtionalized metal nanoparticles are readily taken up by cells, which makes them attractive carrier systems for transporting drugs, biomolecules, or other therapeutic agents into cells [26, 46–51]. Furthermore, the nanoparticles themselves can be used as the therapeutic agents, as, for example, in the use of gold nanoparticles as an intercellular heat source [46]. Trapping the drug molecule between capping ligands attached to the nanoparticle surface is a method that has recently been reported [48]. The drug molecules are held in place through noncovalent intermolecular interactions and are released by diffusion. One of the primary aims of nanoparticle-based drug delivery systems is the targeted delivery of the therapeutic agent at the site of the disease. This can be achieved by synthesizing nanoparticles bearing ligands that are only recognized by specific receptors that are present on the cell surface. A significant amount of attention has focused on the use of nanomaterials in the treatment of cancer and gold nanoparticles have been synthesized that carry folic acid attached to their surface; folate receptors are overexpressed on the surfaces of many cancer cells [49]. Similarly, protein tumor necrosis factor TNFα has been grafted onto the surface of PEG-stabilized gold nanoparticles, which have been shown to be preferentially accumulated by tumors [50]. Methotrexate-functionalized magnetic Fe3O4 nanoparticles have been synthesized (Figure 5.5), the methotrexate interacting with the cellular folate receptors. These materials offer the dual function of diagnostics, acting as a contrast agent for MRI, and drug delivery. The methotrexate was covalently bound to (3-aminopropyl)-trimethoxysilane assembled on the particle surface through amidation between the carboxylic acid end groups on methotrexate and the pendant amine groups on the capping ligand. The methotrexate was shown to be cleaved from the nanoparticles inside cells [51]. An alternative approach to targeting nanoparticles toward specific cellular sites is to use so-called gene guns, where nanoparticle–DNA conjugates are forced inside cells using ballistic acceleration [46]. The use of metal nanoparticles themselves as therapeutic agents stems from their ability to absorb energy, usually light or radiofrequency fields, which causes them to become excited. The excitation energy is eventually dissipated into the surrounding tissues as heat. Since cells are very sensitive to even small variations in temperatures, this can lead to cell death. This approach has been used to destroy cancerous tissues that have been infused with gold nanoparticles. The tissue is illuminated with light that is absorbed by the gold nanoparticles, resulting in cell death. A limitation of this use of gold nanoparticles is that visible and IR light can only penetrate surface tissue. Therefore magnetic

c05.indd 85

7/30/2010 2:18:47 PM

86

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

Fe2O3

MeO OH + MeO Si MeO

NH2

PhMe,sonication

OMe Fe2O3

O Si

NH2

OMe

(i) Methotrexate,DMSO (ii) 1-ethy1-3- (3-(dimethylamino) propylcarbodiimide (EDC), N-hydroxysuccinimide (NHS). O OMe Fe2O3

O Si OMe

N H

NH2 O

HO

N H

N

N

N

O

N

N

NH2

Figure 5.5. Drug molecules can be attached to the surface of nanoparticles using established methodologies, as exemplified by the conjugation of methotrexate with magnetic ferric oxide nanoparticles.

nanoparticles have been developed that are excited by radiofrequency radiation, which are able to reach deep inside the body [46].

5.7

TOXICOLOGY OF METAL NANOPARTICLES

As we have seen, metal nanoparticles offer huge potential benefits to medicine through their applications as diagnostic and therapeutic agents. However, a much greater understanding of the possible risks to human health is also required before nanoparticles are used routinely, and yet this remains a comparatively underexplored aspect of nanomaterials science. A number of recent papers have reviewed aspects of nanoparticle toxicology and cytotoxicity [52– 55]. The paucity of toxicological studies stems, in part, from the fact that nanomaterials are not a uniform group but show great diversity, not only in chemical composition but also in morphology and functionalization. Indeed, the dissociation and subsequent metabolism of capping ligands and other functional molecules appended to the surface of the nanoparticles adds a further level of complexity.

c05.indd 86

7/30/2010 2:18:47 PM

CONCLUSIONS AND FUTURE PROSPECTS

87

The health risks of nanoparticles stem from their small size and high surface area and their ability to penetrate tissues and pass across cellular membranes. Studies show that adverse affects are related to the dosage and concentration and the length of exposure. Much attention has focused on quantum dots due to the risk or releasing of heavy metal ions with known toxicity as the particles are metabolized [55, 56]. A pharmacokinetic study of commercially available QD705 quantum dots, which contain a CdSeTe core surrounded by a ZnS shell and a methoxy-PEG coating, in male ICR mice has been reported. The study reveals that the quantum dots are accumulated in the kidneys, liver, and spleen for at least 28 days but are gradually and partially eliminated by 6 months [56]. Much of the interest in gold nanoparticles results from their stability and perceived biocompatibility. Of particular interest is a report by Connor and co-workers who studied the effects of particle size and capping ligand on the cytotoxicity of human leukemia cells [57]. The gold particle size ranged from 4 to 18 nm. The capping ligands included citrate, glucose, cysteine, CTAB, and biotin. The results indicated that the 18-nm particles functionalized with citrate or biotin were not toxic at concentrations up to 250 μM. In contrast, similar concentration solutions of gold(III) chloride salt are very cytotoxic. Gold particles capped with glucose and cysteine displayed greater cytotoxicty. A comparative study of the toxicology of 2-nm gold nanoparticles functionalized with either cationic (ammonium) or anionic (carboxylate) thiolate ligands toward mammalian and bacterial cell lines has been undertaken [58]. The research revealed that both types of cells show similar levels of uptake and the cationic particles are moderately toxic, whereas the anionic particles are nontoxic, demonstrating the relationship between cytotoxicity and surface functionalization. The studies undertaken to date provide a base for further, more detailed, studies of the pharmacological and toxicological effects of metal nanoparticles, which are almost certainly required before these materials are routinely used in human medical applications.

5.8

CONCLUSIONS AND FUTURE PROSPECTS

Metal nanoparticles offer huge potential in a wide range of biomedical applications. As the field of research has emerged and matured over the past 20 years, the emphasis of much of the research into the synthesis of metal nanoparticles has moved from developing new methodologies to refining and improving existing procedures to make materials for custom applications or with more closely defined morphological parameters. Despite the numerous advances that have been made in recent times, challenges and opportunities remain. The routine production of monodisperse particles with precisely known surface morphology and ligand coverage has still to be achieved. Although we know that metal nanoparticles can be taken up by cells, the

c05.indd 87

7/30/2010 2:18:47 PM

88

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

general mechanistic aspects remain poorly understood. Furthermore, there is a lack of detailed knowledge of the fate of the metal particles once inside cells, which links with the comparatively limited amount of detailed pharmacokinetic and toxicological data on metal nanoparticles. However, it is almost certain that these challenges will be overcome in the near future and that metal nanoparticles will be routinely used for the detection and treatment of disease.

REFERENCES 1. Daniel, M.-C. and Astruc, D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis and nanotechnology. Chem. Rev. 104(1): 293–346 (2004). 2. Pachón, L. D. and Rothenberg, G. Transition-metal nanoparticles: synthesis, stability and the leaching issue. Appl. Organometallic Chem. 22: 288–299 (2008). 3. Kumar, V. and Yadev, S. K. Plant-mediated synthesis of silver and gold nanoparticles and their applications. J. Chem. Technol. Biotechnol. 84(2): 151–157 (2009). 4. Tao, A. R. Habas, S., and Peidong, Y. Shape control of colloidal nanocrystals. Small 4(3): 310–325 (2008). 5. Jadzinsky, P. D., et al. Structure of a thiol monolayer-protected gold nanoparticle at 1.1 Å resolution. Science 318: 430–433 (2007). 6. Turkevitch, J., Stevenson, P. C., and Hillier, J. Nucleation and growth process in the synthesis of colloidal gold. Discuss. Faraday Soc. 11: 55–75 (1951). 7. Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci. 241: 20–22 (1973). 8. Lee, P. C. and Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem. 86(17): 3391–3395 (1982). 9. Munro, C. H., et al. Characterization of the surface of a citrate-reduced colloid optimized for use as a substrate for surface-enhanced resonance Raman-scattering. Langmuir 11(10): 3712–3720 (1995). 10. Brust, M., et al. Synthesis of thiol-derivatized gold nanoparticles in a two-phase liquid-liquid system. J. Chem. Soc. Chem. Commun. 801–802 (1994). 11. Brust, M., et al. Synthesis and reactions of functionalized gold nanoparticles. J. Chem. Soc. Chem. Commun. 1655–1656 (1995). 12. Jana, N. R. and Peng, X. Single-phase and gram-scale routes towards nearly monodisperse Au and other noble metal nanocrystals. J. Am. Chem. Soc. 125(47): 14280–14281 (2003). 13. Trindade, T., O’Brien, P., and Pickett, N. L. Nanocrystalline semiconductors: synthesis, properties and perspectives. Chem. Materials 13(11): 3843–3858 (2001). 14. Green, M., et al. A facile route to CdTe nanoparticles and their use in bio-labelling. J. Materials Chem. 17: 1989–1994 (2007). 15. Reetz, M. T. and Helbig, W. Size-selective synthesis of nanostructured transition metal clusters. J. Am. Chem. Soc. 116(16): 7401–7402 (1994).

c05.indd 88

7/30/2010 2:18:47 PM

REFERENCES

89

16. Amendola, V., Polizzi, S., and Meneghetti, M. Laser ablation synthesis of gold nanoparticles in organic solvents. J. Phys. Chem. 110(14): 7232–7237 (2006). 17. Salmaso, S., et al. Cell up-take control of gold nanoparticles functionalized with a thermoresponsive polymer. J. Materials Chem. 19: 1608–1615 (2009). 18. Wong, K. K. W. and Mann, S. Biomimetic synthesis of cadmium sulfide-ferritin nanocomposites. Adv. Materials 8(11): 929 (1996). 19. Klem, M. T., Young, M., and Douglas, T. Biomimetic magnetic nanoparticles. Materials Today 8(9): 28–37 (2005). 20. Uchida, M., et al. Biological containers: protein cages as multifunctional nanoplatforms. Adv. Materials 19(8): 1025–1042 (2007). 21. Lin, C.-A. J., et al. Synthesis, characterization, and bioconjugation of fluorescent gold nanoclusters towards biological labeling applications. ACS Nano 3(2): 395– 401 (2009). 22. Ingram, R. S., Hostetler, M. J., and Murray, R. W. Poly-hetero-omega-functionalized alkanethiolate-stabilized gold cluster compounds. J. Am. Chem. Soc. 119(39): 9175–9178 (1997). 23. Song, Y., Huang, T., and Murray, R. W. Heterophase ligand exchange and metal transfer between monolayer protected clusters. J. Am. Chem. Soc. 125(38): 11694– 11701 (2003). 24. Caragheorgheopol, A. and Chechik, V. Mechanistic aspects of ligand exchange in Au nanoparticles. Phys. Chem. Chem. Phys. 10: 5029–5041 (2008). 25. Fischler, M. and Simon, U. Metal nanoparticle–DNA hybrids—from assembly towards functional conjugates. J. Materials Chem. 19: 1518–1523 (2009). 26. De, M., Ghosh, P. S., and Rotello, V. M. Applications of nanoparticles in biology. Adv. Materials 20: 4225–4241 (2008). 27. Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382: 607–609 (1996). 28. Alivasatos, A. P., et al. Organization of “nanocrystal molecules” using DNA. Nature 382: 609–6011 (1996). 29. Park, S. Y., et al. DNA-programmable nanoparticle crystallization. Nature 451: 553–556 (2008). 30. Wang, G., Zhang, J., and Murray, R. W. DNA Binding of an ethidium intercalator attached to a monolayer-protected gold cluster. Anal. Chem. 74(17): 4320–4327 (2002). 31. You, C.-C., Chompoosor, A., and Rotello, V. M. The biomacromolecule–nanoparticle interface. Nanotoday 2(3): 34–43 (2007). 32. McIntosh, C. M., et al. Inhibition of DNA transcription using cationic mixed monolayer protected gold clusters. J. Am. Chem. Soc. 123(31): 7626–7629 (2001). 33. Ju-Nam, Y., et al. Phosphonioalkylthiosulfate zwitterions—new masked thiol ligands for the formation of cationic functionalised gold nanoparticle. Organic Biomol. Chem. 4: 4345–4351 (2006). 34. Ju-Nam, Y., et al. ω-Thioacetylalkylphosphonium salts: precursors for the preparation of phosphonium-functionalised gold nanoparticles. J. Organometallic Chem. 693: 3504–3508 (2008).

c05.indd 89

7/30/2010 2:18:47 PM

90

SYNTHESIS OF METAL NANOPARTICLE-BASED INTRACELLULAR BIOSENSORS

35. Weissig, V., Boddapati, S. V., Jabr, L., and D’Souza, G. G. Mitochondria-specific nanotechnology. Nanomedicine 2(3): 275–285 (2007). 36. Murphy, M. P. and Smith, R. J. A. Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu. Rev. Pharmacol. Toxicol. 47: 629–656 (2007). 37. Rideout, D. C. et al. Phosphonium salts exhibiting selective anti-carcinoma activity in vitro. Anti-Cancer Drug Design 4(4): 265–280 (1989). 38. Yang, C.-T., et al. Synthesis and structural characterization of complexes of a DO3A-conjugated triphenylphosphonium cation with diagnostically important metal ions. Inorg. Chem. 46(21): 8988–8997 (2007). 39. Binder, W. H., Sachsenhofer, R., Straif, C. J., and Zirbs, R. Surface-modified nanoparticles via thermal and Cu(I)-mediated “click” chemistry: generation of luminescent CdSe nanoparticles with polar ligands guiding supramolecular recognition. J. Materials Chem. 17: 2125–2132 (2007). 40. Duff, D. G., Baiker, A., and Edwards, P. P. A new hydrosol of gold clusters. 1. Formation and particle size variation. Langmuir 9(9): 2301–2309 (1993). 41. Harnack, O., Ford, W. E., Yasuda, A., and Wessels, J. M. Tris(hydroxymethyl) phosphine-capped gold nanoparticles templated by DNA as nanowire precursors. Nano Lett. 2(9): 919–923 (2002). 42. Qiao, J., Xu, J.-P., Ji, J., and Shen, J.-C. Zwitterionic phosphorylcholine as a better ligand for stabilizing large biocompatible gold nanoparticles. Chem. Commun. 3058–3060 (2008). 43. Ju-Nam, Y., Functionalised Metal Nanoparticles as Novel Reagents for Biomedical Analysis, Ph.D. thesis, Sheffield Hallam University, UK, 2007. 44. Green, M. Semiconductor quantum dots as biological imaging agents. Angew. Chem. Int. Ed. 43: 4129–4131 (2004). 45. Smith, A. M., Duan, H., Mohs, A. M., and Nie, S. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60: 1226–1240 (2008). 46. Sperling, R. A., et al. Biological applications of gold nanoparticles. Chem. Soc. Rev. 37: 1896–1908 (2008). 47. Han, G., Ghosh, P., and Rotello, V. M. Functionalized gold nanoparticles for drug delivery. Nanomedicine 2(1): 113–123 (2007). 48. Kim, C. K., et al. Entrapment of hydrophobic drugs in nanoparticle monolayers with efficient release into cancer cells. J. Am. Chem. Soc. 131(4): 1360–1361 (2009). 49. Dixit, V., et al. Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells. Bioconjug. Chem. 17: 603–609 (2006). 50. Kholer, N., Sun, C., Wang, J., and Zhang, M. Q. Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir 21: 8858–8864 (2005). 51. Paciotti, G. F., et al. Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug Deliv. 11: 169–183 (2004). 52. Oberdorster, G., Stone, V., and Donaldson, K. Toxicology of nanoparticles: a historical perspective. Nanotoxicology 1(1): 2–25 (2007). 53. Yang, W., Peters, J. I., and Williams, R. O. III. Inhaled nanoparticles—a current review. Int. J. Pharmaceutics 356: 239–247 (2008).

c05.indd 90

7/30/2010 2:18:47 PM

REFERENCES

91

54. Murphy, C. J., et al. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc. Chem. Res. 41(12): 1721–1730 (2008). 55. Lewinski, N., Colvin, V., and Drezek, R. Cytotoxicity of nanoparticles. Small 4(1): 26–49 (2008). 56. Lin, P., et al. Computational and ultrastructural toxicology of a nanoparticle, quantum dot 705, in mice. Environ. Sci. Technol. 42(16): 6264–6270 (2008). 57. Connor, E. E., et al. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 1: 325–327 (2005). 58. Goodman, C. M., McCusker, C. D., Yilmaz, T., and Rotello, V. M. Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjug. Chem. 15: 897–900 (2004).

c05.indd 91

7/30/2010 2:18:47 PM

c05.indd 92

7/30/2010 2:18:47 PM

CHAPTER 6

Subcellular Fate of Nanodelivery Systems DUSICA MAYSINGER and SEBASTIEN BORIDY Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada

ELIZA HUTTER Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

6.1 INTRODUCTION Today, nanosciences are experiencing massive investment worldwide. Possible consequences of nanoparticle (NP) entry into biological systems, animals, plants, and humans are not known and a quest to produce safe NPs useful for the electronic industry, food production, artificial paints, and biomedical applications is actively ongoing. The production of manufactured NPs underscores the question: Does long-term cumulative exposure to NPs have deleterious effects on our health? Small size NPs in low nanomolar or picomolar concentrations could reach different tissues including the brain but they cannot be easily detected. Since the induction of intracellular oxidative stress appears to be an essential component of the biological side effects of many metallic and carbon NP types [1–3], a lifetime’s accumulation of NPs in minute quantities could seriously accelerate the onset of inflammatory and degenerative processes in the brain. Therefore, studies to provide information on NP entry into cells and how to reduce their untoward effects at cellular and system levels have been actively pursued in different laboratories. On the other hand, polymeric NPs, which could stimulate repair mechanisms by releasing trophic factors, antioxidants, or chelating agents incorporated into, for example, block copolymer micelles [4–10], are promising candidate NPs for therapeutic interventions. Indeed, several of them are already at different stages of clinical trials. Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

93

c06.indd 93

7/30/2010 2:18:49 PM

94

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

In this chapter we will discuss (1) how the properties of three selected classes of NPs determine their uptake and distribution at the single cell level and in specific organelles, (2) the mechanisms involved in internalization of these NPs, and finally, (3) the possible untoward effects that some (metallic) NPs produce and how to provide the means of preventing and/or protecting cells/tissues from possible adverse effects. Our research is primarily focused on mimicking and activating intrinsic cellular protective mechanisms (e.g., by mimicking and enhancing the capacity of endogenous chaperones, induction of antioxidant systems, and stimulation of endogenous protectins and resolvins) [11–13]. We will focus here on three groups of NPs that are of particular interest in nanomedicine. These NPs are (1) quantum dots, (2) gold NPs, and (3) polymeric artificial chaperones.

6.2

QUANTUM DOTS

6.2.1. Properties of Quantum Dots Quantum dots (QDs) are luminescent NPs with unique optical properties that have been exploited for single cell and whole animal imaging. Excellent reviews summarizing the current status of QDs are available [14–16]. Some of the physicochemical properties of QDs, particularly those used for assessment of intracellular trafficking of therapeutically active molecules, are presented in Chapter 27 of this book. When coated with proteins or biocompatible polymers, QDs are generally not deleterious to cells and organisms. However, when QDs are retained in cells or if they accumulate in the body over a long period of time, their coatings may be degraded, yielding “naked” QDs. We were particularly interested in the effects of “naked” QDs. Our studies showed that they can induce damage to the plasma membrane, mitochondrion, and nucleus, ultimately leading to cell death [17]. Reactive oxygen species (ROSs) are important players in mediating QD-induced cellular damage, for example, with cadmium telluride QDs. QD-induced cytotoxicity can be reduced or even eliminated without covalent binding of protective agents to the QD surface. Among the surfaces that are the most effective are multilayer ZnS capping and polymers such as polyethylene glycol (PEG). Of course, the size and charge of the PEG chain is critical for QD internalization, whole body distribution, and subcellular distribution. The following sections are meant to illustrate these points. 6.2.2 Biodistribution in the Whole Body and Subcellular Distribution of QDs Quantum dots are attractive fluorescent probes extensively investigated in the emerging field of nanomedicine [18]. Recent studies by Choi et al. [19] show

c06.indd 94

7/30/2010 2:18:49 PM

QUANTUM DOTS

95

how size and charge of QDs affect their renal clearance. Selected types of QDs were injected intravenously in rodents and the key results showed that zwitterionic or neutral organic coatings prevented the adsorption of serum proteins and did not markedly impair renal excretion. These studies also show that a final hydrodynamic diameter <5.5 nm provides the appropriate size for urinary excretion and elimination of QDs from the body. Studies related to QDs in whole animals are sparse. One of the reasons for this is that imaging of the whole animal using fluorescent NPs, in general, remains difficult, because of strong autofluorescence of tissues due to endogenous chromophores (e.g., collagens, porphyrins, and flavins), in addition to the stability of the core and corona ligands. In most studies using CdSe/ZnS, the issue of core stability within the time period tested did not seem to pose a problem. However, noncovalently attached surface molecules might have been partly lost en route toward the predicted destination. It is also unknown to which degree QDs are quenched due to interactions with extracellular and cellular proteins. A newer class of QDs with superior photophysical properties, which can at least in part overcome these limitations by emitting in nearinfrared (NIR) regions, were prepared and tested in rodents [20]. Using NIR-emitting QDs avoids interfering noise from the animal autofluorescence. An example of such QDs is QD705 (20–25 nm), which were injected intravenously in mice. Results from these studies show that after 24 hours, they primarily accumulate in the liver and spleen. Analyses of urine and feces did not reveal significant levels, suggesting that most of the QD705 had remained in the body. Concordant with such a notion are results obtained by Fischer et al. [21]. It seems that surface-modified QDs with PEG are biocompatible with both living cells in culture [22] and whole animals. The physical, chemical, and biological properties, in addition to the availability, make PEG an attractive corona-forming candidate for micelles and QDs [23–26]. Reports on distribution and their pharmacokinetic properties [27–29] show that CdSe QDs are sequestered in several organs following intravenous administration, including lymph nodes [30] and solid tumors [23]. Studies by Inoue et al. [31] showed that QD injection allowed for long-term and repeated observation of the reticuloendothelial system (RES) in mice despite gradual decline of signal intensity. Combined fluorescence and luminescence imaging in luciferase-expressing tumor cells provided a way of concomitant detection of tumor cells and QDs. 6.2.3

Nanoparticle Distribution in the Brain

QD distribution in the brain in real time was not extensively explored. We studied glial cells in the nervous system because astrocytes and microglia are most likely the major players in delivering NPs to the central nervous system (CNS) [32, 33]. Importantly, they regulate neuronal signaling and viability; without their biochemical, morphological, and physiological communication, higher mental or emotional functions at the level of the whole

c06.indd 95

7/30/2010 2:18:49 PM

96

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

body would not be achievable. An example of imaging fluorescent CdSe/ZnS and InGaP/ZnS NPs in cells in culture and in vivo in transgenic mice showed functional responses of glia to NPs [34]. Such studies are particularly useful for the assessment of nonfluorescent and degenerate NPs, such as cerium oxide and degenerate “naked” CdTe NPs, which could not be easily detected by common techniques but can still interfere with CNS functions. Similar investigations could provide information on biocompatibility of QDs in live animals, in different glial cell types, especially after repeated imaging sessions. Studies by Maysinger et al. [35] focused on astrocyte response to different types of QDs. Astrocytes are the principal macroglial cell type in the brain and their activation is one of the key components of the cellular responses to stress and brain injuries. The passage from the quiescent to reactive astrocytes is associated with strong upregulation of the intermediate filament, glial fibrillary acidic protein (GFAP) [36, 37]. GFAP upregulation is considered a surrogate marker of neuronal stress and brain inflammatory response. Current methods of astrocyte and microglia detection are mainly based on immunocytochemistry. Using a transgenic mouse that carries the luciferase gene under the transcriptional control of murine GFAP promoter [38], upregulation of GFAP or luciferase expression in response to QDs was analyzed noninvasively in living animals using biophotonic imaging and a high-resolution CCD camera [39]. The principle of generating luciferase-expressing mice and imaging assays in real time are illustrated in Figure 6.1. 6.2.4

NP Distribution and Entry into Cells

Imaging of living cells provided a wealth of knowledge leading seminal findings regarding several types of QDs. These studies answered in part some of the key biological questions related to QD uptake [40], subcellular distribution, and functional consequences. Studies focused primarily on identifying what factors were primarily involved in determining the extent of uptake. Was it the size, charge, or other surface properties that determine the extent of the uptake? Are QDs taken up and retained or eliminated from the cells or do they accumulate intracellularly and ultimately get degraded? Despite the convincing data, these results are limited to particular cell types and particular QDs; much more studies are required to have information for other types of cells and QDs. Nonfunctionalized nanocrystals can exploit cells’ active transport machinery and be delivered into the cells and even the nucleus [41]. These studies show that living human macrophages are able to rapidly internalize and accumulate QDs in distinct cellular compartments, the extent of which depends on the QD’s size and charge. The most remarkable finding is that the smallest CdTe QDs specifically target histones in cell nuclei by a multistep process. The nuclear entry seems to be mediated via the nuclear pore complexes. These studies concur with previous findings by Lovric et al. [42] showing, by confocal

c06.indd 96

7/30/2010 2:18:49 PM

QUANTUM DOTS

TLR2 PROMOTER

Luciferase Reporter

GFAP PROMOTER

Luciferase Reporter

97

cDNA

Inject cDNA into fertilised oocyte

Plant oocyte into a pseudomother and breed transgenic pups

+ Stress + Luciferin

= Bioluminescent signal

Figure 6.1. Transgenic reporter mice with microglial and astroglial activation markers. Luciferase promoter constructs for the glial fibrillary acidic protein (GFAP; astroglial activation marker) and toll-like receptor (TLR2; microglial activation marker) are injected into donor oocytes, which are transplanted into pseudomothers, used to breed transgenic reporter mice. Prior to imaging, mice receive intraperitoneal injections of d-luciferin, the substrate for luciferase. Given that luciferase expression is regulated by stressful stimuli specific to these glial cell types, the resultant conversion of the substrate to oxyluciferin, the biolumiscent product, is proportional to the degree of stress.

microscopy, that this could be the case. More recent studies [43] provide further evidence that minute amounts of small (<5 nm in diameter) QDs can interfere with histones and induce epigenetic changes. Studies in this direction are warranted to uncover possible histone modifications with or without subsequent gene activations. This is an exciting and emerging new branch in nanotoxicology, which we propose to call “nanoepigenetics” [43]. An overview of modes of NP entry is provided in Figure 6.2. The concept of combining bioluminescence and fluorescence in living cells was first illustrated by So et al. [28]. These studies showed self-illuminating QDs tested in living cells and whole animals. The results from these studies provide evidence for the usefulness of QDs covalently bound with luciferase. When the conjugates were exposed to luciferase substrate, coelentrazine, the

c06.indd 97

7/30/2010 2:18:49 PM

98

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

ClathrinMediated

MacroPinocytosis

Amiloride Wortmannin Ly294002

Chlorpromazine Ikarugamycin Phagocytosis EEA-1

Nucleus

EEA-1

Bafilomycin A1 Chloroquine

CaveoleaMediated Clathrin Dynamin

GNPs QDs

Caveolin

CHPs

Methyl-βCyclodextrin Lovastatin

Figure 6.2. Mechanisms of nanoparticle uptake by cells. The four primary modes of NP uptake are illustrated. Clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and phagocytosis. Early endosomes are indicated by the presence of the early endosome antigen 1 (EEA-1), and acidification to the late endosome is indicated by red-yellow gradient, representing a drop in pH. Specifically, it has been shown that via receptor-mediated endocytosis, gold nanoparticles (GNPs), quantum dots (QDs), and nanogels (CHPs) are internalized within clathrin-coated vesicles. Evidence supporting caveolae-dependent uptake has also been demonstrated for GNPs and QDs. Secondary, nonspecific mechanisms are also illustrated, namely, macropinocytosis and phagocytosis. In addition, drugs that selectively inhibit these mechanisms are incorporated in the schematic, some of which have been used to investigate the uptake of NPs discussed in this chapter.

energy released by substrate catabolism was transferred to QDs by bioluminescence energy transfer leading to QD fluorescence. The protocol for making these self-illuminating QDs, provided by So, is widely applicable to other conjugation products, which can be utilized for different biosensing purposes. 6.2.5

Current Status and Future Perspective

Stabilized QDs with high fluorescent yield and specific ligands on their surfaces may eventually become common nanomaterials for the development of highly accurate and sensitive tests for clinical diagnosis. Such tests could

c06.indd 98

7/30/2010 2:18:49 PM

GOLD NANOPARTICLES

99

overcome the limitations of organic fluorophores (e.g., bleaching and wide spectral emission bands). In the meantime, QDs will continue to be very useful tools to explore the mechanisms of cell–NP interactions in experimental animals and living cells in cultures.

6.3 6.3.1

GOLD NANOPARTICLES General

Gold nanoparticles (GNPs) have attracted special attention among the known nanomaterials in the field. The localized surface plasmons (charge density oscillations) provide GNPs with unique optical properties, of which various biological applications (i.e., imaging, sensing, drug and gene delivery, and photothermal therapy) can take advantage. The excitation of localized surface plasmons that occurs for a specific frequency of light results in strong light scattering (an 80-nm diameter metallic NP is estimated to have a scattering flux that corresponds to those of 106 fluorescein molecules [44]), intense absorption, and enhancement of electromagnetic fields. In biological applications, these effects make GNPs particularly useful as labeling agents, signal amplifiers, sensors, or therapeutic agents. Indeed, metallic NPs were shown to be able to replace (or complement) established radioactive, fluorescent, chemiluminescent, or colorimetric labels, used in biochemistry, cell biology, and medical diagnostic applications [45–47]. The ultrabright, nonbleaching NPs can be observed individually and can be prepared with a scattering peak at any color of the visible or near-IR spectrum (by changing their size, shape, or surface), thus enabling the detection of single molecules and/or multiple targets. Rod-shaped GNPs of different lengths, for example, were successfully employed for multiplex identification of cell surface markers [48, 49]. Another advantage of the tunability of the surface plasmon excitation is that it opens the way to work in a desirable spectral range. Gold nanorods, nanoshells, and nanourchins are particularly attractive tools for in vivo applications, since their optical resonance lies in the near-infrared spectral window, away from the region of biomolecular excitation transitions, thus precluding photochemical damage and ensuring deeper penetration of the light into live systems [50–55]. A number of publications demonstrated the feasibility of in vivo imaging using GNPs. For example, gold nanoshells were imaged by photoacoustic tomography in rat brain [56], and PEG-coated spherical GNPs were used to show a clear delineation of cardiac ventricles and great vessels by computer tomography [57]. A twofold contrast was found between hepatoma and normal liver tissue by the same method [57]. In surface-enhanced Raman spectroscopy, large optical enhancement was achieved for the detection of tumors under in vivo conditions using small GNPs [58]. Gold nanorods have been imaged in vivo by two-photon luminescence (TPL) microscopy in epithelial cancer cells [59, 60] and in mouse ear blood vessels [61]. Our group

c06.indd 99

7/30/2010 2:18:49 PM

100

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

explored the fate of gold nanorods in living primary cortical neurons by TPL. We found that gold nanorods with CTAB, PEG, and insulin on their surfaces were internalized by living cortical neurons and could be imaged with excellent signal-to-noise ratio by two-photon luminescence microscopy, as shown in Figure 6.3. These examples of in vivo applications suggest great potential for accurate GNP-based assessment of acute ischemic stroke, lesion development, thermal injury, neovascularization, tumor angiogenesis, tumor necrosis, hepatic function, and regional hemodynamic activities. In addition to imaging, targeted GNPs with near-IR absorbance are excellent candidates for photothermal therapy (the plasmon resonant light-induced heat release is lethal to cancer cells in the proximity of GNPs). Cancer cells, labeled by tumor-specific antibody conjugated gold nanorods or nanoshells, can be selectively destroyed by near-IR laser irradiation [62–65]. Intravenously administered small GNPs were also shown to enhance the effect of radiation therapy in tumor-bearing mice [66, 67]. 6.3.2

Biocompatibility and Biodistribution of GNPs

While the favorable optical and photophysical parameters strongly promote GNPs for in vivo applications, relatively little is known about the interaction of GNPs with living cells and organisms. GNPs were considered inert for many years. However, new findings indicate that even these “noble” NPs are not so noble in dealing with living cells and they can also induce some of undesirable effects. Fortunately, GNP sizes and shapes can be stringently controlled and untoward effects can be reduced by precisely tailoring the size, charge, and ligand on the GNP surface. The longstanding use of colloidal gold in the treatment of rheumatoid arthritis and the use of radioactive GNPs to treat liver cancer [68, 69] suggests that colloidal GNPs are relatively inert and biologically compatible carriers. However, with recent developments in GNP synthesis and application systems, extensive biocompatibility studies are an absolute necessity. Besides the increasing variety of surface modification, a great number of novel shapes have been prepared during the last decade. Gold nanorods [53, 70], nanourchins [50, 54, 55], nanoprisms [71], nanowires [72], nanodisks [73], and nanostars [74, 75] were synthesized with high yield and reproducibility, awaiting their testing in biological systems. The internalization and biocompatibility of GNPs have been assessed in a number of studies employing mostly spherical GNPs and, occasionally, gold nanorods. Most studies revealed that exposure of cell lines or living organisms to spherical GNPs causes no acute cytotoxicity [76–78]. Colloidal GNPs need to be stabilized against aggregation by various ligands, which may be inherently toxic or induce GNP toxicity. For example, Rotello and co-workers reported that small cationic GNPs (2 nm) are more toxic toward red blood cells, Cos-1 cells, and Escherichia coli bacteria than anionic particles [79].

c06.indd 100

7/30/2010 2:18:50 PM

GOLD NANOPARTICLES

101

Figure 6.3. Two-photon luminescence (TPL) imaging of primary cortical neurons. TPL imaging of primary cortical neurons treated 21 days in vitro (DiV 21) with gold nanorods (rod GNP) for 4 hours prior to imaging (CTAB—cetyl-trimethylammonium bromide). Neurons were also stained with mitotracker green FM. Scale bar in the control panel is representative for both panels. (See color insert.)

Chan’s team work indicates that the size of the GNPs can also modulate their toxicity, small spherical GNPs being more toxic than their larger counterparts [80]. In addition, cell-specific toxicity has also been reported: the same 33-nm citrate-capped gold nanospheres that were found to be cytotoxic, at certain concentrations, to a human carcinoma lung cell line, proved to be innocuous toward baby hamster kidney and human hepatocellular liver carcinoma cells [81]. We tested different morphologies (spherical, rod-shaped, and urchin-like) for GNPs in primary neurons (Figure 6.3) in neuroblastoma cells (SH-SY5Y), and in microglial cultures, considered to be the macrophages of the nervous system (Figure 6.4). Results from these studies show that within the concentration range examined (up to 57.9 μM) GNPs do not harm neurons or glia, regardless of their shape and surface chemistry [81a]. Upon administration in animals, GNPs were found to accumulate in the liver and the spleen, but without causing apparent cytotoxic effects [66]. In a study evaluating the influence of particle size on the biodistribution of GNPs, rats were intravenously injected in the tail vein with GNPs with a diameter of 10, 50, 100, and 250 nm, respectively [82]. After 24 h, the rats were sacrificed and blood and various organs were collected for gold determination. The presence of gold was measured quantitatively with inductively coupled plasma mass spectrometry (ICP-MS) [82]. For all GNP sizes the majority of the gold was found to be present in liver and spleen. A clear difference was observed between the distribution of the 10-nm particles and the larger particles. The 10-nm particles were present in various organ systems including blood, liver, spleen, kidney, testis, thymus, heart, lung, and brain, whereas the larger particles were only detected in blood, liver, and spleen. The results demonstrate that tissue distribution of GNPs is size dependent with the smallest 10-nm NPs

c06.indd 101

7/30/2010 2:18:50 PM

102

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

Figure 6.4. Cellular internalization of GNPs. Microglial cells (N9) were treated for 4 hours with two different functionalized rod GNPs (rod GNP-CTAB, rod GNP-INS) and urchin-like GNPs (GNP-URCHIN). Cells were fixed with paraformaldehyde and subsequently analyzed for the internalization of GNPs. (*Nanoscale hyperspectral images, where red signals denote a positive match with the spectral profile of the respective GNP, were performed using Cytoviva Image Analysis Software.) (See color insert.)

showing the most widespread organ distribution [82]. Similar findings were reported by Sonavane et al. [83], with 15-, 50-, 100-, and 200-nm GNPs. Interestingly, 15- and 50-nm GNPs were able to pass the blood–brain barrier [83]. After oral administration of metallic colloidal GNPs of decreasing size (58, 28, 10, and 4 nm) to mice, an increased distribution to other organs was observed [84]. The smallest particle (4 nm) administered orally resulted in an increased presence of gold particles in kidney, liver, spleen, lungs, and even the brain. The biggest particle (58 nm) tested was detected almost solely inside the gastrointestinal tract [84]. For 13-nm sized colloidal gold beads the highest amount of gold was observed in liver and spleen after intraperitoneal administration [67]. In another study, intravenously injected gold nanorods revealed that within 30 minutes these particles accumulated predominantly in the liver [85]. The PEGylation (coating with polyethylene glycol) of these gold nanorods resulted in a prolonged circulation [85]. Naturally, the surface molecules attached to the GNPs play a determining role in the final localization of the

c06.indd 102

7/30/2010 2:18:50 PM

GOLD NANOPARTICLES

103

particles. By choosing a suitable ligand for surface modification, GNPs may be concentrated at certain locations in the living organism. Targeting tumorspecific surface receptors, for example, will bring the majority of the GNPs to the tumor cell membrane. There is not enough data for a complete overview of the effect of size, shape, and surface properties of GNPs on the cellular uptake, biodistribution, and cytotoxicity, but from the modest number of studies available to date one can conclude that these parameters are of great importance for biocompatibility and that different cell types have to be tested because of their different sensitivities toward GNPs. 6.3.3 Mechanism of Cellular Entry and Subcellular Distribution of Gold Nanoparticles Since the interaction between cells and GNPs depends strongly on the cell type and size and surface of the GNPs, there are cases when GNPs do not enter into the cells at all. PEG-modified GNPs, even after prolonged incubation for 24 h or after tenfold increase in concentration, were not found inside HeLa cells by electron microscopy, and no gold could be detected by absorption emission spectroscopy [86]. Under the right conditions, however, GNPs enter cells by a number of different routes. Not all of these ways are well understood, and it is not yet clear which factors favor any particular uptake mechanism (Figure 6.2). Receptor/clathrin-mediated endocytosis, followed by confinement to the endosomes, is the most commonly observed route of internalization [80, 86–88]. This process was studied for transferrin-coated GNPs and was found to be highly dependent on size, with the most efficient uptake occurring around the 50-nm size range [80, 88]. Shape dependency was also described, with spherical GNPs being internalized more readily than rodshaped GNPs [88]. Many factors, such as ratio of adhesion and membrane stretching and the membrane’s bending energy, may be involved in why size affects the uptake of the NPs. These parameters determine the so-called wrapping time, which describes how a membrane enclosed a particle [89]. Gao et al. [89] suggested that NPs that were ∼55 nm would have the fastest wrapping time and the receptor–ligand interaction can produce enough free energy to drive the NPs into the cell. This minimum wrapping time leads to a higher accumulation of the 55-nm NPs into the cell [89]. For the smaller NPs to go in, they must be clustered together. While a 50-nm GNP can enter the cell as a single NP, at least 6 of the 14-nm GNPs are required to cluster together before uptake [80]. For bigger NPs (>50 nm), the wrapping time is slower because of the slower receptor diffusion kinetics (more receptors were taken up during the receptor–ligand binding process and are not available for binding). Different proteins coated on the GNPs may lead to different results. The surface composition of the GNP can influence both the extent of internalization and the mechanism of uptake. For example, GNPs protected with

c06.indd 103

7/30/2010 2:18:50 PM

104

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

tiopronin induce either endocytosis or adhesion to the cell membrane depending on the chemical composition of the NP surface. The tiopronin GNPs derivatized with ethylenediamine induce endocytosis, and the same NPs functionalized with polyethylene glycol derivative promote particle-cell adhesion [90]. The endosomal pathway can be avoided if the NPs are delivered to the cells via liposomes or if they are chemically modified with membrane-penetrating peptide sequences, such as the oligopeptide TAT from HIV or Pntn protein from Drosophila Antennapedia [86]. There are a number of reports on successful nuclear targeting and transfection [86, 91–93] in which case the particles appear to enter the cytosol either directly through the cell membrane or by endosomal escape [86]. In these cases, particles can be observed in the cytosol and/or in the nucleus [86]. For gene delivery, in particular, but also to label designated sites, the ability to direct NPs toward targets inside the cell is of great interest.

6.3.4

Concluding Remarks

More studies are necessary for a complete overview of the effect of size, shape, and surface properties of GNPs on the cellular uptake, biodistribution, and cytotoxicity. To date, the modest number of studies available indicate that these parameters play a very important role in the GNP–cell interactions. Also, it is likely that the different cell types have to be tested individually for their sensitivity toward GNPs. The long-term effect of GNPs on living organisms remains obscure.

6.4

NANOGELS AND ARTIFICIAL CHAPERONES

Polymeric delivery systems currently dominate the nanomedicinal field [94, 95]. Polymeric NPs are matrix-like, characterized by the ability to provide targeted and predictable controlled release of a “drug” of interest [96, 97]. The drug of interest either can be encapsulated within the polymer matrix or can be adsorbed or conjugated onto the surface of the NP. Intrinsic to this model of delivery, cargo release is mediated by two processes that take place over time: diffusion of the drug out of the polymeric matrix and/or degradation and digestion of the polymer. A number of different polymers, both natural (gelatin, alginate, chitosan, etc.) and synthetic (polylactide–polyglycolide copolymers, polyacrylates, polycaprolactones, etc.), have been utilized for the formulation of polymeric NPs. By changing the formulation composition or method of formulation, the NP size, charge, hydrophobicity/hydrophilicity, and drug release can easily be controlled. A diverse array of therapeutic and diagnostic agents, such as lipophilic or hydrophilic drugs, oligonucle-

c06.indd 104

7/30/2010 2:18:50 PM

NANOGELS AND ARTIFICIAL CHAPERONES

105

otides, DNA, peptides, proteins, and antigens or even imaging agents, such as quantum dots, can be encapsulated in polymeric NPs [98]. Nanogels, which constitute polymeric NPs, are strictly defined as crosslinked polymer networks that form stable nanosized hydrogel particles upon self-association, commonly observed in an aqueous milieu. Nanogels, or hydrogel NPs, are another example of nanomaterials with applications in drug delivery, specifically to the CNS because of their ability to cross the blood– brain barrier (BBB) [6, 99]. The BBB is known to be the most restrictive barrier in the body, preventing the entry of many small molecules and most macromolecular drugs into the CNS [100]. Given the prevalence and debilitating nature of neurodegenerative diseases such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS), systems and strategies that improve drug delivery to specific intracellular sites within the CNS are fervently sought after. In many respects, nanogels have shown significant promise toward this avenue. The following addresses the most prominent of these advancements, with a particular emphasis on nanogels with the capacity to act as artificial chaperones and nanodelivery devices with potential therapeutic application in the diagnosis and treatment of neurodegenerative diseases, such as AD. 6.4.1 Nanodelivery and Artificial Chaperoning Properties of Nanogels Several different nanogel formulations have been developed for varying purposes, including the intracellular delivery of DNA [101] and proteins [102, 103] in a controlled and targeted manner, as well as for mediating chaperonelike activity [104]. In light of the fact that neurodegenerative diseases are commonly characterized by the accumulation of toxic protein aggregates [105], nanogels with the capacity to act both as artificial chaperones and drug delivery systems (DDS) hold a lot of promise as effective therapeutic strategies. In the early 1990s, long pullulan chains hydrophobized by the addition of cholesterol moieties at a frequency of 1–2 residues per 100 glucose units, termed cholesterol-bearing pullulans (CHPs), were shown to self-associate in water [106]. The self-aggregation of these amphiphilic polymers later led to the development of one of the first hydrogel NPs [107], which spontaneously forms due to noncovalent crosslinking between hydrophobic cholesterol moieties. In an in vitro model of protein refolding, carbonic anhydrase and citrate synthase were chemically denatured using guanidium chloride [108]. Interestingly, these nanogels were capable of preventing protein aggregation, while simultaneously stimulating the refolding of enzymes upon β-cyclodextrin binding in vitro, as evidenced by the recovery of activity. The mechanism of protein refolding was stipulated to be similar to that of an endogenous chaperone; that is, by catch and release, allowing the protein several new occasions to refold properly. Akiyoshi’s group was ultimately capable of demonstrating

c06.indd 105

7/30/2010 2:18:50 PM

106

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

similar refolding capacities of GroEL, the main bacterial chaperone, and CHP using acid-denatured green fluorescence protein (GFP) as an in vitro model [109]. The direct therapeutic application of artificial chaperones to neurodegenerative diseases was first evidenced by the ability of CHP nanogels to bind and prevent the fibrillation of β-amyloid in vitro [110], a property later shown to be common among other polymeric NPs in vitro [111, 112]. Nanogels with artificial chaperoning capacity also have the capacity to encapsulate proteins for targeted release as nanodelivery devices, already shown for bovine serum albumin (BSA) protein and β-galactosidase using cationic amino-group (NH2) derivatives of these nanogels [102], in addition to interleukin-12 (IL-12) with implications in tumor immunotherapy [103]. Our group proposes the use of nanogel DDS for the encapsulation of not only small charged or hydrophobic proteins, but for hydrophobic drugs as well. The ability to specifically target toxic hydrophobic aggregates extra- and intracellularly, while synergistically resulting in the controlled release of drug at the site of insult or injury, is especially advantageous in diseases that require effective targeting of several processes, simultaneously for successful intervention, such as cancer and neurodegeneration. 6.4.2

Intracellular Delivery and Fate of Nanogels

The first study conducted in cells with CHP nanogels employed the cationic derivatives (CHPNH2) encapsulating protein conjugated QD655, a hybrid NP with implications in bioimaging [113]. It was first shown that nanogels of CHPNH2 with 15 amino groups per 100 glucose residues encapsulating QD655 conjugated with protein A (CHPNH2(15)-QD) were the most efficiently uptaken by HeLa cells, a cervical cancer cell line, after 3 hours of incubation when compared with CHP-QD and CHPNH2(9)-QD derivatives. The fate of CHPNH2(15)-QDs were further investigated using confocal laser scanning fluorescence microscopy (CLSFM), and Hasegawa et al. [113] clearly show that they are more efficiently uptaken within 3 hours compared with QDs on their own or liposomes encapsulating QDs. This same group has recently shown that CHPNH2-QDs remained detectable in rabbit mesenchymal stem cells (MSCs) for at least 2 weeks following internalization and had minimal effects on the chondrogenic activity of MSCs [114], in addition to labeling periodontal ligament cells for at least 3 weeks [115]. Using flow cytometry, Ayame et al. [102] demonstrated that CHPNH2 consisting of 17 amino groups per 100 glucose units resulted in the most effective delivery of fluorescein isothiocyanate (FITC) labeled BSA (FITC-BSA) to HeLa cells, supporting the use of nanogels for intracellular delivery of proteins. They further illustrated that CHPNH2-FITC-BSA internalized the most efficiently compared with FITC-BSA in the absence of carrier, or in the presence of a protein transduction domain (PTD)–based carrier or even using cationic liposomes as an alternate NP-based carrier system. This was shown to be the case in three other cell lines, NIH-3T3, cos-7, and CHO-K1, as well

c06.indd 106

7/30/2010 2:18:50 PM

NANOGELS AND ARTIFICIAL CHAPERONES

107

as for FITC-β-gal, in the place of FITC-BSA. Given that the mode of entry into cells will dictate the subcellular fate of nanogels (e.g., endosomes, cytosol, and nucleus), Ayame et al. [102] investigated potential mechanisms of entry for cationic nanogel protein carriers. Cellular uptake was shown to drop by over 60% upon placing cells at 4 °C, a method known to inhibit general endocytic mechanisms. Further investigation with inhibitors of clathrin-mediated endocytosis (i.e., ikarugamycin) and macropinocytosis (i.e., amiloride) suggested the primary implication of macropinocytic mechanisms in the uptake of CHPNH2. We studied the uptake and intracellular fate of nanogels (i.e., CHP and CHPNH2) in N9 cells, a microglial cell line. Microglia can act as carriers for drug delivery systems and can result in specific targeting to the site of injury where microglia tend to accumulate, as is observed in AD at the site of senile plaques [116, 117]. Using plate-based fluorometric studies, fluorescence microscopy (Figure 6.5), and fluorescence activated cell sorter (FACS) analysis, we have shown that tetramethyl rhodamine isothiocyanate (TRITC)– labeled cationic nanogels (CHPNH2) readily accumulate intracellularly within 60 minutes in microglial cells, whereas CHP nanogels slowly accumulate over 3 hours [118]. However, CHPNH2 in the absence of negatively charged interacting species (i.e., oligonucleotide cargo) causes toxicity to microglial cells, whereas CHP nanogels are nontoxic, which is in agreement with other studies regarding cationic nanogel delivery systems [99]. These observations tend to suggest that neutral nanogels are useful for the incorporation of hydrophobic, uncharged molecules, whereas cationic nanogels require the incorporation of charged species. Our present studies indicate that CHPNH2, although readily taken up by primary murine neural cells, does not colocalize with lysosomes, labeled with lysotracker green DND-22, within this time period. Further studies are being conducted in microglial cells to demonstrate whether or not, and at what time point, cationic and neutral nanogels colocalize with lysosomes. Importantly, our group has clearly shown that nanogels favorably interact with Aβ, in the monomeric and oligomeric forms, the latter of which has recently been shown to be the most toxic species [119]. Using Förster resonance energy transfer (FRET) studies, nanogels, both cationic and neutral, bind Aβ monomers and oligomers in vitro. Moreover, cationic and neutral nanogels prevent Aβ-mediated toxicity in mouse microglia (N9) and primary murine cells, respectively, as evidenced using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. 6.4.3 Current Status in Clinical Trials and Future Perspectives of Nanogels Nanogels have already been employed as DDS in vivo and in clinical trials, primarily for cancer therapy. In mice with subcutaneous fibrosarcoma, subcutaneous injections of recombinant murine interleukin-12 (IL-12) encapsulated

c06.indd 107

7/30/2010 2:18:50 PM

108

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

CHP

CHPNH2

0

1h

2h

3h

Figure 6.5. Cellular uptake of TRITC-labelled CHP-nanoparticles. N9 microglial cells were treated with tetramethyl rhodamine isothiocyanate (TRITC)-labeled CHP nanogels (12 nM) for 3 hours. Fluorescent images were taken at each time point: before treatment (T = 0 min) and at 3 time points therafter (T = 1, 2, 3 hours post-treatment). Scale bar in top left panel (20 μm) is representative for all images. (See color insert.)

in CHP nanogels, via incubation at room temperature, led to a prolonged elevation of IL-12 in the sera and resulted in significant growth retardation of the tumor [103]. Clinical trials for a vaccine incorporating the HER2 tumor antigen into CHP nanogels as an effective delivery system to antigen presenting cells (APCs), such as denditic cells and macrophages, has also been developed. It was shown that with biweekly subcutaneous administration of CHP-HER2, patients not only tolerated the vaccine, but induced CD8+ and CD4+ T-cell responses [120]. They further illustrated that HER2specific IgG antibodies were elevated in the sera of patients vaccinated

c06.indd 108

7/30/2010 2:18:50 PM

BIOLOGICAL RESPONSES TO INTERNALIZED METALLIC NANOPARTICLES

109

with CHP-HER2 relative to controls and demonstrated that granulocytemacrophage colony-stimulating factor accelerated this humoral response to HER2 [121]. Given the minimal amount of information available presently with regard to the effects of nanogels on cellular functioning and more importantly on the subcellular targeting of nanogels for greater specificity, more studies need to be conducted at the single cell level. Importantly, nanogels appear to be excellent candidates for brain delivery, thus indicating that the majority of studies should focus on cell lines pertaining to brain cells, and more importantly on primary cultures, both human and rodent. The cellular uptake of specific types of nanogels will no doubt depend on the size, shape, and most importantly the composition. An investigation into the mechanisms of uptake not only at the blood–brain barrier, but also at the level of neurons and/or glial cells within the central nervous, will demonstrate which nanogels favor a cytosolic destination over an endosomal or nuclear, for example. Such studies are necessary if nanogels are ever to be proposed as specific drug delivery systems for targeting at the subcellular level.

6.5 BIOLOGICAL RESPONSES TO INTERNALIZED METALLIC NANOPARTICLES When metallic NPs enter cells they can induce inflammation in different cells [22] and, conceivably, there are several ways that NPs could exert their effects leading to or accelerating neurodegeneration: (1) by generating reactive oxygen species (ROS) and reactive nitrogen species (RNS) [17, 122–128], thereby altering the cellular redox status; (2) by induction or translocation of redox-sensitive transcription factors to the nucleus (e.g., NF-κB), which regulate proinflammatory genes, such as TNF-α and inducible nitric oxide synthase (iNOS) [129]; (3) by modification of proteins, lipids, and nucleic acids, which further stimulate the antioxidant defense system or even lead to cell death; (4) by excessively activating microglia and astrocytes; and (5) by promoting abnormalities in communications between organelles and post-translational modifications of proteins. Many diseases have been linked to oxidative stress and increased formation of ROS/RNS. Among these are lung, cardiovascular, and autoimmune diseases, as well as brain inflammation and aging [35]. As an underlying principle, cells exposed to inflammatory stimuli have a decreased capacity to deal with ROS/RNS, thus magnifying the extent of NP-induced injury. We are only now beginning to explore and understand the specific mechanisms underlying how low-dose NP exposure exerts these actions and consequently contributes to an enhanced rate and extent of neuroinflammation and neurodegeneration in the long term [34]. Oxidative stress is a state of redox disequilibrium that develops when ROS/ RNS production overwhelms the antioxidant defense system of the cell, thereby setting in motion the activation of redox signaling cascades that lead

c06.indd 109

7/30/2010 2:18:51 PM

110

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

to toxicity [1, 130–133]. The extent of oxidative stress can vary from the very mild to the most extensive depending on the insulting agent(s) (i.e., type and size of NP), their concentration(s), as well as the duration of exposure and the cell types exposed [134]. Three major levels of oxidative stress are proposed by Nel et al. [3], known as the “hierarchical oxidative stress model.” In this model minor levels of oxidative stress are regulated mainly by the transcription/nuclear factor, erythroid 2-related factor 2 (Nrf2), which controls the transcriptional activation of about 200 antioxidant and detoxification enzymes [135, 136]. Among these genes are heme oxygenase 1 (HO-1), glutathione S-transferase (GST) isoenzymes, NADPH quinine oxyreductase (NQO1) catalase, superoxide dismutase, and glutathione peroxidase (GPx) [135–138]. Interestingly, our recent studies show remarkable changes in antiapoptotic and pro-apoptotic genes induced by QDs [43]. Among the antioxidant defense systems, GPx was the most susceptible to the treatment with QDs and was completely abolished after 24 hours. When this preventative antioxidant defense system is insufficient, for instance, if long-term NP exposure is overwhelming the system, a pronounced increase in ROS can result and subsequently cause the release of proinflammatory cytokines [139–141]. This proinflammatory stage is mediated by the redox-sensitive MAPK and NF-κB cascades. Further increase in the severity of oxidative damage leads to morphological changes in mitochondria and also, as we have recently demonstrated, to an enlargement of lysosomes and activation of lysosomal enzymes [122]. Our pilot studies using a mouse-derived microglia cell line (N9 cells) show that a large number of NPs are internalized by these cells and, subsequently, glial cells are activated. Enzymatic activity in this activated state is significantly enhanced, supporting the view that activation of microglia leads to acidification of lysosomes and consequent activation of lysosomal proteases [142]. Evidence has been accumulating that among other free radicals, reactive nitrogen species (RNS) have been associated with the etiology and progression of inflammation in different neurodegenerative disorders and environmental insults [143, 144]. Nitric oxide (NO), a bioactive free radical, plays an important role in the development of neurodegenerative diseases such as Alzheimer disease (AD), Huntington disease, Pick disease, epilepsy, schizophrenia, and cerebral ischemia [145]. At low concentrations, NO plays a role in neurotransmission, synaptic plasticity, and vasodilatation, while at higher concentrations it is implicated in neurodegenerative disease pathologies [146, 147]. NO is enzymatically formed from l-arginine by the enzyme nitric oxide synthase (NOS). The calcium-dependent NOS present in neurons is constitutively expressed, whereas inducible nitric oxide (iNOS) expressed in astrocytes and microglia is inducible on the transcriptional level by various stimuli [143, 148]. Resident glia cells of the brain express iNOS and produce high levels of NO in response to a wide variety of proinflammatory and degenerative stimuli [149, 150]. Few cytokines, such as IL-β and IFN-γ, alone can induce iNOS; TNF-α is usually required in conjunction with IL-β and IFN-γ. In fact,

c06.indd 110

7/30/2010 2:18:51 PM

REFERENCES

111

the inducibility of NO depends on the cytokine, the cell type, and the species, as well as where the cells are derived from [149]. When NPs enter the cell, mechanisms involved in cell and tissue repair [151], including enzymatic systems maintaining normal cellular redox status, can be activated. These defensive systems include glutathione (glutathione/ glutathione peroxidase or glutathione/glutaredoxin) and thioredoxin (thioreduxin/thioredoxin reductase or thioredoxin/peroxiredoxin) systems. These systems complement each other and sometimes overlap in cytoprotection. Thioredoxin reductase is a member of the selenium-containing oxidoreductase family [152] and its role in regulating levels of oxidative stress is well recognized [153].

6.6

CONCLUSION

Inadequate drug delivery is one of the most important factors limiting the utilization of molecular medicines today. Effective targeting of drugs toward specific cell types and subcellular compartments has proved difficult but significant advances were made in achieving this goal, mainly in cancer therapy. Quantum dots seem to be particularly promising components of the highly sensitive and relatively simple diagnostic devices for the analyses of biological specimens ex vivo and in living cells due to their unique photophysical properties. GNPs are not only excellent markers of subcellular site distribution but also serve as extremely useful components of sensors for testing functional cellular responses and delivering anti-inflammatory and regeneration-promoting drugs. We should carefully examine possible side effects of NPs, particularly those with metallic cores. We are looking forward to new clinical trials with biocompatible and biodegradable polymeric NPs, delivering therapeutics with improved efficacy, as well as additional synergistic chaperoning activity, ultimately promoting the recovery of the compromised central nervous system.

ACKNOWLEDGMENTS We gratefully acknowledge the granting agencies National Science Research Council and Canadian Institutes of Health Research for support of the research carried out by the authors of this chapter.

REFERENCES 1. Xia, T., Kovochich, M., and Nel, A. The role of reactive oxygen species and oxidative stress in mediating particulate matter injury. Clin. Occup. Environ. Med. 5: 817–836 (2006).

c06.indd 111

7/30/2010 2:18:51 PM

112

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

2. Maysinger, D. Nanoparticles and cells: good companions and doomed partnerships. Org. Biomol. Chem. 5: 2335–2342 (2007). 3. Nel, A., Xia, T., Madler, L., and Li, N. Toxic potential of materials at the nanolevel. Science N.Y. 311: 622–627 (2006). 4. Bae, Y. and Kataoka, K. Intelligent polymeric micelles from functional poly(ethylene glycol)-poly(amino acid) block copolymers. Adv. Drug. Deliv. Rev. 61: 768–784 (2009). 5. Aliabadi, H. M., Shahin, M., Brocks, D. R., and Lavasanifar, A. Disposition of drugs in block copolymer micelle delivery systems: from discovery to recovery. Clin. Pharmacokinet. 47: 619–634 (2008). 6. Gilmore, J. L., Yi, X., Quan, L., and Kabanov, A. V. Novel nanomaterials for clinical neuroscience. J. Neuroimmune Pharmacol. 3: 83–94 (2008). 7. Mikhail, A. S. and Allen, C. Block copolymer micelles for delivery of cancer therapy: Transport at the whole body, tissue and cellular levels. J. Control. Release 138: 214–223 (2009). 8. Savic, R., Eisenberg, A., and Maysinger, D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle–cell interactions. J. Drug Target. 14: 343– 355 (2006). 9. Torchilin, V. P. Micellar nanocarriers: pharmaceutical perspectives. Pharm. Res. 24: 1–16 (2007). 10. Gaucher, G., Dufresne, M. H., Sant, V. P., Kang, N., Maysinger, D., and Leroux, J. C. Block copolymer micelles: preparation, characterization and application in drug delivery. J. Control. Release 109: 169–188 (2005). 11. Jain, M. P., Choi, A. O., Neibert, K. D., and Maysinger, D. Probing and preventing quantum dot-induced cytotoxicity with multimodal alpha-lipoic acid in multiple dimensions of the peripheral nervous system. Nanomedicine 4: 277–290 (2009). 12. Kang, J. X. and Weylandt, K. H. Modulation of inflammatory cytokines by omega-3 fatty acids. Subcell. Biochem. 49: 133–143 (2008). 13. Serhan, C. N., Chiang, N., and Van Dyke, T. E. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat. Rev. Immunol. 8: 349–361 (2008). 14. Alivisatos, A. P., Gu, W., and Larabell, C. Quantum dots as cellular probes. Annu. Rev. of Biomed. Eng. 7: 55–76 (2005). 15. Delehanty, J. B., Mattoussi, H., and Medintz, I. L. Delivering quantum dots into cells: strategies, progress and remaining issues. Anal. Bioanal. Chem. 393: 1091– 1105 (2009). 16. Medintz, I. L., Mattoussi, H., and Clapp, A. R. Potential clinical applications of quantum dots. Int. J. Nanomed. 3: 151–167 (2008). 17. Lovric, J., Cho, S. J., Winnik, F. M., and Maysinger, D. Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death. Chem. Biol. 12: 1227–1234 (2005). 18. Giepmans, B. N., Adams, S. R., Ellisman, M. H., and Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science N.Y. 312: 217–224 (2006).

c06.indd 112

7/30/2010 2:18:51 PM

REFERENCES

113

19. Choi, H. S., Liu, W., Misra, P., Tanaka, E., Zimmer, J. P., Itty Ipe, B., Bawendi, M. G., and Frangioni, J. V. Renal clearance of quantum dots. Nat. Biotechnol. 25: 1165–1170 (2007). 20. Choi, H. S., Ipe, B. I., Misra, P., Lee, J. H., Bawendi, M. G., and Frangioni, J. V. Tissue- and organ-selective biodistribution of NIR fluorescent quantum dots. Nano Lett. 9: 2354–2359 (2009). 21. Fischer, H. C., Liu, L., Pang, K. S., and Chan, W. C. W. Pharmacokinetics of nanoscale quantum dots: in vivo distribution, sequestration, and clearance in the rat. Adv. Funct. Mater. 16: 1299–1305 (2006). 22. Ryman-Rasmussen, J. P., Riviere, J. E., and Monteiro-Riviere, N. A. Surface coatings determine cytotoxicity and irritation potential of quantum dot nanoparticles in epidermal keratinocytes. J. Invest. Dermatol. 127: 143–153 (2007). 23. Ballou, B., Ernst, L. A., Andreko, S., Harper, T., Fitzpatrick, J. A., Waggoner, A. S., and Bruchez, M. P. Sentinel lymph node imaging using quantum dots in mouse tumor models. Bioconjug. Chem. 18: 389–396 (2007). 24. Bentzen, E. L., Tomlinson, I. D., Mason, J., Gresch, P., Warnement, M. R., Wright, D., Sanders-Bush, E., Blakely, R., and Rosenthal, S. J. Surface modification to reduce nonspecific binding of quantum dots in live cell assays. Bioconjug. Chem. 16: 1488–1494 (2005). 25. Boulmedais, F., Bauchat, P., Brienne, M. J., Arnal, I., Artzner, F., Gacoin, T., Dahan, M., and Marchi-Artzner, V. Water-soluble pegylated quantum dots: from a composite hexagonal phase to isolated micelles. Langmuir 22: 9797–9803 (2006). 26. Yu, W. W., Chang, E., Drezek, R., and Colvin, V. L. Water-soluble quantum dots for biomedical applications. Biochem. Biophys. Res. Commun. 348: 781–786 (2006). 27. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W., and Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22: 969–976 (2004). 28. So, M. K., Xu, C., Loening, A. M., Gambhir, S. S., and Rao, J. Self-illuminating quantum dot conjugates for in vivo imaging. Nat. Biotechnol. 24: 339–343 (2006). 29. Kim, S., Lim, Y. T., Soltesz, E. G., et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 22: 93–97 (2004). 30. Frangioni, J. V., Kim, S. W., Ohnishi, S., Kim, S., and Bawendi, M. G. Sentinel lymph node mapping with type-II quantum dots. Methods Mol. Biol. (Clifton, N.J.) 374: 147–160 (2007). 31. Inoue, Y., Izawa, K., Yoshikawa, K., Yamada, H., Tojo, A., and Ohtomo, K. In vivo fluorescence imaging of the reticuloendothelial system using quantum dots in combination with bioluminescent tumour monitoring. Eur. J. Nucl. Med. Mol. Imaging 34: 2048–2056 (2007). 32. Banati, R. B. Neuropathological imaging: in vivo detection of glial activation as a measure of disease and adaptive change in the brain. Br. Med. Bull. 65: 121–131 (2003). 33. Perry, V. H., Andersson, P. B., and Gordon, S. Macrophages and inflammation in the central nervous system. Trends Neurosci. 16: 268–273 (1993). 34. Behrendt, M., Sandros, M., McKinney, R., McDonald, K., Kriz, J., Przybytkowski, E., Tabrizian, M., and Maysinger, D. Cell imaging in real time and organelle

c06.indd 113

7/30/2010 2:18:51 PM

114

35.

36.

37. 38.

39.

40. 41.

42.

43.

44.

45.

46.

47.

48.

49.

c06.indd 114

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

distribution of fluorescent InGaP/ZnS nanoparticles. Nanomedicine 4: 747–761 (2009). Maysinger, D., Behrendt, M., Lalancette-Hebert, M., and Kriz, J. Real-time imaging of astrocyte response to quantum dots: in vivo screening model system for biocompatibility of nanoparticles. Nano Lett. 7: 2513–2520 (2007). Ridet, A., Guillouf, C., Duchaud, E., Cundari, E., Fiore, M., Moustacchi, E., and Rosselli, F. Deregulated apoptosis is a hallmark of the Fanconi anemia syndrome. Cancer Res. 57: 1722–1730 (1997). Pekny, M. and Nilsson, M. Astrocyte activation and reactive gliosis. Glia 50: 427–434 (2005). Zhu, L., Ramboz, S., Hewitt, D., Boring, L., Grass, D. S., and Purchio, A. F. Noninvasive imaging of GFAP expression after neuronal damage in mice. Neurosci. Lett. 367: 210–212 (2004). Kadurugamuwa, J. L., Modi, K., Coquoz, O., Rice, B., Smith, S., Contag, P. R., and Purchio, T. Reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous imaging, in vivo, of the pathogen and host response. Infect. Immunity 73: 7836–7843 (2005). Zhang, L. W. and Monteiro-Riviere, N. A. Mechanism of quantum dot nanoparticle cellular uptake. Toxicol. Sci. 110: 138–155 (2009). Nabiev, I., Mitchell, S., Davies, A., et al. Nonfunctionalized nanocrystals can exploit a cell’s active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nano Lett. 7: 3452–3461 (2007). Lovric, J., Bazzi, H. S., Cuie, Y., Fortin, G. R., Winnik, F. M., and Maysinger, D. Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. J. Mol. Med. (Berlin) 83: 377–385 (2005). Choi, A. O., Brown, S. E., Szyf, M., and Maysinger, D. Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells. J. Mol. Med. (Berlin) 86: 291–302 (2008). Ekgasit, S., Thammacharoen, C., Yu, F., and Knoll, W. Evanescent field in surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopies. Anal. Chem. 76: 2210–2219 (2004). Englebienne, P. Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes. Analyst 123: 1599–1603 (1998). Englebienne, P., Van Hoonacker, A., and Verhas, M. High-throughput screening using the surface plasmon resonance effect of colloidal gold nanoparticles. Analyst 126: 1645–1651 (2001). Watanabe, S., Sonobe, M., Arai, M., Tazume, Y., Matsuo, T., Nakamura, T., and Yoshida, K. Enhanced optical sensing of anions with amide-functionalized gold nanoparticles. Chem. Commun. 2866–2867 (2002). Yu, C. X., Nakshatri, H., and Irudayaraj, J. Identity profiling of cell surface markers by multiplex gold nanorod probes. Nano Lett. 7: 2300–2306 (2007). Yu, C. X. and Irudayaraj, J. Multiplex biosensor using gold nanorods. Anal. Chem. 79: 572–579 (2007).

7/30/2010 2:18:51 PM

REFERENCES

115

50. Lu, L. H., Ai, K., and Ozaki, Y. Environmentally friendly synthesis of highly monodisperse biocompatible gold nanoparticles with urchin-like shape. Langmuir 24: 1058–1063 (2008). 51. Jain, P. K., Lee, K. S., El-Sayed, I. H., and El-Sayed, M. A. Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine. J. Phys. Chem. B 110: 7238–7248 (2006). 52. Sau, T. K. and Murphy, C. J. Room temperature, high-yield synthesis of multiple shapes of gold nanoparticles in aqueous solution. J. Am. Chem. Soc. 126: 8648– 8649 (2004). 53. Nikoobakht, B. and El-Sayed, M. A. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Materials 15: 1957– 1962 (2003). 54. Bakr, O. M., Wunsch, B. H., and Stellacci, F. High-yield synthesis of multi-branched urchin-like gold nanoparticles. Chem. Materials 18: 3297–3301 (2006). 55. Yu, K. F., Kelly, K. L., Sakai, N., and Tatsuma, T. Morphologies and surface plasmon resonance properties of monodisperse bumpy gold nanoparticles. Langmuir 24: 5849–5854 (2008). 56. Wang, Y. W., Xie, X. Y., Wang, X. D., Ku, G., Gill, K. L., O’Neal, D. P., Stoica, G., and Wang, L. V. Photoacoustic tomography of a nanoshell contrast agent in the in vivo rat brain. Nano Lett. 4: 1689–1692 (2004). 57. Kim, D., Park, S., Lee, J. H., Jeong, Y. Y., and Jon, S. Antibiofouling polymercoated gold nanoparticles as a contrast agent for in vivo X-ray computed tomography imaging. J. Am. Chem. Soc. 129: 7661–7665 (2007). 58. Qian, X. M., Peng, X. H., Ansari, D. O., et al. In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nat. Biotechnol. 26: 83–90 (2008). 59. Durr, N. J., Larson, T., Smith, D. K., Korgel, B. A., Sokolov, K., and Ben-Yakar, A. Two-photon luminescence imaging of cancer cells using molecularly targeted gold nanorods. Nano Lett. 7: 941–945 (2007). 60. Huff, T. B., Hansen, M. N., Zhao, Y., Cheng, J. X., and Wei, A. Controlling the cellular uptake of gold nanorods. Langmuir 23: 1596–1599 (2007). 61. Wang, H. F., Huff, T. B., Zweifel, D. A., He, W., Low, P. S., Wei, A., and Cheng, J. X. In vitro and in vivo two-photon luminescence imaging of single gold nanorods. Proc. Nat. Acad. Sci. U.S.A. 102: 15752–15756 (2005). 62. Bernardi, R., Lowery, A., Thompson, P., Blaney, S., and West, J. Immunonanoshells for targeted photothermal ablation in medulloblastoma and glioma: an in vitro evaluation using human cell lines. J. Neuro-Oncol. 86: 165–172 (2008). 63. El-Sayed, I. H., Huang, X. H., and El-Sayed, M. A. Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Lett. 239: 129–135 (2006). 64. O’Neal, D. P., Hirsch, L. R., Halas, N. J., Payne, J. D., and West, J. L. Photothermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Lett. 209: 171–176 (2004).

c06.indd 115

7/30/2010 2:18:51 PM

116

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

65. Huang, X. H., El-Sayed, I. H., Qian, W., and El-Sayed, M. A. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 128: 2115–2120 (2006). 66. Gannon, C., Patra, C., Bhattacharya, R., Mukherjee, P., and Curley, S. Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells. J. Nanobiotechnol. 6: 2 (2008). 67. Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 49: N309–N315 (2004). 68. Rubin, P. and Levitt, S. H. The response of disseminated reticulum cell sarcoma to the intravenous injection of colloidal radioactive gold. J. Nucl. Med. 5: 581–594 (1964). 69. Alfonso, A. E., Hassan, A., Gardner, B., Stein, S., Patti, J., Solomon, N. A., McCarthy, J., and Steigman, J. Prevention of hepatic metastases by intravenous radioactive gold. Cancer Res. 38: 2740–2744 (1978). 70. Sau, T. K. and Murphy, C. J. Seeded high yield synthesis of short Au nanorods in aqueous solution. Langmuir 20: 6414–6420 (2004). 71. Millstone, J. E., Metraux, G. S., and Mirkin, C. A. Controlling the edge length of gold nanoprisms via a seed-mediated approach. Adv. Functional Materials 16: 1209–1214 (2006). 72. Lu, X. M., Yavuz, M. S., Tuan, H. Y., Korgel, B. A., and Xia, Y. N. Ultrathin gold nanowires can be obtained by reducing polymeric strands of oleylamine– AuCl complexes formed via aurophilic interaction. J. Am. Chem. Soc. 130: 8900 (2008). 73. Shen, Z. R., Miyabayashi, K., Higashimoto, M., Shimoda, T., and Miyake, M. Single-crystalline gold nanodisks prepared by the shape transformation under UV irradiation from nanoparticles protected with discotic liquid crystalline ligands. Chem. Lett. 37: 1276–1277 (2008). 74. Khoury, C. G. and Vo-Dinh, T. Gold nanostars for surface-enhanced Raman scattering: synthesis, characterization and optimization. J. Phys. Chem. C 112: 18849–18859 (2008). 75. Kumar, P. S., Pastoriza-Santos, I., Rodriguez-Gonzalez, B., Garcia de Abajo, F. J., and Liz-Marzan, L. M. High-yield synthesis and optical response of gold nanostars. Nanotechnology 19: DOI:10.1088/0957-4484/19/01/015606 (2008). 76. Brown, C., Whitehouse, M., Tiekink, E., and Bushell, G. Colloidal metallic gold is not bio-inert. Inflammopharmacology 16: 133–137 (2008). 77. Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J., and Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 1: 325–327 (2005). 78. Murphy, C. J., Gole, A. M., Stone, J. W., Sisco, P. N., Alkilany, A. M., Goldsmith, E. C., and Baxter, S. C. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc. Chem. Res. 41: 1721–1730 (2008). 79. Goodman, C. M., McCusker, C. D., Yilmaz, T., and Rotello, V. M. Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjug. Chem. 15: 897–900 (2004). 80. Jiang, W., Kim, B. Y. S., Rutka, J. T., and Chan, W. C. W. Nanoparticle-mediated cellular response is size-dependent. Nat. Nanotechnol. 3: 145–150 (2008).

c06.indd 116

7/30/2010 2:18:51 PM

REFERENCES

117

81. Patra, H. K., Banerjee, S., Chaudhuri, U., Lahiri, P., and Dasgupta, A. K. Cell selective response to gold nanoparticles. Nanomed. Nanotechnol. Biol. Med. 3: 111–119 (2007). 81a.Hutter, E., Boridy, S., Labrecque, S., Lalancette-Hébert, M., Kriz, J., Winnik, F. M., and Maysinger, D. ACS Nano. 4: 2595–2606 (2010). 82. De Jong, W. H., Hagens, W. I., Krystek, P., Burger, M. C., Sips, A. J. A. M., and Geertsma, R. E. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 29: 1912–1919 (2008). 83. Sonavane, G., Tomoda, K., and Makino, K. Biodistribution of colloidal gold nanoparticles after intravenous administration: effect of particle size. Colloids Surfaces B Biointerfaces 66: 274–280 (2008). 84. Julián F. and Hillyer, R. M. A. Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles. J. Pharm. Sci. 90: 1927–1936 (2001). 85. Niidome, T., Yamagata, M., Okamoto, Y., Akiyama, Y., Takahashi, H., Kawano, T., Katayama, Y., and Niidome, Y. PEG-modified gold nanorods with a stealth character for in vivo applications. J. Control. Release 114: 343–347 (2006). 86. Nativo, P., Prior, I. A., and Brust, M. Uptake and intracellular fate of surfacemodified gold nanoparticles. ACS Nano 2: 16390–1644 (2008). 87. Chithrani, B. D. and Chan, W. C. W. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett. 7: 1542–1550 (2007). 88. Chithrani, B. D., Ghazani, A. A., and Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6: 662–668 (2006). 89. Gao, H., Shi, W., and Freund, L. B. Mechanics of receptor-mediated endocytosis. Proc. Nat. Acad. Sci. U.S.A. 102: 9469–9474 (2005). 90. de la Fuente, J. M., Berry, C. C., Riehle, M. O., and Curtis, A. S. G. Nanoparticle targeting at cells. Langmuir 22: 3286–3293 (2006). 91. Berry, C. C., de la Fuente, J. M., Mullin, M., Chu, S. W. L., and Curtis, A. S. G. Nuclear localization of HIV-1 Tat functionalized gold nanoparticles. NanoBioscience IEEE Trans. 6: 262–269 (2007). 92. Tkachenko, A. G., Xie, H., Liu, Y., Coleman, D., Ryan, J., Glomm, W. R., Shipton, M. K., Franzen, S., and Feldheim, D. L. Cellular trajectories of peptidemodified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjug. Chem. 15: 482–490 (2004). 93. Oyelere, A. K., Chen, P. C., Huang, X. H., El-Sayed, I. H., and El-Sayed, M. A. Peptide-conjugated gold nanorods for nuclear targeting. Bioconjug. Chem. 18: 1490–1497 (2007). 94. Wagner, V., Dullaart, A., Bock, A. K., and Zweck, A. The emerging nanomedicine landscape. Nat. Biotechnol. 24: 1211–1217 (2006). 95. Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., and Farokhzad, O. C. Nanoparticles in medicine: therapeutic applications and developments. Clin. Pharmacol. Ther. 83: 761–769 (2008). 96. Moghimi, S. M., Hunter, A. C., and Murray, J. C. Long-circulating and targetspecific nanoparticles: theory to practice. Pharmacol. Rev. 53: 283–318 (2001).

c06.indd 117

7/30/2010 2:18:51 PM

118

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

97. Panyam, J. and Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55: 329–347 (2003). 98. Bertin, P. A., Gibbs, J. M., Shen, C. K., Thaxton, C. S., Russin, W. A., Mirkin, C. A., and Nguyen, S. T. Multifunctional polymeric nanoparticles from diverse bioactive agents. J. Am. Chem. Soc. 128: 4168–4169 (2006). 99. Vinogradov, S. V., Batrakova, E. V., and Kabanov, A. V. Nanogels for oligonucleotide delivery to the brain. Bioconjug. Chem. 15: 50–60 (2004). 100. Patel, M. M., Goyal, B. R., Bhadada, S. V., Bhatt, J. S., and Amin, A. F. Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs 23: 35–58 (2009). 101. Lemieux, P., Vinogradov, S. V., Gebhart, C. L., et al. Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell. J. Drug Target. 8: 91–105 (2000). 102. Ayame, H., Morimoto, N., and Akiyoshi, K. Self-assembled cationic nanogels for intracellular protein delivery. Bioconjug. Chem. 19: 882–890 (2008). 103. Shimizu, T., Kishida, T., Hasegawa, U., Ueda, Y., Imanishi, J., Yamagishi, H., Akiyoshi, K., Otsuji, E., and Mazda, O. Nanogel DDS enables sustained release of IL-12 for tumor immunotherapy. Biochem. Biophys. Res. Commun. 367: 330– 335 (2008). 104. Akiyoshi, K., Sasaki, Y., and Sunamoto, J. Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B. Bioconjug. Chem. 10: 321–324 (1999). 105. Muchowski, P. J. and Wacker, J. L. Modulation of neurodegeneration by molecular chaperones. Nat. Rev. Neurosci. 6: 11–22 (2005). 106. Akiyoshi, K., Deguchi, S., Moriguchi, N., Yamaguchi, S., and Sunamoto, J. Selfaggregates of hydrophobized polysaccharides in water. Formation and characteristics. Macromolecules 26: 3062–3068 (1993). 107. Akiyoshi, K., Deguchi, S., Tajima, H., Nishikawa, T., and Sunamoto, J. Microscopic structure and thermoresponsiveness of a hydrogel nanoparticle by self-assembly of a hydrophobized polysaccharide. Macromolecules 30: 857–861 (1997). 108. Nomura, Y., Ikeda, M., Yamaguchi, N., Aoyama, Y., and Akiyoshi, K. Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone. FEBS Lett. 553: 271–276 (2003). 109. Asayama, W., Sawada, S., Taguchi, H., and Akiyoshi, K. Comparison of refolding activities between nanogel artificial chaperone and GroEL systems. Int. J. Biol. Macromol. 42: 241–246 (2008). 110. Ikeda, K., Okada, T., Sawada, S., Akiyoshi, K., and Matsuzaki, K. Inhibition of the formation of amyloid beta-protein fibrils using biocompatible nanogels as artificial chaperones. FEBS Lett. 580: 6587–6595 (2006). 111. Cabaleiro-Lago, C., Quinlan-Pluck, F., Lynch, I., Lindman, S., Minogue, A. M., Thulin, E., Walsh, D. M., Dawson, K. A., and Linse, S. Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles. J. Am. Chem. Soc. 130: 15437– 15443 (2008). 112. Heegaard, P. M., Boas, U., and Otzen, D. E. Dendrimer effects on peptide and protein fibrillation. Macromol. Biosci. 7: 1047–1059 (2007).

c06.indd 118

7/30/2010 2:18:51 PM

REFERENCES

119

113. Hasegawa, U., Nomura, S. M., Kaul, S. C., Hirano, T., and Akiyoshi, K. Nanogelquantum dot hybrid nanoparticles for live cell imaging. Biochem. Biophys. Res. Commun. 331: 917–921 (2005). 114. Toita, S., Hasegawa, U., Koga, H., Sekiya, I., Muneta, T., and Akiyoshi, K. Protein-conjugated quantum dots effectively delivered into living cells by a cationic nanogel. J. Nanosci. Nanotechnol. 8: 2279–2285 (2008). 115. Fukui, T., Kobayashi, H., Hasegawa, U., Nagasawa, T., Akiyoshi, K., and Ishikawa, I. Intracellular delivery of nanogel-quantum dot hybrid nanoparticles into human periodontal ligament cells. Drug Metab. Lett. 1: 131–135 (2007). 116. Frautschy, S. A., Yang, F., Irrizarry, M., Hyman, B., Saido, T. C., Hsiao, K., and Cole, G. M. Microglial response to amyloid plaques in APPsw transgenic mice. Am. J. Pathol. 152: 307–317 (1998). 117. McGeer, P. L., Itagaki, S., Tago, H., and McGeer, E. G. Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR. Neurosci. Lett. 79: 195–200 (1987). 118. Boridy, S., Takahashi, H., Akiyoshi, K., and Maysinger, D. The binding of pullulan modified cholesterol nanogel to Abeta oligomers and their suppression of cytotoxicity Biomaterials 30: 5583–5591 (2009). 119. Selkoe, D. J. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav. Brain. Res. 192: 106–113 (2008). 120. Kitano, S., Kageyama, S., Nagata, Y., et al. HER2-specific T-cell immune responses in patients vaccinated with truncated HER2 protein complexed with nanogels of cholesteryl pullulan. Clin. Cancer Res. 12: 7397–7405 (2006). 121. Kageyama, S., Kitano, S., Hirayama, M., et al. Humoral immune responses in patients vaccinated with 1–146 HER2 protein complexed with cholesteryl pullulan nanogel. Cancer Sci. 99: 601–607 (2008). 122. Cho, S. J., Maysinger, D., Jain, M., Roder, B., Hackbarth, S., and Winnik, F. M. Long-term exposure to CdTe quantum dots causes functional impairments in live cells. Langmuir 23: 1974–1980 (2007). 123. Choi, A. O., Cho, S. J., Desbarats, J., Lovric, J., and Maysinger, D. Quantum dot-induced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells. J. Nanobiotechnol. 5: 1 (2007). 124. Mamelak, M. Alzheimer’ s disease, oxidative stress and gammahydroxybutyrate. Neurobiol. Aging, 28: 1340–1360 (2007). 125. Moreira, P. I., Santos, M. S., and Oliveira, C. R. Alzheimer’s disease: a lesson from mitochondrial dysfunction. Antioxidants & Redox Signaling 9: 1621–1630 (2007). 126. Morinaga, A., Hirohata, M., Ono, K., and Yamada, M. Estrogen has antiamyloidogenic effects on Alzheimer’s beta-amyloid fibrils in vitro. Biochem. Biophys. Res. Commun. 359: 697–702 (2007). 127. Zhu, X., Su, B., Wang, X., Smith, M. A., and Perry, G. Causes of oxidative stress in Alzheimer disease. Cell Mol. Life Sci. 64: 2202–2210 (2007). 128. Li, N., Sioutas, C., Cho, A., et al. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ. Health Perspect. 111: 455–460 (2003). 129. Castranova, V. Signaling pathways controlling the production of inflammatory mediators in response to crystalline silica exposure: role of reactive oxygen/ nitrogen species. Free Radic. Biol. Med. 37: 916–925 (2004).

c06.indd 119

7/30/2010 2:18:51 PM

120

SUBCELLULAR FATE OF NANODELIVERY SYSTEMS

130. Bell, A. T. The impact of nanoscience on heterogeneous catalysis. Science N.Y. 299: 1688–1691 (2003). 131. Halliwell, B. and Gutteridge, J. M. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch. Biochem. Biophys. 246: 501–514 (1986). 132. Li, N., Hao, M., Phalen, R. F., Hinds, W. C., and Nel, A. E. Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin. Immunol. 109: 250–265 (2003). 133. Xiao, G. G., Wang, M., Li, N., Loo, J. A., and Nel, A. E. Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line. J. Biol. Chem. 278: 50781–50790 (2003). 134. Foged, C., Brodin, B., Frokjaer, S., and Sundblad, A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int. J. Pharm. 298: 315–322 (2005). 135. Chan, K. and Kan, Y. W. Nrf2 is essential for protection against acute pulmonary injury in mice. Proc. Nat. Acad. Sci. U.S.A. 96: 12731–12736 (1999). 136. Li, N., Alam, J., Venkatesan, M. I., et al. Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. J. Immunol. 173: 3467–3481 (2004). 137. Talalay, P., Dinkova-Kostova, A. T., and Holtzclaw, W. D. Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis. Adv. Enzyme Regul. 43: 121–134 (2003). 138. Dinkova-Kostova, A. T., Liby, K. T., Stephenson, K. K., et al. Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress. Proc. Nat. Acad. Sci. U.S.A. 102: 4584–4589 (2005). 139. Fine, J. M., Gordon, T., Chen, L. C., Kinney, P., Falcone, G., and Beckett, W. S. Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. J. Occup. Environ. Med. 39: 722–726 (1997). 140. Ho, G. J., Drego, R., Hakimian, E., and Masliah, E. Mechanisms of cell signaling and inflammation in Alzheimer’s disease. Curr. Drug Targets Inflamm. Allergy 4: 247–256 (2005). 141. Sayes, C. M., Wahi, R., Kurian, P. A., Liu, Y., West, J. L., Ausman, K. D., Warheit, D. B., and Colvin, V. L. Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol. Sci. 92: 174–185 (2006). 142. Majumdar, A., Cruz, D., Asamoah, N., Buxbaum, A., Sohar, I., Lobel, P., and Maxfield, F. R. Activation of microglia acidifies lysosomes and leads to degradation of Alzheimer amyloid fibrils. Mol. Biol. Cell. 18: 1490–1496 (2007). 143. Jaffrey, S. R. and Snyder, S. H. Nitric oxide: a neural messenger. Annu. Rev. Cell Dev. Biol. 11: 417–440 (1995). 144. Nathan, C. and Xie, Q. W. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915–918 (1994).

c06.indd 120

7/30/2010 2:18:51 PM

REFERENCES

121

145. Aksenova, M. V., Aksenov, M. Y., Mactutus, C. F., and Booze, R. M. Cell culture models of oxidative stress and injury in the central nervous system. Curr. Neurovasc. Res. 2: 73–89 (2005). 146. Garthwaite, J. and Boulton, C. L. Nitric oxide signaling in the central nervous system. Annu. Rev. Physiol. 57: 683–706 (1995). 147. Saha, R. N. and Pahan, K. Regulation of inducible nitric oxide synthase gene in glial cells. Antioxid. Redox Signal. 8: 929–947 (2006). 148. Galea, E., Feinstein, D. L., and Reis, D. J. Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. Proc. Natl. Acad. Sci. U.S.A. 89: 10945–10949 (1992). 149. Jana, M., Anderson, J. A., Saha, R. N., Liu, X., and Pahan, K. Regulation of inducible nitric oxide synthase in proinflammatory cytokine-stimulated human primary astrocytes. Free Radic. Biol. Med. 38: 655–664 (2005). 150. Trajkovic, V., Stosic-Grujicic, S., Samardzic, T., Markovic, M., Miljkovic, D., Ramic, Z., and Mostarica Stojkovic, M. Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes. J. Neuroimmunol. 119: 183–191 (2001). 151. Perry, G., Avila, J., Kinoshita, J., and Smith, M. A. Alzheimer’s Disease: A Century of Scientific and Clinical Research. IOS Press, Amsterdam, 2006. 152. Mustacich, D. and Powis, G. Thioredoxin reductase. Biochem. J. 346(Pt 1): 1–8 (2000). 153. Venardos, K. M. and Kaye, D. M. Myocardial ischemia-reperfusion injury, antioxidant enzyme systems, and selenium: a review. Curr. Med. Chem. 14: 1539–1549 (2007).

c06.indd 121

7/30/2010 2:18:51 PM

c06.indd 122

7/30/2010 2:18:51 PM

CHAPTER 7

Intracellular Fate of Plasmid DNA Polyplexes KEVIN MAIER and ERNST WAGNER Pharmaceutical Biotechnology, Munich Center for System-Based Drug Research, and Center for NanoScience, Ludwig-Maximilians-Universität, Munich, Germany

7.1

INTRODUCTION

Gene delivery by synthetic vectors started almost 50 years ago, when Smull and Ludwig [1] demonstrated that uptake of poliovirus ribonucleic acid is enhanced by interaction with plaque-forming basic proteins. From this point of time, up to date many cationic polymers were developed with the ability to condense plasmid DNA (pDNA) and promote its cellular uptake (e.g., diethylaminomodified dextran, poly-l-lysine (PLL), polyethylene imine (PEI), chitosan, cationic dendrimers). These complexes, built up from synthetic polymers and DNA, have been named polyplexes [2]. Shortly after their discovery it became clear that polyplexes possess advantages and disadvantages at the same time as compared to viral gene vectors. Flexibility concerning the payload is one of the major benefits of polyplexes. The most severe handicap of the polyplexes is their far lower transfection efficiency. To improve this, it is necessary to understand the barriers these polyplexes are confronted with, during their journey from the extracellular environment to the site of action—the nucleus in the case of pDNA, the cytosol in the case of RNA and siRNA. Cell entry, endolysosomal escape, cytoplasmic trafficking, DNA polyplex dissociation, and transport to the nucleus have been identified as the main hurdles that have to be overcome by the polyplexes. Numerous efforts were taken to address all these problems. Polymers used at the beginning of synthetic gene delivery were quite simple but tend to become more and more complex and chemically dynamic in structure. According to the different cellular compartments (each with a different chemical environment) in which the polyplexes Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

123

c07.indd 123

7/30/2010 2:18:53 PM

124

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

find themselves during the delivery of their cargo, they are supposed to change their chemical properties to react in the required manner. Since these new polyplexes behave almost like their natural viral counterparts, they are already called “synthetic viruses.” This chapter reviews state-of-the-art concepts and insights into the intracellular behavior of various polyplexes used for gene delivery.

7.2

CELLULAR BINDING AND UPTAKE

The first step of gene delivery by synthetic vectors is binding of the polyplex to the plasma membrane. If no targeting ligand is used, this binding is mediated through electrostatic interaction between the negatively charged cell surface and the positively charged surface of the polyplex. Little is known about the exact role of different cell surface components like glycosaminoglycans (GAGs), proteoglycans, integrins, and phospholipids. Mislick and Baldeschwieler [3] showed in 1996 that binding of PLL polyplexes to proteoglycan-deficient CHO mutant cells is strongly reduced and therefore suggested that heparin sulfate proteoglycans are involved in binding. Another evidence for their hypothesis was the limited binding of polyplexes in the presence of competitive soluble heparin and heparan sulfate in the medium. Chondroitin sulfate seems to play a minor role and the addition of hyaluronic acid had no effect on binding. A special role is supposed for adhesion of the polyplexes to syndecans (transmembrane heparan sulfate proteoglycans). Binding should effect enclosing the particle by lateral diffusion of the syndecans, clustering into cholesterol-rich rafts. Furthermore, these clusters cause activation of protein kinase C (PKC) and linker mediated binding of actin to the cytoplasmatic end of the syndecan molecule. The resulting fibers were supposed to pull the particle into the cell [4].

7.2.1

Uptake Mechanisms

Due to their size and charged surface, polyplexes cannot diffuse passively through the plasma membrane. All types of polyplexes share endocytosis as a common uptake means [5–7]. In literature at least five different ways of endocytosis are described—phagocytosis, clathrin-mediated endocytosis, macropinocytosis, caveolae-mediated endocytosis, and clathrin-caveolae-independent endocytosis [8]. It is necessary to understand these internalization pathways, as they vary in size, coating, and most importantly in further processing of the internalized vesicles after uptake. The different uptake mechanisms and further processing of the vesicles are shown in Figure 7.1. For example, uptake through caveolae-mediated endocytosis has the advantage that the polyplex is not internalized in vesicles, which can lead to lysosomal degradation [9]. Which endocytotic mechanism by which the polyplexes are

c07.indd 124

7/30/2010 2:18:53 PM

CELLULAR BINDING AND UPTAKE

125

Figure 7.1. Schematic illustration of polyplex uptake pathways and further processing of the internalized vesicles. Except for caveolae-mediated endocytosis, all other cellular entries lead at least partly to fusion of the vesicles with lysosomes and therefore to acidic degradation of the cargo.

internalized is dependent on many parameters, like particle size, the polymer employed, and the cell line [10–13]. 7.2.1.1 Nonspecific Uptake As described above, standard polyplexes are bound to the plasma membrane by electrostatic interaction. They are supposed to be taken up mainly by clathrin-dependent endocytosis (CDE) and caveolae-mediated endocytosis (CME). This assumption is based on many transfection experiments with different chemicals inhibiting different uptake pathways. Colocalization studies with transferrin (evidence for CDE) and choleratoxin B (evidence for CME) also revealed that linear PEI (LPEI) and branched PEI (BPEI) polyplexes are included via both mechanisms [13]. Moreover, by inhibiting one specific pathway, for example, CDE with chlorpromazine or caveolae-mediated endocytosis with filipin III, cell line dependence for the favored uptake mechanism could be shown. The main player for successful transcription in COS-7 cells with LPEI and BPEI was CDE, whereas in HUH-7 cells both pathways are equally important for transfection with BPEI. For LPEI polyplexes again CDE was the main contributor to the transcription process. In HeLa cells, independent of the polymer employed, both internalization pathways led to effective transcription with a small preference for the caveolae-mediated route. Only a few studies have been done to

c07.indd 125

7/30/2010 2:18:53 PM

126

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

reveal the mode of endocytosis for PLL polyplexes. Lühmann et al. [14] showed that in COS-7 cells the internalized PLL-g-PEG polyplex did not colocalize with the early endosome antigen-1 (EEA-1), which is a marker for the endolysosomal pathway. Although the cellular uptake of the nanoparticle was inhibited about 50% with genistein (inhibitor of CME), there was no decrease in transfection efficiency. Furthermore, no colocalization of the polyplexes with caveolin 1 protein could be observed. This result led to the assumption that the impact of caveolin-mediated uptake is low. As inhibition of the clathrin-mediated endocytosis and macropinocytosis decreased transfection efficacy about 17% and 24%, they assumed that several pathways were participating in the uptake of the polyplexes and led to transfection. Cholesterol depletion experiments demonstrated the main uptake mechanism for the commercially available polyamidoamine (PAMAM) dendrimer superfect (Qiagen) in an endothelial cell line. Without cholesterol the binding of the dendriplexes as well as the transfection efficiency was strongly decreased [15]. After reinstatement of the membrane cholesterol the efficacy was rebuilt. In addition, confocal microscopy studies showed colocalization with the membrane raft ganglioside GM1. There is strong evidence that the uptake is provided mainly through caveolae-dependent processes. Another widely used polymer for gene delivery is the polysaccharide chitosan, which is built up from repeating units of glucosamine. In 2009 Nam et al. [16] suggested after endocytotic inhibitor experiments and colocalization studies in HeLa cells that at least three uptake mechanisms (macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis) are involved in the internalization of hydrophobically modified glycol chitosan polyplexes (HGC). The uptake of alginate chitosan polyplexes seems to be cell line dependent [17]. HEK 293T cells internalize the particles through CDE, whereas the uptake in CHO cells is predominantly provided through CME. Interestingly, the latter led to no gene expression, because the cargo was entrapped in caveosomes and could not escape. In contrast, gene expression could be observed in HEK 293T cells, consistent with release from acidified endosome provided through the socalled proton sponge effect (see Section 7.3.1). Another important parameter influencing the uptake mode is the size of the nanoparticles. Medium sized particles (50–200 nm) are internalized through a clathrin-dependent and/or a caveolae-mediated mechanism, whereas nanoparticles larger than 200 nm are taken up mainly by macropinocytosis. For very small particles a caveolaemediated pathway could be observed [10–13, 18]. 7.2.1.2 Targeted Uptake In contrast to untargeted polyplexes, a targeting ligand causes binding of the polyplex to a special membrane region, where the appropriate receptor is located. This should lead to a selective uptake pathway as well as a special further processing and intracellular trafficking. Indeed, in general, ligands, except for a few, result in clathrin-dependet endocytosis [19, 20]. So far many different endogenous and exogenous ligands for targeting of polyplexes have been investigated (Table 7.1). Lactose targeted PLL is inter-

c07.indd 126

7/30/2010 2:18:53 PM

CELLULAR BINDING AND UPTAKE

127

TABLE 7.1 Targeting Ligands Used for Polyplexes Ligand Carbohydrates Lactose Galactose Mannose Peptides Tet1 HIV-TAT Penetratin Antibodies anti-EGFR anti-CD3 anti-Her2 Endogenous ligands EGF Transferrin TGF-α

Reference 21 23 24 25 26 27 28 29 30 22 6 31

nalized through clathrin-dependent processes, whereas glycosylated BPEI is taken up mainly through caveolae-mediated mechanisms [21]. To avoid unspecific binding of polyplexes (caused by electrostatic interaction of the cationic polyplexes with the overall negatively charged cell surface) and trigger ligand– receptor mediated binding, the polyplexes have to be shielded. The commonly used reagent for shielding is polyethylene glycol. While the binding of the shielded polyplexes to the cell surface without a targeting ligand is highly reduced, targeting is rebuilding effective binding in the presence of shielding. A widely used targeting ligand is transferrin. It was shown that transferrin targeting increases cellular binding and the uptake rate as well as transfection efficiency in K562 cells [6]. The 7-kDa protein EGF is another well-studied endogenous targeting ligand, which enhances cellular binding and therefore transgene expression in human adenocarcinoma (KB) cells [6]. EGF targeting leads to faster binding and uptake compared to untargeted polyplexes in HUH-7 cells. As detected by novel single-particle tracking microscopy, 5 minutes after incubation 50% of targeted polyplexes were internalized, whereas only 5% of untargeted polyplexes were taken up at this time point [22]. 7.2.2 Phases of Polyplex Internalization and Further Intracellular Routing Single-particle experiments revealed an internalization process that can be divided into three different phases. During phase 1, which includes the extracellular binding and internalization process, the particles moved slowly with a drift mediated by the actin cytoskeleton. In this phase the endosomes are

c07.indd 127

7/30/2010 2:18:53 PM

128

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

(A) targeted Polyplex

Membrane protein

(B)

3 μm Phases 1+2

Phases 3 Actin

Endosome

Microtubule Phase 1

Phase 2

Phase 3

instantaneous velocity [μm/s]

(C) Phases 3 3 2,5 2 1,5 Phases 1 Phases 2 1 0,5 0 0 50 100 150 200 250 time [s]

Figure 7.2. Cell binding, cellular entry, and further trafficking of an EGF-PEG-PEI polyplex. (A) Scheme of the three internalization phases. Phase 1 includes cellular binding and uptake, mediated by the actin cytoskeleton. Phase 2 shows free diffusion of the internalized vesicle. Phase 3 is characterized by fast directed motion along microtubules. (B) Trajectory plot of the particle with a frame rate of 300 ms. (C) This panel shows velocity of the vesicle during the different phases. The corresponding video can be found at http://www.nature.com/mt/journal/v15/n7/suppinfo/6300176s1 .html. (Adapted from de Bruin et al. [22].)

formed and tensioned by actin over membrane proteins. In phase 2, the vesicles are waiting to combine with the microtubule transport system. Normal diffusion in the cytoplasm with random movement is characteristic for this phase. During phase 3, a dramatic increase in particle velocity and active, directed transport of the vesicles along microtubules could be observed. Except for a shortening of phase 1 the same phases for EGF targeted as well as for untargeted polyplexes could be monitored [22]. Figure 7.2 shows the trajectory of the vesicles during these three different phases. These observations are in accordance with previous studies done by Kulkarni et al. [32] in 2005. They showed two different phases of intracellular polyplex movement by spatiotemporal image correlation spectroscopy: a long term phase (about 60 seconds) with low velocity and random motions of the particles, and a short-term phase (about 10 seconds) characterized by a high velocity (0,05 μm/s) and directed motion of the vesicles. They also suggest that this directed movement is correlated with transport of the endosomes along microtubules. After the polyplexes have overcome the hurdle of the cytoplasmic membrane and the vesicles have reached the cytoplasm, further processing can involve

c07.indd 128

7/30/2010 2:18:53 PM

ENDOSOMAL ESCAPE

129

recycling back to the cell surface, fusion to degenerative vesicle forms like lysosomes, as well as transport to organelles like endoplasmic reticulum or Golgi apparatus. As shown in Figure 7.1, this is primarily dependent on the internalization pathway.

7.3

ENDOSOMAL ESCAPE

In Figure 7.1 it becomes apparent that four of the five internalization pathways for polyplexes lead at least partly to fusion of the internalized vesicles with lysosomes. In this cell compartiment with a typically acidic pH between 4 and 5, the cargo of the vesicles is degraded. It is generally believed that cellular uptake of polyplexes has a lesser impact on transfection efficacy than further processing [33]. Dinh et al. [34] predicted when using PEI polyplexes that only 1% of the internalized DNA molecules reach the nucleus, the final aim for DNA in gene delivery. Endosomal escape was identified as one of the major bottlenecks in gene delivery by synthetic vectors [35, 36]. In contrast to lipoplexes, classical cationic polyplexes do not exhibit hydrophobic regions and therefore cannot release their cargo from the endosomes by membrane fusion processes. A lot of different strategies that help the polyplexes to get over this hurdle have been investigated so far [37]. The most common ones are presented in the next sections. 7.3.1

Protonable Amines

Polyplexes built up from cationic polymers like BPEI, LPEI, and histidinylated PLL possess protonable primary, secondary, or tertiary amines. Such protonable amines possess a pKa between 5 and 7 and therefore can buffer acidification of the late endosomes by taking up protons. This buffered acidification of late endosomes goes along with concurrent influx of chloride to maintain charge neutrality and results in an increased ionic strength inside the endosome. To balance this, water is accumulating passively inside the endosome. Therefore the pressure inside the endosome increases more and more until the membrane bursts and the content of the endosome is released into the cytosol [38]. Today this mechanism is known as the “proton sponge effect.” Akinc et al. [39] showed that the transfection efficacy of N-quarternized PEI (lacks protonable amines) is reduced by approximately 100-fold. The same observation was made by using the vacuolar proton pump inhibitor bafilomycin A1. Chloroquine is an osmotically active lysosomal buffer reagent, which increases the transfection efficiency of PLL polyplexes between twofold and more than 100-fold [40, 41]. Other neutralizing but not osmotically active inhibitors did not enhance gene transfer. This reveals that blocked lysosomal degradation of DNA (due to prevention of acidification) and endosomal escape contributed to the beneficial effect. No effect of chloroquine could be observed with PEI polyplexes that can buffer the endosomal pH by

c07.indd 129

7/30/2010 2:18:53 PM

130

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

themselves. More recent findings suggest that the osmotic effect by the proton sponge action of PEI is not sufficient to explain endosomal escape; the protonated cationic PEI residues appear to be directly involved in the membrane disruption process. For example, irreversible shielding of PEI polyplexes by PEGylation strongly interferes with this process and pH-sensitive PEG attachment recovers the activity [42]. 7.3.2

Photoinduced Endosomal Release

Photochemical disruption of the endosomal membrane [43, 44] is another strategy to overcome lysosomal degradation. In this case the polyplexes are internalized by the cells together with amphiphilic photosensitive reagents (e.g., disulfonated meso-tetraphenylporphine [TPPS2a]) that were present in the transfection medium (Figure 7.3). After internalization the membranebound photosensitizer (PS) is activated by illuminating the cells. The PS mediates energy transfer to molecular oxygen, generating radical singlet oxygen inside the endosomes. Subsequently, these radicals lead to oxidative damage of the endosomal membrane, resulting in rupture of the vesicular membrane and release of the polyplexes into the cytosol. Using this so-called photochemical internalization (PCI) technique, the transfection efficiency of EGF targeted PEI polyplexes could be increased in HUH-7, HepG2 and A431 cells between twofold and 600-fold [45]. For in vivo application the PS could be

Photosensitizer (PS)

Polyplex

early Endosome

light

h*v

1

PS*

PS 1O 2 3PS*

O2

Figure 7.3. Photochemical internalization and operating mode of a photosensitizer for promoting endosomal escape. After light activation, the photosensitizer mediates energy transfer to molecular oxygen, resulting in radical singlet oxygen inside the endosome. The built radicals attack the endosome membrane and lead to its rupture. Thus liberation of the polyplex into the cytosol is accomplished.

c07.indd 130

7/30/2010 2:18:53 PM

ENDOSOMAL ESCAPE

131

internalized inside the polyplexes [44]. In the case of PLL polyplexes, which lack proton sponge mediated liberation, de Bruin et al. [46] showed a fast release of the cargo into the cytosol (within about 50 seconds) after activation of the photosensitizer. 7.3.3 Membrane Disruptive Peptides The use of membrane destabilizing peptides was the first technique to promote effective release of polyplexes entrapped in endosomal vesicles. Up to date a lot of natural and synthetic peptides have been used (Table 7.2) to trigger this endosomal release. These peptides can be divided into two classes. Group one contains peptides with an inherent membrane lytic activity (e.g., melittin, KALA), whereas the other peptides only show lytic activity at acidic pH (e.g., GALA, INF). The selective lytic activity of the latter class is explained by pH-dependent conformational changes in peptide structure. Only at acidic pH can the peptides form an alpha-helix, which is required for lytic activity. The alpha-helical conformation is necessary for amphiphilic interaction with the membrane phospholipid bilayer and building pores in it that lead to rupture [47]. Wagner et al. [48] showed that the transfection efficiency of PLL-transferrin polyplexes could be enhanced about 100 times when coupling influenza virus hemagglutinin peptide HA2 to the polymers. Furthermore, Rittner et al. [49] designed new basic amphiphilic peptides (ppTG1, ppTG20) that can be used as singlecomponent vectors by combining DNA complexing ability and endosomal release function in one molecule. The bee venom peptide melittin also enabled release of polyplexes into the cytoplasm [50]. As melittin has an inherent membrane disruptive property, not only at acidic pH inside the endosome but also at neutral pH inside the cytosol, it increases cellular toxicity of the polyplexes. To overcome this problem investigators either introduced acidic residues into melittin [51] or composed a chemically modified melittin by coupling dimethylmaleic anhydride (DMMAn) to the lysine residues of melittin [52, 53]. When the polyplexes are acidified inside the endosome, this pH labile

TABLE 7.2

Membrane Disruptive Peptides

Peptide Natural peptides Melittin INF-HA2 HRV2-VP1 PFO Synthetic peptides NMA-3 KALA GALA ppTG1 ppTG20

c07.indd 131

Reference 50 48 56 57 51 58 47 49 49

7/30/2010 2:18:54 PM

132

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

binding of the protection groups is cleaved and the lytic activity is regained. This resulted in less toxicity compared to PLL-melittin polyplexes and even to single PLL polyplexes. Gene expression was enhanced about 1800-fold compared to simple PLL polyplexes [53]. The concept has also been adapted for efficient cytosolic delivery of siRNA [54, 55].

7.4

DNA POLYPLEX DISSOCIATION

For various reasons the release of the DNA from the polyplex is necessary for successful transcription. It remains controversial whether this has to occur before or after nuclear entry. By using quantum dot/FRET assays, the kinetics of unpackaging has been monitored and occurs in several compartments [59]. On the one hand, flexible small size for nuclear import and accessibility of transgene DNA in the nucleus by the transcription machinery (transcription factors, polymerases) is important. For example, a standard 50–100-nm polyplex would be too large to enter the nucleus through a nuclear pore, either passively (10-nm pore limit) or actively (maximum pore diameter of around 30 nm) [60]. On the other hand, the dissociation of the vector from the DNA in the cytosol is a critical step in efficient gene delivery, because after release free cytosolic DNA has no protection against degradation enzymes like nucleases. Therefore the dissociation step should occur as late as possible, optimally just before penetration of the DNA into the nucleus; this happens, for example, in the case of adenoviral infection. The release of the electrostatic bound nucleic acid from positively charged polymers is provided by anionic cellular components like overall positively charged proteins, lipids, and last but not least RNA molecules [61–63]. Microinjection experiments revealed that the dissociation of PEI polyplexes in contrast to lipoplexes can also happen in the nucleus, presumably because of the exchange of the transgene with cellular DNA [64]. Schaffer et al. [65] demonstrated an enhancement of transfection efficiency when using low molecular weight PLL for transfection in comparison with the high molecular weight counterpart. They predicted that this enhancement arises from easier dissociation of the polyplexes in the cytosol. This has also been postulated for the more effective LPEI in comparison to BPEI [66]. Most recently, intranuclear FRET analysis has been used for analysis of pDNA decondensation from nonviral carriers [67]. pDNA polyplex decondensation after nuclear entry was the major determining factor for transgene expression. Besides better DNA release, lower cytotoxicity is another great advantage of smaller or degradable polymers. Again, different strategies have been developed to overcome these obstacles. 7.4.1

pH-Sensitive Polymers

Polyplexes that are cleaved into smaller metabolites after acidification of the endosome were developed by Knorr et al. [68,69]. These polymers are based

c07.indd 132

7/30/2010 2:18:54 PM

DNA POLYPLEX DISSOCIATION

133

on small 800-Da PEI subunits. These subunits were chemically covalently conjugated to larger molecules over pH-sensitive acetone ketal crosslinkers such as 2,2-bis(N-maleimidoethyloxy)propane (MK) or 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). These polyplexes showed better transfection efficiency than analogs built up with noncleavable linkers at high vector to plasmid ratios. Moreover, the cytotoxicity of the pH-sensitive polyplexes was greatly reduced as shown with methylthiazoletetrazolium (MTT)/thiazolyl blue assay in Neuro 2a and B16F10 cells. 7.4.2

Biodegradable Esters

Linkage of small subunits to larger polymers over biodegradeable ester bonds is another promising strategy [70–72]. Ester bonds are hydrolyzed by water perhaps and even intracellular esterases can cleave the bonds. In contrast to the above-mentioned pH-sensitive acetal bonds, ester bonds are more stabile with a longer half-life. Many parameters can be varied (e.g., changes in structure and molecular weight of the subunits) to obtain the desired degradation kinetic. Furthermore, amine density of the subunits as well as the grade of crosslinking can influence the release of DNA [73–75]. Russ et al. [74] described a polymer based on generation two PPI dendrimers as the core, grafted with PEI 800 Da and crosslinked with hexane-1,6-diol diacrylate, that exhibits a transfection efficiency comparable to or even better than PEI, but with lower cytotoxicity. 7.4.3

Intracellular Reducible Polyplexes

These polyplexes capitalize for dissociation on the different redox potentials between the oxidizing extracellular environment on the one hand and the reducing cytosolic environment (mediated by glutathione) on the other hand. The polyplexes are stable until they are released into the cytosol. Once they reach the cytosol the disulfide bonds between the subunits are cleaved and thus the DNA release is facilitated and toxicity is reduced. For cleavable PEI (CLPEI50%) polymers built up from smaller 1800-Da PEI subunits and crosslinked with 3,3′-dithiopropionimidate dihydrochloride (DTBP), cleavage in the presence of 3-mM glutathione—comparable to intracellular concentrations—was evident [76]. Furthermore, these conjugates exhibited lower toxicity than PEI 25 kDa. These data are in accordance with that of another group that reveals high transfection efficacy of an analogous biodegradeable polyethylene imine sulfide polyplex and also low cytotoxicity [77]. Moreover, Lee et al. visualized the intracellular degradation of the polyplexes in HeLa cells using fluorescence quenching of a conjugated BODIPY-dye. All these studies to enhance the cytosolic release of the DNA into the cytoplasm are quite promising, but as already mentioned, once free in the cytosol, the DNA is no longer protected against degredation enzymes like proteases. At this stage, perhaps an even more important bottleneck for synthetic gene

c07.indd 133

7/30/2010 2:18:54 PM

134

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

delivery by cationic polyplexes is reached—the transport of the DNA into the nucleus.

7.5

CYTOSOLIC TRAFFICKING TOWARD THE NUCLEUS

The short half-life of free cytosolic DNA makes clear why a fast transport of the DNA into the nucleus is desired. In HeLa and COS-1 cells pDNA halflives are between 50 and 90 minutes, as monitored by fluorescent in situ hybridization (FISH) experiments [78]. Thus it is not surprising that intranuclear microinjection of plasmids encoding for β-galactosidase into COS-7 cells results in much higher gene expression than when the same amount of plasmid is injected into the cytosol [79]. Highly impeded velocity of the DNA in the cytoplasm is one obstacle for fast nuclear import [80]. As microinjected oligonucleotides are distributed homogeneously in the cytoplasm, size is the main factor of impeded velocity rather than interaction with cytosolic proteins [81]. The cytoskeleton built up by microtubules, intermediate filaments, and microfilaments forms a dense network with narrow meshes, which prevents free diffusion of plasmids larger than 250 base pairs [82]. For this reason strategies to actively transport the DNA toward the nucleus (either within vesicles or after intracytosolic release) [83] are highly desired.

7.6

NUCLEAR IMPORT

The next hurdle that has to be overcome by the plasmids on their way into the nucleus is the nuclear envelope. This double membrane is interspersed by nuclear pore complexes (NPCs) with a diameter of about 10 nm. Molecules, smaller than 40 kDa, can diffuse passively through these pores. Larger molecules have to be transported by an active energy-dependent process, which goes along with conformational changes of the NPCs and widening of the pores to a diameter of about 30 nm [84]. Now the pores are able to transport compact macromolecules as large as 50 MDa [85]. Plasmids used for gene delivery have a molecular weight between 2 and 10 MDa and a flexible size, with an extended length of around 1 μm but a small helix diameter of 2 nm. To initiate this energy-dependent uptake mechanism, a targeting signal is necessary [86]. For all these reasons it becomes clear why only 1 out of 1000 cytosolic cDNA molecules is estimated to reach the karyoplasm [78]. A number of efforts were made to facilitate this critical step in gene delivery by polyplexes. 7.6.1

Cell Cycle Dependence

Microinjection of naked DNA in HeLa cells revealed that transfection efficiency was dependent on the cell cycle stage [87]. After injection of 10 ng naked plasmid DNA, 28% of the nondividing cells showed transgene expres-

c07.indd 134

7/30/2010 2:18:54 PM

NUCLEAR IMPORT

135

sion, whereas 50% of the cells that divided after injection expressed the gene encoded on the plasmid. Although even growth arrested cells could be transfected with PEI, the main entry of the transgene into the nucleus happens passively, when the nuclear envelope is temporarily degraded during mitotic cell division [88]. Brunner et al. [89] demonstrated that transfection efficiency for transferrin-polylysine and transferrin-polyethylenimine is strongly dependent on the cell cycle stage at the time of transfection. Compared to cells transfected during G1 phase, cells transfected during S or G2 phase exhibited 30–500-fold higher transgene expression in K562 cells. Further studies showed that this cell cycle dependence is not valid for all cationic polymers. In contrast to polyplexes formed with BPEI, polyplexes built up with LPEI do not display such strong cell cycle dependence in transfection efficiency [90]. Therefore it can be suggested that LPEI polyplexes exhibit improved nuclear import characteristics and/or improved nuclear unpackaging compared to BPEI and PLL formulations. 7.6.2

Nuclear Localization Signal (NLS)

As already described, macromolecules over 40 kDa can pass the NPCs only in an energy-dependent process. This mechanism is activated by nuclear localization signals, built up from small, mostly cationic, peptide sequences. Large numbers of different NLS sequences are known (e.g., SV40 large T-antigen, M9 human nuclear ribonucleoprotein A1, HIV type-1 viral protein R [Vpr], human T cell leukemia virus type 1 [HTLV]). Macromolecules containing such sequences are bound to import proteins (importins) that mediate the translocation of the complexes into the karyoplasm. In contrast to the passive cellcycle-dependent nuclear import of DNA, this is an active transport process. Peptides containing such NLS sequences can be conjugated either to the polymer vector [91, 92] or, alternatively, to the plasmid. Covalent conjugation of an NLS sequence derived from simian virus 40 to the DNA over a cyclopropapyrroloindole crosslinker induced nuclear accumulation of the plasmid DNA in digitonin permeabilized HeLa cells. It seems that the amount of NLSs per kilo base pair is an important factor [93]. Sebestyen et al. [93] used at least 100 NLS sequences per kilo base pair to achieve high nuclear accumulation of the plasmids. Because of that high modification grade, transgene expression was completely abolished [93]. A second binding possibility is mediated just by electrostatic interactions between cationic NLS sequences and the negatively charged nucleic acid [94]. Although it is a promising idea to enhance nuclear import with the usage of NLSs containing peptides, it now becomes clear that its application is limited by many different factors [95, 96]. 7.6.3

DNA Sequences

In contrast to the above described technique to transport DNA via coupling to NLS peptides (trans-acting), a further mechanism of nuclear DNA import is DNA sequence dependent (cis-acting). Some sequences of plasmids are

c07.indd 135

7/30/2010 2:18:54 PM

136

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

supposed to have a certain affinity to proteins like transcription factors. Such proteins often carry NLS sequences, which lead to an energy-dependent nuclear import of the associated DNA [97]. An example for such a DNA sequence is the 10 base pair long κB binding site that can be found in the promotor and enhancer regions of many plasmids. NF-κB is an NLS containing transcription factor with high affinity to this κB sequence. By microinjection of plasmids into the cytoplasm of HeLa cells, Mesika et al. [98] demonstrated that these κB sites not only facilitated the nuclear penetration of the plasmids but enhanced their migration through the cytoplasm toward the nucleus in a dynein-dependent manner. Futhermore, sequences from the SV40 viral genome containing the SV40 origin of replication, early and late promoters, supported nuclear import [99]. This sequence-specific import could be inhibited by energy depletion as well as by the addition of NPC inhibitory agents like wheat germ agglutinin. Moreover, Dean [99] showed that before nuclear uptake the DNA is transported and accumulated in the nuclear periphery. The uptaken DNA was colocalized inside the karyoplasms with SC-35 splicing complex antigen and thus it is suggested that the DNA uptake is linked to transcription. Indeed, the inhibition of transcription blocked nuclear uptake. Despite these interesting results, nuclear import of DNA into the nucleus is one of the least understood mechanisms of synthetic gene delivery and remains a major bottleneck.

7.7

CONCLUSION

Although due to new techniques, like single-particle tracking, great advances in elucidating the different steps of internalization and intracellular processing of polyplexes were made, some mechanisms still remain quite unclear. These not well understood steps, for example, the penetration of DNA into the nucleus, seem to be major limiting factors in efficient gene delivery. No polyplex has been designed so far to address all known hurdles at the same time, while combining high efficiency (independent from cell line) with low cytotoxicity. Nevertheless, substantial progress has been made for reaching this final aim in the last decade.

REFERENCES 1. Smull, C. E. and Ludwig, E. H. Enhancement of the plaque forming capacity of poliovirus ribonucleic acid with basic proteins. J. Bacteriol. 84: 1035–1040 (1962). 2. Felgner, P. L., et al. Nomenclature for synthetic gene delivery systems. Hum. Gene Ther. 8: 511–512 (1997). 3. Mislick, K. A. and Baldeschwieler, J. D. Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93: 12349–12354 (1996).

c07.indd 136

7/30/2010 2:18:54 PM

REFERENCES

137

4. Kopatz, I., Remy, J. S., and Behr, J.P. A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J. Gene Med. 6: 769–776 (2004). 5. Labat, M., et al. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 3: 1010–1017 (1996). 6. Ogris, M., et al. DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression. AAPS Pharm. Sci. 3: E21 (2001). 7. Remy-Kristensen, A., et al. Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim. Biophys. Acta 1514: 21–32 (2001). 8. Conner, S. D. and Schmid, S.L. Regulated portals of entry into the cell. Nature 422: 37–44 (2003). 9. Ferrari, A., et al. Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol. Ther. 8: 284–294 (2003). 10. Lai, S. K., et al. Characterization of the intracellular dynamics of a non-degradative pathway accessed by polymer nanoparticles. J. Control. Release 125: 107–111 (2008). 11. Rejman, J., et al. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem. J. 377: 159–169 (2004). 12. Rejman, J., Bragonzi, A., and Conese, M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol. Ther. 12: 468–474 (2005). 13. von Gersdorff, K., et al. The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol. Ther. 14: 745–753 (2006). 14. Lühmann, T., et al. Cellular uptake and intracellular pathways of PLL-g-PEGDNA nanoparticles. Bioconjug. Chem. 19: 1907–1916 (2008). 15. Manunta, M., et al. Gene delivery by dendrimers operates via a cholesterol dependent pathway. Nucleic Acids Res. 32: 2730–2739 (2004). 16. Nam, H. Y., et al. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J. Control. Release 135: 259–267 (2009). 17. Douglas, K. L., Piccirillo, C. A., and Tabrizian, M. Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors. Eur. J. Pharm. Biopharm. 68: 676–687 (2008). 18. Grosse, S., et al. Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations. J. Gene Med. 7: 1275–1286 (2005). 19. Parton, R. G., Joggerst, B., and Simons, K. Regulated internalization of caveolae. J. Cell Biol. 127: 1199–1215 (1994). 20. Subtil, A., Hemar, A., and Dautry-Varsat, A. Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited. J. Cell Sci. 107(Pt 12): 3461–3468 (1994). 21. Grosse, S., et al. Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes. J. Gene Med. 6: 345–356 (2004).

c07.indd 137

7/30/2010 2:18:54 PM

138

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

22. de Bruin, K., et al. Cellular dynamics of EGF receptor-targeted synthetic viruses. Mol. Ther. 15: 1297–1305 (2007). 23. Plank, C., et al. Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjug. Chem. 3: 533–539 (1992). 24. Engering, A.J., et al. Mannose receptor mediated antigen uptake and presentation in human dendritic cells. Adv. Exp. Med. Biol. 417: 183–187 (1997). 25. Park, I. K., et al. Neuron-specific delivery of nucleic acids mediated by Tet1modified poly(ethylenimine). J. Gene Med. 9: 691-702 (2007). 26. Rudolph, C., et al. Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells. J. Biol. Chem. 278: 11411– 11418 (2003). 27. Christiaens, B., et al. Enhancement of polymethacrylate-mediated gene delivery by Penetratin. Eur. J. Pharm. Sci. 24: 525–537 (2005). 28. Suzuki, M., Takayanagi, A., and Shimizu, N. Targeted gene delivery using humanized single-chain antibody with negatively charged oligopeptide tail. Cancer Sci. 95: 424–429 (2004). 29. Kircheis, R., et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4: 409–418 (1997). 30. Germershaus, O., et al. Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors. Bioconjug. Chem. 17: 1190–1199 (2006). 31. Fominaya, J., Uherek, C., and Wels, W. A chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the EGF receptor. Gene Ther. 5: 521-530 (1998). 32. Kulkarni, R. P., et al. Quantitating intracellular transport of polyplexes by spatiotemporal image correlation spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 102: 7523– 7528 (2005). 33. Breunig, M., et al. Mechanistic insights into linear polyethylenimine-mediated gene transfer. Biochim. Biophys. Acta 1770: 196–205 (2007). 34. Dinh, A. T., et al. Understanding intracellular transport processes pertinent to synthetic gene delivery via stochastic simulations and sensitivity analyses. Biophys. J. 92: 831–846 (2007). 35. Curiel, D. T., et al. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci. U.S.A. 88: 8850–8854 (1991). 36. Wagner, E., et al. Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. U.S.A. 89: 6099–6103 (1992). 37. Wagner, E. Application of membrane-active peptides for nonviral gene delivery. Adv. Drug Deliv. Rev. 38: 279–289 (1999). 38. Behr, J. P. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 51: 34–36 (1997). 39. Akinc, A., et al. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J. Gene Med. 7: 657–663 (2005). 40. Cotten, M., et al. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected

c07.indd 138

7/30/2010 2:18:54 PM

REFERENCES

41. 42.

43. 44.

45. 46. 47.

48.

49. 50.

51.

52.

53.

54.

55. 56. 57.

c07.indd 139

139

DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87: 4033–4037 (1990). Cotten, M., Wagner, E., and Birnstiel, M. L. Receptor-mediated transport of DNA into eukaryotic cells. Methods Enzymol. 217: 618–644 (1993). Walker, G. F., et al. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol. Ther. 11: 418–425 (2005). Hogset, A., et al. Light directed gene transfer by photochemical internalisation. Curr. Gene Ther. 3: 89–112 (2003). Nishiyama, N., et al. Photochemical enhancement of transgene expression by polymeric micelles incorporating plasmid DNA and dendrimer-based photosensitizer. J. Drug Target. 14: 413–424 (2006). Kloeckner, J., et al. Photochemically enhanced gene delivery of EGF receptortargeted DNA polyplexes. J. Drug Target. 12: 205–213 (2004). de Bruin, K.G., et al. Dynamics of photoinduced endosomal release of polyplexes. J. Control. Release 130: 175–182 (2008). Li, W., Nicol, F., and Szoka, F. C. Jr. GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv. Drug Deliv. Rev. 56: 967–985 (2004). Wagner, E., et al. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferring–polylysine–DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. U.S.A. 89: 79347938 (1992). Rittner, K., et al. New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo. Mol. Ther. 5: 104–114 (2002). Ogris, M., et al. Melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors. J. Biol. Chem. 276: 47550–47555 (2001). Boeckle, S., et al. Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes. J. Control. Release 112: 240–248 (2006). Rozema, D. B., et al. Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules. Bioconjug. Chem. 14: 51–57 (2003). Meyer, M., et al. A dimethylmaleic acid–melittin–polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential. J. Gene Med. 9: 797–805 (2007). Meyer, M., et al. Breathing life into polycations: functionalization with pH-responsive endosomolytic peptides and polyethylene glycol enables siRNA delivery. J. Am. Chem. Soc. 130: 3272–3273 (2008). Meyer, M., et al. Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA–polymer conjugate. Mol. Pharm. 6: 752–762 (2009). Zauner, W., et al. Rhinovirus-mediated endosomal release of transfection complexes. J. Virol. 69: 1085–1092 (1995). Gottschalk, S., et al. Efficient gene delivery and expression in mammalian cells using DNA coupled with perfringolysin O. Gene Ther. 2: 498–503 (1995).

7/30/2010 2:18:54 PM

140

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

58. Wyman, T. B., et al. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 36: 3008–3017 (1997). 59. Chen, H. H., et al. Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET. Mol. Ther. 16: 324– 332 (2008). 60. Melchior, F. and Gerace, L. Mechanisms of nuclear protein import. Curr. Opin. Cell Biol. 7: 310–318 (1995). 61. Huth, S., et al. Interaction of polyamine gene vectors with RNA leads to the dissociation of plasmid DNA-carrier complexes. J. Gene Med. 8: 1416–1424 (2006). 62. Iida, T., et al. Overall interaction of cytosolic proteins with the PEI/DNA complex. J. Control. Release 118: 364–369 (2007). 63. Wolff, J.A. and Rozema, D.B. Breaking the bonds: non-viral vectors become chemically dynamic. Mol. Ther. 16: 8–15 (2008). 64. Zabner, J., et al. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol Chem. 270: 18997–19007 (1995). 65. Schaffer, D. V., et al. Vector unpacking as a potential barrier for receptormediated polyplex gene delivery. Biotechnol. Bioeng. 67: 598–606 (2000). 66. Itaka, K., et al. In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J. Gene Med. 6: 76–84 (2004). 67. Matsumoto, Y., et al. Intranuclear fluorescence resonance energy transfer analysis of plasmid DNA decondensation from nonviral gene carriers. J. Gene Med. 11: 615–623 (2009). 68. Knorr, V., et al. Acetal linked oligoethylenimines for use as pH-sensitive gene carriers. Bioconjug. Chem. 19: 1625–1634 (2008). 69. Knorr, V., Ogris, M., and Wagner, E. An acid sensitive ketal-based polyethylene glycol-oligoethylenimine copolymer mediates improved transfection efficiency at reduced toxicity. Pharm. Res. 25: 2937–2945 (2008). 70. Zhong, Z., et al. A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine)s. J. Control. Release 109: 317–329 (2005). 71. Forrest, M. L., Koerber, J. T., and Pack, D. W. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug. Chem. 14: 934–940 (2003). 72. Akinc, A., et al. Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery. Bioconjug. Chem. 14: 979–988 (2003). 73. Wong, S. Y., Pelet, J. M., and Putnam, D. Polymer systems for gene delivery—past, present, future. Prog. Polym. Sci. 32: 799–837 (2007). 74. Russ, V., et al. Novel degradable oligoethylenimine acrylate ester-based pseudodendrimers for in vitro and in vivo gene transfer. Gene Ther. 15: 18–29 (2008). 75. Russ, V., et al. Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. J. Control. Release 132: 131–140 (2008). 76. Wang, Y., Chen, P., and Shen, J. The development and characterization of a glutathione-sensitive cross-linked polyethylenimine gene vector. Biomaterials 27: 5292–5298 (2006).

c07.indd 140

7/30/2010 2:18:54 PM

REFERENCES

141

77. Lee, Y., et al. Visualization of the degradation of a disulfide polymer, linear poly(ethylenimine sulfide), for gene delivery. Bioconjug. Chem. 18: 13–18 (2007). 78. Lechardeur, D., et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6: 482–497 (1999). 79. Pollard, H., et al. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273: 7507–7511 (1998). 80. Dauty, E. and Verkman, A. S. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. J. Biol. Chem. 280: 7823–7828 (2005). 81. Leonetti, J. P., et al. Intracellular distribution of microinjected antisense oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 88: 2702–2706 (1991). 82. Lukacs, G. L., et al. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275: 1625–1629 (2000). 83. Ng, C. P., et al. Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility. Biomaterials 30, 951–958 (2009). 84. Dworetzky, S. I., Lanford, R. E., and Feldherr, C. M. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J. Cell Biol. 107: 1279–1287 (1988). 85. Lechardeur, D., Verkman, A. S., and Lukacs, G. L. Intracellular routing of plasmid DNA during non-viral gene transfer. Adv. Drug Deliv. Rev. 57: 755–767 (2005). 86. Dingwall, C., Sharnick, S. V., and Laskey, R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30: 449-458 (1982). 87. Ludtke, J. J., Sebestyen, M. G., and Wolff, J. A. The effect of cell division on the cellular dynamics of microinjected DNA and dextran. Mol. Ther. 5: 579–588 (2002). 88. Grosse, S., et al. Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives? J. Gene Med. 8: 845–851 (2006). 89. Brunner, S., et al. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7: 401–407 (2000). 90. Brunner, S., et al. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5: 80–86 (2002). 91. Moffatt, S., Wiehle, S., and Cristiano, R. J. A multifunctional PEI-based cationic polyplex for enhanced systemic p53-mediated gene therapy. Gene Ther. 13: 1512– 1523 (2006). 92. Talsma, S. S., et al. Development and in vitro validation of a targeted delivery vehicle for DNA vaccines. J. Control. Release 112: 271–279 (2006). 93. Sebestyen, M. G., et al. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat. Biotechnol. 16: 80–85 (1998). 94. Subramanian, A., Ranganathan, P., and Diamond, S. L. Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat. Biotechnol. 17: 873– 877 (1999). 95. van der Aa, M. A., et al. An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems. J. Gene Med. 7: 208–217 (2005).

c07.indd 141

7/30/2010 2:18:54 PM

142

INTRACELLULAR FATE OF PLASMID DNA POLYPLEXES

96. van der Aa, M. A., et al. The nuclear pore complex: the gateway to successful nonviral gene delivery. Pharm. Res. 23: 447–459 (2006). 97. Wilson, G. L., et al. Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J. Biol. Chem. 274: 22025–22032 (1999). 98. Mesika, A., et al. Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein. Hum. Gene Ther. 16: 200–208 (2005). 99. Dean, D. A. Import of plasmid DNA into the nucleus is sequence specific. Exp. Cell Res. 230: 293–302 (1997).

c07.indd 142

7/30/2010 2:18:54 PM

CHAPTER 8

Intracellular Trafficking of Membrane Receptor-Mediated Uptake of Carbon Nanotubes BIN KANG and YAODONG DAI Nanjing University of Aeronautics and Astronautics, Nanjing, Peoples Republic of China

8.1

INTRODUCTION

Over the last decade, newly synthesized nanomaterials (NMs), exhibiting distinctive optical, magnetic, and conducting properties, have been found to exhibit fascinating potential applications in biomedical fields. Their small size, increased surface area, and reactivity allow them to easily penetrate cell membranes, efficiently bind molecular species, and catalyze chemical reactions [1–3]. Due to their enhanced properties, NMs have been proposed for use in the fabrication of various devices such as catalysts, sensors, composite fillers, actuators, transistors, drug and gene deliverers, biosensors, virus inhibitors, and protein immobilizers [4–13]. Among such nanomaterials, carbon nanotubes (CNTs) are among the most promising materials for such applications because CNTs have better biocompatibility and easy cell internalization [14– 18]. Also, CNTs can easily be functionalized and linked to fluorescent beacons, nucleic acids, or other small molecules, which make CNTs an ideal candidate for a nanoparticle-based system for drug delivery and bioimaging [19–22]. The cellular uptake of CNTs is a very fundamental issue that needs to be understood before using CNTs in various biological applications. Although a lot of research has been done on the interaction of CNTs and living cells, current published data on cell penetration mechanisms and the intracellular dynamic are still particularly inconsistent and widely disputed. Some studies suggest that CNTs can pass directly through the cell membrane via an energyindependent nonendocytotic mechanism [23, 24]: that is, the CNTs may cause Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

143

c08.indd 143

7/30/2010 2:18:56 PM

144

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

localized damage on the cell membrane through which cargos enter the cells. The one-dimensional structure of CNTs has been shown to enhance this “holepunching” process and improve the efficiency of delivery into mammalian cells [24]. On the other hand, other groups propose that cells take up the CNTs through an energy-requiring endocytosis process [8]. The underlying reasons for the discrepancies might be attributed to two causes: first, the wide variability in sample preparation and “purification” methods, including incomplete characterization of the CNT materials following purification, such as minimal description about their postpurification solution behavior; and second, the use of nonuniform characterization methods and materials with different preparative protocols, viability assessment methods, and cell populations. For CNTs, the different physical characteristics, such as tube diameter, length, surface chemistry, and biological coatings, may result in various interactions in the biological context. Definitive discrimination of relative and synergistic effects with respect to these differences will continue to be impossible without implementation of precise measurements, complete characterization, and the use of well-defined materials. Another unaddressed question is the intracellular distribution of internalized CNTs after penetrating the cytoplasm membrane and further fate. Previous reports focused mainly on the uptake of the CNTs and little work has been done with respect to the behaviors of CNTs after their penetration into cells. For example, how long can the CNTs stay in the cells? Can CNTs be released from the cells through exocytosis? Can CNTs pass through the cellular nuclear envelope? All these questions are still being debated. While most studies have shown that CNTs can penetrate through the cell membrane and be internalized into the cell cytoplasm, few studies show conclusive evidence that CNTs can or cannot get into the cell nucleus, even through some studies have given indirect evidence, such as a G-band Raman signal in the nuclear region or successful use for plasmid DNA delivery. Single-particle tracking (SPT) using fluorophores is a recently developed technique for answering the above questions in cellular systems, although with small fluorophores and QDs, photobleaching is a major limitation to real-time measurements. The photobleaching time constrains the observation window during tracking so that events occurring on the order of several hours must be observed through multiple and distinct incubation periods, with each observation starting at a different time after incubation. However, SPT is considered a promising tool to study the uptake of nanoparticles; in particular, special SPT methods using the NIR fluorescence of CNTs show potential to achieve a better understanding of the cellular uptake dynamics of nanotubes. Therefore the focus of this chapter is to summarize briefly the important results of cellular uptake and tracking of CNTs conducted to date, with emphasis on the uptake pathway and the subcelluar distribution as well as the recent status on uptake dynamics by using single-particle tracking. A description of the current in vitro analysis techniques and materials characterization

c08.indd 144

7/30/2010 2:18:56 PM

RELATION OF NANOTUBE SIZE AND FUNCTIONALIZATION

145

methods is given within the context of the ongoing challenges, and recommendations are made for achieving more reliable measures of CTN–living cell interactions.

8.2

RELATION OF NANOTUBE SIZE AND FUNCTIONALIZATION

There is broad agreement that biological studies should put more emphasis on detailed characterization of test nanomaterials. For cellular uptake of CNTs, one of the main sources of the variation in the published data is the wide variability in CNT dispersion from different preparative protocols and methods. The differences in dispersion ranging from clusters, bundles of multiple nanotubes, or individually dispersed nanotubes will dramatically affect the absolute size and amount of surface area of the nanotube material to which the cells are exposed. Dispersion protocols involving surfactants are attractive, as the incorporation of the nanotubes inside a surfactant shell does not appreciably alter the graphitic structure and desirable physicochemical properties of the single-walled carbon nanotubes (SWCNTs) [25]. Recently, DNA, peptides, and carbohydrates have been used in this surfactant/ wrapping polymer role and have been demonstrated to suspend SWCNTs, with high individual dispersion of the nanotubes [26, 27]. These dispersions, in the case of DNA, are even stable enough to allow for separation of the dispersed material into well-defined subpopulations of the SWCNTs. Another point is the physical properties of the materials used for CNTs, especially the length. Recent reports have highlighted successes in separating polydisperse SWCNT populations into well-defined length and chirality fractions using gel chromatography [25, 28–30], size exclusion chromatography (SEC) [31–35], ion chromatography (IC) [36, 37], or various forms of electrophoresis [28, 38–40]. This sorting affords opportunities to explore the uptake and fate of CNTs with different lengths. Some typical work has been done by Becker et al. [41]. They used well-dispersed, separated-length CNT fractions by SEC, and gave an exhaustive characterization of these fraction populations. Their results indicated a threshold on the length and corresponding toxicity of SWCNTs that are taken up into cells. They found that the longer tubes are excluded from the cell interior while the shorter SWCNTs are able to access the cytosol. Through the length-selective uptake of nanotubes by the cell populations, they determined an approximate uptake threshold of approximately 189 ± 17 nm, indicating that nanotubes shorter than this length are consumed and likely induce more toxicity. Also, the size threshold concept has been suggested for multiwalled carbon nanotubes, which influence cell uptake, retention, and biodistribution in other nanoparticle systems [42–44]. Another fundamental question is whether CNTs with different functionalized groups have the same behavior and are capable of cell binding, uptake, and internalization. Note that the functionalization here only means general chemical groups, in the absence of any coating or conjugation with biological

c08.indd 145

7/30/2010 2:18:56 PM

146

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE NH+3Cl– O

O

HO2C

(FITC) H N O

HN

O

OH OH O

O

1

N

2

OH H N

N

O

O

O O

OH O N

N

OH

O

O

4 O

6

S HN

NH N H

O

O

N

O

O

O N Glu-NH H O

O

OH

N

H HN N S HO2C (FITC)

O O

N N NH2 N N (MTX) NH2

OH

(FITC)

HO2C O

NH+3Cl–

O

O

H2N

S

3 OH

HO

NH

(FITC)

NH+3Cl–

O

OH

(AmB)

O

NH

7

HO

H N

N

O

O O OH OH

CO2H

O

N

O

S

N

O

O

O

O

HO

NHCOCH3

O

OH

N

O

5

O

H HN N S HO2C

O (FITC) O

Figure 8.1. Molecular structures of CNT covalently functionalized with different types of small molecules: 1, ammonium-functionalized CNT; 2, acetamido-functionalized CNT; 3, CNT functionalized with fluorescein isothiocyanate (FITC); 4, CNT bifunctionalized with ammonium groups and FITC; 5, CNT bifunctionalized with methotrexate (MTX) and FITC; 6, shortened CNT bifunctionalized with amphotericin B (AmB) and FITC; 7, shortened CNT bifunctionalized with ammonium groups and FITC (through an amide linkage). (Copyright Nature Publish Group. Reproduced with permission from Ref. 45.)

macromolecules such as antibodies and others. This question highlights the systemic investigation of the behavior of different functionalized CNTs (fCNTs). Kostarelos and co-workers [45] did excellent work showing the effect of different functionalized groups on the interaction with cells. They carried out a series of experiments using functionalized single-walled (f-SWCNTs) and multiwalled (f-MWCNTs) CNTs with a wide variety of functional groups (Figure 8.1). Intrinsically luminescent or fluorescently labeled f-CNTs were directly tracked and imaged intracellularly by epifluorescence and confocal laser scanning microscopy (CLSM). The imaging of f-CNTs and not their adsorbed macromolecules, as previously reported by other groups [11, 46], is imperative in order to elucidate their interaction with cellular compartments.

c08.indd 146

7/30/2010 2:18:56 PM

UPTAKE PATHWAY AND MECHANISM

147

Even in studies tracking the intracellular localization of nonfunctionalized pristine CNTs (pCNTs) solubilized by polymer or single-stranded DNA molecules by the IR spectral characteristics of the pCNT backbone [47–49], one should be very cautious when extrapolating conclusions about pCNT–cellular compartment interactions. Indeed, the effect of the macromolecules acting as their solubilizing agent can play a critical role in determining the type of ensuing interactions with cells and the mechanisms of cellular uptake, as has been shown for other polymer-coated nanostructures [50]. The functionalization of SWCNTs and MWCNTs in the Kostarelos et al. [45] study was mainly performed using the 1,3-dipolar cycloaddition of azomethine ylides [51–53]. This approach allows insertion of amino functions around the side walls and at the tips of the CNTs, which renders the tubes highly soluble in aqueous environments. The amino groups were further modified by covalently linking a variety of small molecules including fluorescent probes and anticancer and antibiotic agents [18, 54]. The addition of functional groups was carried out in a modular fashion, gradually increasing the molecular complexity of the groups covalently linked onto the nanotube side walls (Figure 8.1). In agreement with other studies that used differently functionalized CNTs, f-CNTs 1 and 2 were found to be intrinsically luminescent in the ultraviolet/visible region [55, 56]. Meanwhile, f-CNTs 3–7 were conjugated with fluorescein isothiocyanate (FITC) to obtain high levels of fluorescence signal. The interaction between f-CNTs and a wide variety of live cells was studied. All f-CNTs were allowed to interact with different cell types. It could be observed that the nature of the functional group on the CNT surface did not determine whether f-CNTs were internalized or not. Even in cases where the functional groups were electrostatically neutral or negatively charged in physiological conditions, nanotubes were consistently taken up by cells. The intracellular trafficking of individual or small bundles of f-CNTs occurred, and the transportation of nanotubes toward the perinuclear region was observed a few hours following initial contact with the cells, even under endocytosis-inhibiting conditions. Other mechanisms (such as phagocytosis)—depending on cell type, size of nanotube, and extent of bundling—may also be contributing to or be triggered by the ability of f-CNTs to penetrate the plasma membrane, and therefore be directly involved in the intracellular trafficking of the f-CNTs. From the systemic research of cellular uptake of CNTs with different kinds of groups, they draw a conclusion that the cellular uptake of functionalized carbon nanotubes is independent of functional group.

8.3

UPTAKE PATHWAY AND MECHANISM

Generally, eukaryotic cells internalize extracellular materials inside the cytoplasm through two uptake pathways: an energy-independent pathway and an energy-dependent pathway. Low molecular weight solutes directly transport through the plasma membrane via the energy-independent pathway without

c08.indd 147

7/30/2010 2:18:56 PM

148

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

energy consumption. The energy-dependent pathway, also known as endocytosis for uptake of relatively large particles, can be hindered when incubations are taken at 4 °C or in ATP (adenosine triphosphate)-free medium. The ATP production in cells is disturbed with a well-known reagent, NaN3. The endocytosis is divided into at least two subcategories, that is, clathrin-dependent and clathrin-independent endocytosis. Extracellular materials or solutes that were taken into cells through the clathrin-dependent pathway were first enclosed within clathrin-coated vesicles derived from folds or invaginations of the plasma membrane, and then were brought inside the cells. On the other hand, clatherin-independent endocytosis can occur through the caveolae or lipid-raft pathway. The two subcategories can be separated by hindering one of them. Such treatment consisted of pretreating the cells with sucrose to disturb the formation of clathrin-coated vesicles on the cell membrane, thereby hindering clathrin-dependent endocytosis. As the caveolae-dependent or lipid-rafts-dependent cell internalization relies on the presence of cholesterol domain, the chemical filipin III can be used to disturb the cholesterol distribution within the cell membrane, thus hindering the formation of caveolae. For CNTs, detailed work to establish the cellular uptake mechanism and pathway is still lacking. Pantarotto et al. [16] have suggested an energy-independent nonendocytotic mechanism that involves insertion and diffusion of nanotubes through the lipid bilayer of the cell membrane. However, the Dai group [8] proposed a different mechanism, that is, energy-dependent endocytosis progress. They have systematically investigated the cellular uptake mechanism and pathway for carbon nanotubes. They present evidence showing that clathrindependent endocytosis is the pathway for the uptake of various SWCNT conjugates with proteins and DNA. They also discuss the differences between the nanotube materials and the experimental procedures used in their work and by Pantarotto et al. [16], who suggested an energy-independent nonendocytotic uptake of nanotubes. Their work seems to clearly establish the intracellular uptake mechanism of SWCNTs in the form of individual and small bundles with lengths of <1 μm and is very helpful to avoid any confusion and controversy over the cellular uptake mechanism for these materials. Next, they carried out a systematic investigation of the cellular internalization mechanism and pathway for SWCNT conjugates. Cellular incubations were carried out at 4 °C and with cells pretreated with NaN3 in parallel with our regular incubation conditions. They found that the fluorescence levels observed with confocal microscopy from cells, after incubation in SWCNT conjugates at 4 °C (Figure 8.2B) and ATP depletion by NaN3 (Figure 8.2C), were low. This therefore indicates endocytosis as the internalization mechanism for the uptake of SWCNT conjugates at 37 °C. This was further confirmed by flow cytometry measurements, which indicated a significant reduction in cellular uptake at 4 °C and under the azide ATP depletion conditions (Figure 8.2D). To assess the role of clathrin in the internalization of SWCNTs, they carried out incubations under conditions that are known to disrupt the formation of clathrin-coated vesicles on the cell membrane. Their

c08.indd 148

7/30/2010 2:18:56 PM

UPTAKE PATHWAY AND MECHANISM

149

(A)

(B)

(C)

150

100

50

200 0 150 100 50

DN DN A ASW CN T pr pr ot ot ei ei n nSW pr CN ot ei T nSW 4 ° pr CN C ot T ei nSW az CN ide T

0

(F)

protein-SWCNT

sucrose Cell treatment

(G) Mean fluorescence

Mean fluorescence

(D) 250

Mean fluorescence

(E) 200

K+ depletion

600

400 200

0 Control

Filipin Nystatin

Figure 8.2. Confocal microscopy images of HeLa cells after incubation in fluorescently labeled DNA–SWCNT at (A) 37 °C, (B) 4 °C, and (C) after pretreatment with NaN3. (D) Flow cell cytometry data for HL-60 cells that were incubated in fluorescently labeled pure DNA or protein solutions without nanotubes (labeled “DNA” and “protein,” respectively), DNA–SWCNT and BSA–SWCNT at 37 °C, and for protein– SWCNT at 37 °C in cells incubated at 4 °C or pretreated with sodium azide. (E) Flow cell cytometry data obtained after incubation in protein–SWCNT solutions for untreated cells, cells pretreated with 0.45 M sucrose, and K+-depleted medium, respectively. (F) A confocal image that shows cellular uptake of a rhodamine-labeled transferring protein in HL-60 cells at 37 °C. The inset shows the lack of uptake of the transferrin protein after pretreatment of cells in sucrose. (G) Flow cytometry data of cells after incubation in choleratoxin B (black bars) and BSA-SWCNT (gray bars) for HeLa cells without any pretreatment (control) and cells pretreated with filipin and nystatin, respectively. (Copyright Wiley-VCH. Reproduced with permission from Ref. 8.)

experimental data suggest that cellular internalization of SWCNT conjugates with proteins and DNA is through the clathrin-dependent endocytosis pathway (Figure 8.2). Receptor-mediated endocytosis through clathrin-coated pits is the most common pathway of endocytosis. It provides a means for the selective and efficient uptake of macromolecules and particles that may be present at relatively low concentrations in the extracellular medium. Cells have receptors for the uptake of many different types of ligands, including hormones, growth factors, enzymes, and plasma proteins. For active targeting to cancer cells, nanomaterials were functionalized with ligands such as antibodies, peptides, nucleic acid aptamers, carbohydrates, and small molecules. Among these, folic acid (FA) is appealing as a small molecular ligand for targeting cell membrane and allowing nanomaterial endocytosis via a folate–folate receptor-mediated

c08.indd 149

7/30/2010 2:18:56 PM

150

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

3h

5h

+folate blocking

Fluor channel

FA-SWCNTs 1h

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

20 μm

Merged

TD channel

20 μm

Figure 8.3. Multichannel confocal images of Hep G2 cells after incubation with FASWCNTs for 1 h (left, first column), 3 h (left, second column), and 5 h (left third column). The green fluorescence from the outside to the inside of cells shows the uptake pathway of FA-SWCNTs into cancer cells. Right-hand column: multichannel confocal images of Hep G2 cells incubated in FA-SWCNTs for 3 h with pretreatment of free FA blocking. The concentrations of FA-SWCNTs in all groups are 20 μg/mL. (Copyright IOP. Reproduced with permission from Ref. 57.) (See color insert.)

pathway for higher uptake yields. Also, the high affinity of folate to bind its receptor allows its use for specific cell targeting. A typical result of cellular uptake of folate conjugated CNTs is shown in Figure 8.3 [57]. After incubation in FA-SWCNTs for 1 hour, the SWCNTs initially bonded on the plasma membrane of the cells. After 3 hours, stronger fluorescence was observed in the cytoplasm, indicating that SWCNTs entered into the cells. For 5 hours, confocal images revealed decreased and spotted fluorescence inside cells, corresponding to redistribution and discharge of SWCNTs out of the cells. To further elucidate this pathway, we blocked the folate receptor on the surface of Hep G2 cell membrane with free folate acid before incubation with FA conjugated nanotubes. The uptake of FA-SWCNTs was blocked, as evidenced by the small amount of fluorescence in the cells with the pretreatment of free folate acid. These results demonstrated that Hep G2 cells were able to internalize folate-conjugated SWCNTs via the folate receptor-mediated pathway.

c08.indd 150

7/30/2010 2:18:56 PM

SUBCELLULAR DISTRIBUTION

8.4

151

SUBCELLULAR DISTRIBUTION

After cell penetration, the intracellular fate of the internalized CNTs appears to vary. The subcellular distribution of SWCNTs inside Hep G2 cells have shown that the FA-SWCNTs are located in the cytoplasm but not in the nuclei, indicating that the nanotubes under study cannot transport through the nuclear envelope. It is well known that the nuclear pore on the nuclear envelope allows the mRNA and tRNA to move across the nuclear envelope, and the diameter of the pore is estimated to be about 20 nm in various papers. After functionalization of SWCNTs, the diameter of the nanotubes might be larger than that, or the nanotubes might be too long to pass the nuclear pore, resulting in failure to transport inside the nuclei. The lack of nanotubes in nuclei indicates that studying the subcellular distribution of SWCNTs is very important for understanding the uptake and toxicity of carbon nanotubes. In particular, since SWCNTs are currently under consideration as transporters of DNA and RNA for gene therapy, the presence or absence of carbon nanotubes in nuclei is a vital question that needs to be addressed before the practice of gene therapy. Although most related studies have reported that water-soluble CNTs modified with functional groups resided in the cytoplasm upon incubation, nuclear accumulation was observed. One study has reported that an intense G-band signal was observed in the nuclear region in exposed HeLa cells using confocal Raman imaging analysis; however, the nuclear accumulation of CNTs was not inferred because of the lack of conclusive evidence of CNT-like structures in the nucleus through ultrasections analysis under transmission electron microscopy. The nuclear accumulation of CNTs has been demonstrated by Bianco and co-workers [58]; furthermore, they reported that peptide functionalized multiwalled CNTs could cross the nuclear membrane and be successfully used for plasmid DNA delivery [16]. A better understanding of the intracellular properties of CNTs is therefore essential for the development of this nanomaterial as a drug delivery vector. Recent work by Cheng et al. [59] revealed a new insight into the nuclear accumulation of SWCNTs conjugated with polyethylene glycol (PEG) and labeled with fluorescent dye FITC (FITC-PEG-SWCNTs) in several mammalian cell lines. Cellular uptake and release of FITC-PEG-SWCNTs were observed using time-lapse fluorescence microscopy. This work extends our current understanding of the interactions between CNTs and biological systems, such as the dynamic fate, intracellular behavior, and the uptake and release mechanism. They investigated the cell-penetrating property of PEG-SWCNTs by incubating human cervical carcinoma HeLa cells with FITC-PEG-SWCNTs for 48 h. After extensive washing, the treated cells were analyzed by flow cytometry. As shown in Figure 8.4A, FITC fluorescence was detected in virtually all treated cells, suggesting that a substantial amount of FITC-PEG-SWCNTs had become associated with HeLa cells. Free FITC did not associate with

c08.indd 151

7/30/2010 2:18:56 PM

152

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

Figure 8.4. The cell penetration of FITC-PEG-SWCNTs and its nuclear accumulation in mammalian cells. (A) FACS analysis of HeLa cells treated with FITC-PEG-SWCNTs for 48 h showed that the treated cells took up the FITC-PEG-SWCNTs uniformly. (B) Live cell imaging showed that FITC-PEG-SWCNTs accumulated in the nucleus, mainly in the nucleolus (arrows), of HeLa cells. (C) DIC image of panel B. (D–G) U2OS cells (D, E) and MEF (F, G) were incubated with FITC-PEG-SWCNTs and then organeller markers. Signals from PEGylated SWCNTs (green) did not coincide with mitochondria (red, D–G) but did with nuclei (blue, E, G). Note that the distributions of FITC-PEG-SWCNTs between the nucleolus and the nucleoplasm are different in these three cell lines. Scale bars: (B, C) 20 μm; (D–G) 10 μm. (Copyright ACS. Reproduced with permission from Ref. 59.) (See color insert.)

HeLa cells. The cellular association of FITC-PEG-SWCNTs was time and dose dependent, with cellular green fluorescence detectable as soon as after 1 h of incubation. The detection of cellular FITC by flow cytometry suggested that PEGylated SWCNTs could either enter the cells or adhere to the cell surface. The intracellular distribution of FITC-PEG-SWCNTs in a number of mammalian cell lines was revealed by live cell fluorescence microscopy. Figure 8.4 panels B and C, show HeLa cells incubated with FITC-PEG-SWCNTs. The green fluorescence signal was predominately in the cell nucleus with a weaker cytoplasmic staining in some cells. In most cells, FITC-PEG-SWCNTs were observed to be enriched in a few intranuclear structures. Bright-field DIC images indicated that these intranuclear structures were nucleoli (Figure 8.4B,C, arrows). To further characterize the subcellular distribution of FITCPEG-SWCNTs, CNT-treated cells were counterstained by a mixture of cellpermeable dyes, Hoechst 33342 and MitoTracker, to label the nucleus and mitochrondria, respectively. The dyes were used at concentrations that did not cause detectable cell toxicity and did not interfere with normal mitosis for at least 24 h [16, 17]. FITC-PEG-SWCNT was observed to coincide with Hoechst

c08.indd 152

7/30/2010 2:18:56 PM

SINGLE-PARTICLE TRACKING

153

33342 (Figure 8.4E,G), indicating the internalized SWCNT was predominately nuclear. This confirms that the green fluorescence detected by flow cytometry represented intracellular SWCNTs and supports previous observations that modified SWCNTs can effectively cross cell membranes. While the nuclear targeting mechanism of CNTs is not known, it is possible that the internalized PEGylated SWCNTs are retained in the nucleus through binding with specific nuclear proteins or nucleic acids. Future investigations will include the study of the effect of the molecular weight and surface coverage of PEG on the cellular uptake, intracellular distribution, and dynamic fate of CNTs. It seems PEGylated CNTs are one of the few nanomaterials that accumulate in mammalian cell nuclei without the need of cell-penetrating and nuclearlocalizing tags. Several types of nanoparticles can also enter cells without any conjugation, but these nanomaterials are trapped in the endosome [60]. Many studies reported a predominantly cytoplasmic localization of CNTs [46, 61] instead of a nuclear accumulation. The intracellular location of different kinds of CNTs has not been systematically studied and the cause of this discrepancy is not clear.

8.5

SINGLE-PARTICLE TRACKING

Although much effort has been expended to understand the pathway and mechanism of cellular uptake of nanoparticles, some central questions are still unclear: How is this process carried out? What are the dynamics? How long can the particles stay inside the cells? Does exocytosis occur for nanoparticles? A discussion on these issue is nearly absent from the literature. In recently published works on Au nanoparticles, exocytosis was observed after the extracellular nanoparticle concentration gradient was removed. Studying exocytosis involves incubations where separate specimens are prepared at various time points after incubation, largely to avoid photobleaching [62–66]. There are several limitations on this approach, particularly in capturing nanoparticle dynamics and precisely quantifying endocytosis or exocytosis rates simultaneously. Single-particle tracking (SPT) using fluorophores is a recently developed technique for answering the above questions in cellular systems [67, 68], although with small fluorophores and QDs, photobleaching is a major limitation on real-time measurements. The photobleaching time constrains the observation window during tracking so that events occurring on the order of several hours must be observed through multiple and distinct incubation periods, with each observation starting at a different time after incubation. Strano’s group developed a new technique for SPT of carbon nanotubes, that is, use of the NIR fluorescence signal of SWCNTs [69]. By using this method, they tracked over 10,000 individual trajectories of nonphotobleaching SWCNTs as they are incorporated into and expelled from NIH-3T3 cells in real time on a perfusion microscope stage. Also, an analysis of mean square

c08.indd 153

7/30/2010 2:18:57 PM

154

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

displacement allows the complete construction of the mechanistic steps involved from single duration experiments. They observed the first conclusive evidence of SWCNT exocytosis and showed that the rate closely matches the endocytosis rate with negligible temporal offset. They identified and studied the endocytosis and exocytosis pathway that leads to the previously observed aggregation and accumulation of SWCNTs within the cells. By using image processing algorithms [70, 71], over 10,000 SWCNT trajectories have been tracked from the sequence of images from both data sets. A snapshot of the detected trajectories from ImageJ is shown in Figure 8.5A. Direct proof of endocytosis (Figure 8.5B–I) and exocytosis (Figure 8.5J–M) is repeatedly observed from the NIR image sequences, represented as independent trajectories, confirmed by the MSD features of endocytosis and exocytosis. For instance, MSD of endocytosis has the signature presented later in this chapter. By using a halogen illuminator (100 W), they could observe

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

(K)

(L)

(M)

(A)

(N)

(O)

(P)

(Q)

Figure 8.5. (A) A subset of observed trajectories extracted using ParticleTracker. (B–I) Endocytosis (confirmed by MSD feature) of a single particle (arrow) is observed between t = 1029 and 1112 s, identified as RME (see text). (J–M) Evidence of exocytosis (confirmed by MSD feature) of a single particle (arrow) observed from t = 2218 to 2250 s. (N–Q) Aggregation and movement inside the cell. The cell is illuminated by the halogen lamp and DNA-SWCNTs is illuminated by laser at 785 nm. (Copyright ACS. Reproduced with permission from Ref. 69.)

c08.indd 154

7/30/2010 2:18:57 PM

REFERENCES

155

the scattering of the cell periphery and the PL signal from DNA-SWCNTs simultaneously, which allows for unambiguous classification. An example image sequence of the movement of internalized particles is shown in Figure 8.5N–Q (the dark spot in the middle is from the shade of the light filter). This technique can also verify the overlap between phase contrast cell images and measured trajectories. In order to explain endocytosis and exocytosis mathematically, recurring events of temporal and spatial coordinates of each particle are then analyzed using an algorithm we have written in Matlab to classify the trajectories using their starting and ending locations relative to the cell, as well as the functional form of its MSD. Briefly, the coordinates of the particle on the stage relative to the location of the membrane, cell interior, and the flow direction along the stage, together with the functional form of its MSD, assist in classifying the trajectories according to a number of commonly observed steps within the complete pathway. While 49.2% of trajectories were purely convective diffusion in the flow field with no cellular interaction, the remaining 50.8% demonstrated membrane surface adsorption (6.2%), surface diffusion (18.4%), endocytosis (12.7%), exocytosis (5.9%), or desorption (7.4%).

8.6

CONCLUSION

Increased efforts should be made on the basic characterization of well-defined CNTs, including the diameter, length, surface chemistry, dispersity, and functionalization procedure. It is necessary to develop a standardized process for the characterization and application procedure of CNTs, even though there is a long, long way to go. Also, we emphasize the need for research on the fate of CNTs inside live cells, that is, “what will happen when CNTs are inside cells” not only just “if CNTs can get into cells or not.” New techniques like single-particle tracking should be continuously developed and improved to provide appropriate tools for studying the dynamics of cell–CNT interaction in real time. Only if these central issues are addressed can we have a good understanding of the cellular effect of CNTs; thus application of CNTs in biomedical fields such as DNA transporter and drug delivery will be more reasonable and safer.

REFERENCES 1. Heath, J. R. and Davis, M. E. Nanotechnology and cancer. Annu. Rev. Med. 59: 251–265 (2008). 2. Hartman, K. B., Wilson, L. J., and Rosenblum, M. G. Detecting and treating cancer with nanotechnology. Mol. Diagnosis Ther. 12(1), 1–14 (2008). 3. Alexis, F., et al. New frontiers in nanotechnology for cancer treatment. Urol. Oncol. Semin. O I 26(1): 74–85 (2008).

c08.indd 155

7/30/2010 2:18:57 PM

156

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

4. Wu, Y. R., Phillips, J. A., Liu, H. P., Yang, R. H., and Tan, W. H. Carbon nanotubes protect DNA strands during cellular delivery. ACS Nano 2(10): 2023–2028 (2008). 5. De La Zerda, A., et al. Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nat. Nanotechnol. 3(9): 557–562 (2008). 6. Nakayama-Ratchford, N., Bangsaruntip, S., Sun, X. M., Welsher, K., and Dai, H. J. Noncovalent functionalization of carbon nanotubes by fluorescein-polyethylene glycol: supramolecular conjugates with pH-dependent absorbance and fluorescence. J. Am. Chem. Soc. 129(9): 2448 (2007). 7. Lu, Y. R., et al. DNA functionalization of carbon nanotubes for ultrathin atomic layer deposition of high kappa dielectrics for nanotube transistors with 60 mV/ decade switching. J. Am. Chem. Soc. 128(11): 3518–3519 (2006). 8. Kam, N. W. S., Liu, Z. A., and Dai, H. J., Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway. Angew. Chem. Int. Ed. 45(4): 577–581 (2006). 9. Kam, N. W. S. and Dai, H. J. Single walled carbon nanotubes for transport and delivery of biological cargos. Phys. Status Solidi B 243(13): 3561–3566 (2006). 10. Kam, N. W. S., O’Connell, M., Wisdom, J. A., and Dai, H. J. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci. U.S.A. 102(33): 11600–11605 (2005). 11. Kam, N. W. S., Liu, Z., and Dai, H. J. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 127(36): 12492–12493 (2005). 12. Chen, R. J., et al. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Natl. Acad. Sci. U.S.A. 100(9): 4984–4989 (2003). 13. Singh, R., et al. Internalization of glycosphingolipid analogs in multiple cell types is caveolin-1 dependent and clathrin-independent. Mol. Biol. Cell 13: 230a–231a (2002). 14. Zanello, L. P., Zhao, B., Hu, H., and Haddon, R. C. Bone cell proliferation on carbon nanotubes. Nano Lett. 6(3): 562–567 (2006). 15. Dumortier, H., et al. Functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells. Nano Lett. 6(7): 1522–1528 (2006). 16. Pantarotto, D., et al. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew. Chem. Int. Ed. 43(39): 5242–5246 (2004). 17. Kam, N. W. S., Jessop, T. C., Wender, P. A., and Dai, H. J. Nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into mammalian cells. J. Am. Chem. Soc. 126(22): 6850–6851 (2004). 18. Wu, W., et al., Targeted delivery of amphotericin B to cells by using functionalized carbon nanotubes. Angew. Chem. Int. Ed. 44(39): 6358–6362 (2005). 19. Wang, X., Yang, L. L., Chen, Z., and Shin, D. M. Application of nanotechnology in cancer therapy and imaging. CA Cancer J. Clin. 58(2): 97–110 (2008). 20. Welsher, K., Liu, Z., Daranciang, D., and Dai, H. Selective probing and imaging of cells with single walled carbon nanotubes as near-infrared fluorescent molecules. Nano Lett. 8(2): 586–590 (2008).

c08.indd 156

7/30/2010 2:18:57 PM

REFERENCES

157

21. Zavaleta, C., et al., Noninvasive Raman spectroscopy in living mice for evaluation of tumor targeting with carbon nanotubes. Nano Lett. 8(9): 2800–2805 (2008). 22. Liu, Z., Sun, X. M., Nakayama-Ratchford, N., and Dai, H. J. Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery. ACS Nano 1(1): 50–56 (2007). 23. Rojas-Chapana, J., Troszczynska, J., Firkowska, I., Morsczeck, C., and Giersig, M. Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells. Lab Chip 5(5): 536–539 (2005). 24. Cai, D., et al. Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing. Nat. Methods 2(6): 449–454 (2005). 25. Zhou, W., et al. Small angle neutron scattering from single-wall carbon nanotube suspensions: evidence for isolated rigid rods and rod networks. Chem. Phys. Lett. 384(1-3): 185–189 (2004). 26. Bauer, B. J., Hobbie, E. K., and Becker, M. L. Small-angle neutron scattering from labeled single-wall carbon nanotubes. Macromolecules 39(7): 2637–2642 (2006). 27. Zheng, M., et al. DNA-assisted dispersion and separation of carbon nanotubes. Nat. Mater. 2(5): 338–342 (2003). 28. Heller, D. A., et al. Concomitant length and diameter separation of single-walled carbon nanotubes. J. Am. Chem. Soc. 126(44): 14567–14573 (2004). 29. Heller, D. A., Barone, P. W., Swanson, J. P., Mayrhofer, R. M., and Strano, M. S. Using Raman spectroscopy to elucidate the aggregation state of single-walled carbon nanotubes. J. Phys. Chem. B 108(22): 6905–6909 (2004). 30. Dyke, C. A., Stewart, M. P., and Tour, J. M. Separation of single-walled carbon nanotubes on silica gel. Materials morphology and Raman excitation wavelength affect data interpretation. J. Am. Chem. Soc. 127(12): 4497–4509 (2005). 31. Chattopadhyay, D., Lastella, S., Kim, S., and Papadimitrakopoulos, F. Length separation of zwitterion-functionalized single wall carbon nanotubes by GPC. J. Am. Chem. Soc. 124(5): 728–729 (2002). 32. Duesberg, G. S., et al. Experimental observation of individual single-wall nanotube species by Raman microscopy. Chem. Phys. Lett. 310(1-2): 8–14 (1999). 33. Duesberg, G. S., Muster, J., Byrne, H. J., Roth, S., and Burghard, M. Towards processing of carbon nanotubes for technical applications. Appl. Phys. Mater. 69(3): 269–274 (1999). 34. Duesberg, G. S., et al. Chromatography of carbon nanotubes. Synthetic Met. 103(13): 2484–2485 (1999). 35. Farkas, E., Anderson, M. E., Chen, Z. H., and Rinzler, A. G. Length sorting cut single wall carbon nanotubes by high performance liquid chromatography. Chem. Phys. Lett. 363(1-2): 111–116 (2002). 36. Lustig, S. R., Jagota, A., Khripin, C., and Zheng, M. Theory of structure-based carbon nanotube separations by ion-exchange chromatography of DNA/CNT hybrids. J. Phys. Chem. B 109(7): 2559–2566 (2005). 37. Zheng, M., et al. Structure-based carbon nanotube sorting by sequence-dependent DNA assembly. Science 302(5650): 1545–1548 (2003). 38. Krupke, R., Hennrich, F., von Lohneysen, H., and Kappes, M. M. Separation of metallic from semiconducting single-walled carbon nanotubes. Science 301(5631): 344–347 (2003).

c08.indd 157

7/30/2010 2:18:57 PM

158

INTRACELLULAR TRAFFICKING OF MEMBRANE RECEPTOR-MEDIATED UPTAKE

39. Doorn, S. K., et al. Geometry-based separations of carbon nanotubes using capillary electrophoresis. Abstr. Pap. Am. Chem. Soc. 224: U404 (2002). 40. Doorn, S. K., et al. High resolution capillary electrophoresis of carbon nanotubes. J. Am. Chem. Soc. 124(12): 3169–3174 (2002). 41. Becker, M. L., et al. Length-dependent uptake of DNA-wrapped single-walled carbon nanotubes. Adv. Mater. 19(7): 939 (2007). 42. Pitsillides, C. M., Joe, E. K., Wei, X. B., Anderson, R. R., and Lin, C. P. Selective cell targeting with light-absorbing microparticles and nanoparticles. Biophys. J. 84(6): 4023–4032 (2003). 43. Sun, X. K., et al. An assessment of the effects of shell cross-linked nanoparticle size, core composition, and surface PEGylation on in vivo biodistribution. Biomacromolecules 6(5): 2541–2554 (2005). 44. Moghimi, S. M., Hunter, A. C., and Murray, J. C. Long-circulating and targetspecific nanoparticles: theory to practice. Pharmacol. Rev. 53(2): 283–318 (2001). 45. Kostarelos, K., et al. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nat. Nanotechnol. 2(2): 108–113 (2007). 46. Kam, N. W. S. and Dai, H. J. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J. Am. Chem. Soc. 127(16): 6021–6026 (2005). 47. Govaert, F. and Bernard, M. Discriminating red spray paints by optical microscopy, Fourier transform infrared spectroscopy and X-ray fluorescence. Forensic Sci. Int. 140(1): 61–70 (2004). 48. Graff, R. A., et al. Achieving individual-nanotube dispersion at high loading in single-walled carbon nanotube composites. Adv. Mater. 17(8): 980 (2005). 49. Tsyboulski, D. A., Bachilo, S. M., and Weisman, R. B. Versatile visualization of individual single-walled carbon nanotubes with near-infrared fluorescence microscopy. Nano Lett. 5(5): 975–979 (2005). 50. Kostarelos, K. and Miller, A. D. Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors. Chem. Soc. Rev. 34(11): 970–994 (2005). 51. Lu, X., Tian, F., Wang, N. Q., and Zhang, Q. N. Organic functionalization of the sidewalls of carbon nanotubes by Diels–Alder reactions: a theoretical prediction. Org. Lett. 4(24): 4313–4315 (2002). 52. Georgakilas, V., et al. Organic functionalization of carbon nanotubes. J. Am. Chem. Soc. 124(5): 760–761 (2002). 53. Dent, W. H., Erickson, W. R., Fields, S. C., Parker, M. H., and Tromiczak, E. G. 9-BBN: an amino acid protecting group for functionalization of amino acid side chains in organic solvents. Org. Lett. 4(8): 1249–1251 (2002). 54. Pan, H., et al. Strong green luminescence of Mg-doped ZnO nanowires. J. Nanosci. Nanotechnol. 6(8): 2529–2532 (2006). 55. Guldi, D. M., Holzinger, M., Hirsch, A., Georgakilas, V., and Prato, M. First comparative emission assay of single-wall carbon nanotubes—solutions and dispersions. Chem. Commun. 10: 1130–1131 (2003). 56. Lacerda, L., et al. Luminescence of functionalized carbon nanotubes as a tool to monitor bundle formation and dissociation in water: the effect of plasmid-DNA complexation. Adv. Funct. Mater. 16(14): 1839–1846 (2006).

c08.indd 158

7/30/2010 2:18:57 PM

REFERENCES

159

57. Kang, B., et al. Intracellular uptake, trafficking and subcellular distribution of folate conjugated single walled carbon nanotubes within living cells. Nanotechnology 19(37): 375103–375111 (2008). 58. Pantarotto, D., Briand, J. P., Prato, M., and Bianco, A. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem. Commun. 1: 16–17 (2004). 59. Cheng, J. P., et al. Reversible accumulation of PEGylated single-walled carbon nanotubes in the mammalian nucleus. ACS Nano 2(10): 2085–2094 (2008). 60. Duan, H. W. and Nie, S. M. Cell-penetrating quantum dots based on multivalent and endosome-disrupting surface coatings. J. Am. Chem. Soc. 129(11): 3333–3338 (2007). 61. Monteiro-Riviere, N. A., Nemanich, R. J., Inman, A. O., Wang, Y. Y. Y., and Riviere, J. E. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol. Lett. 155(3): 377–384 (2005). 62. Chithrani, B. D. and Chan, W. C. W. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett. 7(6): 1542–1550 (2007). 63. Panyam, J., et al. Polymer degradation and in vitro release of a model protein from poly(d,l-lactide-co-glycolide) nano- and microparticles. J. Control. Release 92(1-2): 173–187 (2003). 64. Panyam, J., Sahoo, S. K., Prabha, S., Bargar, T., and Labhasetwar, V. Fluorescence and electron microscopy probes for cellular and tissue uptake of poly(d,l-lactideco-glycolide) nanoparticles. Int. J. Pharm. 262(1-2): 1–11 (2003). 65. Panyam, J. and Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55(3): 329–347 (2003). 66. Panyam, J. and Labhasetwar, V. Dynamics of endocytosis and exocytosis of poly(d,l-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm. Res. 20(2), 212–220 (2003). 67. Jin, S., Haggie, P. M., and Verkman, A. S. Single-particle tracking of membrane protein diffusion in a potential: simulation, detection, and application to confined diffusion of CFTR Cl-channels. Biophys. J. 93(3): 1079–1088 (2007). 68. Jin, S. and Verkman, A. S. Single particle tracking of complex diffusion in membranes: simulation and detection of barrier, raft, and interaction phenomena. J. Phys. Chem. B 111(14): 3625–3632 (2007). 69. Jin, H., Heller, D. A., and Strano, M. S. Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Lett. 8(6): 1577–1585 (2008). 70. Sbalzarini, I. F., Mezzacasa, A., Helenius, A., and Koumoutsakos, P. Effects of organelle shape on fluorescence recovery after photobleaching. Biophys. J. 89(3): 1482–1492 (2005). 71. Sbalzarini, I. F. and Koumoutsakos, P. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151(2): 182–195 (2005).

c08.indd 159

7/30/2010 2:18:57 PM

c08.indd 160

7/30/2010 2:18:57 PM

CHAPTER 9

Real-Time Particle Tracking for Studying Intracellular Transport of Nanotherapeutics CLIVE CHEN and JUNGHAE SUH Department of Bioengineering, Rice University, Houston, TX

9.1

INTRODUCTION

The use of nanotherapeutic agents, such as drug and gene carriers, has offered distinct advantages, such as targeted delivery and controlled release, over systemic infusion of therapeutic drugs [1]. The efficacy of these nanotherapeutics is often affected by their ability to reach target organelles upon cellular entry. Intracellular transport of nano-sized carriers can be a difficult process as the cytoplasm, packed with a dense network of cytoskeletal filaments and organelles, is a highly complex biological medium to traverse [2, 3]. There is great interest in understanding the intracellular transport mechanisms involved in the trafficking of nanotherapeutics so that current designs can be improved to facilitate the delivery of cargo drugs and genes to their sites of action [4, 5]. Through advances in fluorescence microscopy, the movement of fluorescently labeled components can be monitored in real time in live cells. Various techniques have been developed to quantify the mobility of labeled molecules and among these, single-particle tracking (SPT) has emerged as a particularly powerful tool for measuring the transport properties of individual transporting entities [6]. In SPT, the positions of tens to hundreds of particles are monitored simultaneously over time and subsequent image analysis provides information on the transport properties for each individual particle. In contrast to other widely used mobility measurement techniques, such as fluorescence recovery after photobleaching (FRAP) and fluorescence correlation Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

161

c09.indd 161

7/30/2010 2:19:02 PM

162

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

spectroscopy (FCS), SPT not only can be used to measure the ensembleaveraged transport properties of a large number of particles, but it can also quantitatively delineate the mobility of individual particles. The ability to obtain detailed quantitative information on the transport properties of individual particles adds greatly to our understanding of the complex intracellular trafficking faced by our engineered nanotherapeutics. In this chapter, we will first discuss the two major stages involved in SPT: locating and tracking of individual particles over time, and analysis of tracking data. Since the majority of SPT studies on nanotherapeutics have been performed in two dimensions, our discussion will be limited to two-dimensional (2D) particle tracking. Readers are referred to a review elsewhere [6] for a discussion on how calculations involved in 2D particle tracking can be translated to three dimensions. In the second part of the chapter, we will review and discuss studies conducted using SPT to investigate the intracellular transport of a variety of nanoscale platforms, including polymeric nanoparticles, quantum dots, and viruses.

9.2

LOCATING AND TRACKING OF INDIVIDUAL PARTICLES

In SPT, the motions of particles are recorded as movies, which are a series of microscopy images taken at a fixed time interval. The trajectories of individual particles are then determined by tracing their changes in position over time. Spatial resolution, or the minimum distance required between two particles in order to resolve them as being separate, is the well-known diffraction limited resolution of about 200 nm. Tracking resolution, or the minimum displacement that can be measured with SPT, can be much smaller [7]. Tracking algorithms are used to locate individual particles precisely and to monitor their trajectories in a high-throughput manner. While many tracking algorithms have been developed over the years, they generally revolve around two basic approaches. The first approach focuses on approximating the absolute positions of particles within individual images while the second approach is correlation-based and requires the comparison of signal intensity patterns between successive frames of images [8, 9]. The absolute position of a particle can be estimated by the center of its point spread function (PSF) when imaged with a microscope. The PSF of the particle can in turn be approximated with a Gaussian function [10, 11]. The central peak of the Gaussian fit is taken to be the coordinates of the particle. Alternatively, a center of mass, or centroid, is identified by weighing the signal intensity within a region of interest selected from a microscopic image [7, 12], and its location is taken to be the position of a particle. The centroid of a spherical particle of uniform fluorescence intensity will also be the center of its PSF. After a particle has been located in every frame of the recorded movie, its change in position between successive frames is traced to determine the trajectory of the particle.

c09.indd 162

7/30/2010 2:19:02 PM

ANALYSIS OF TRACKING DATA

163

In the correlation-based approach, images of a particle between consecutive frames are assumed to have the most similar intensity patterns [13, 14]. An image frame with an intensity pattern that represents a particle is selected and overlaid on top of its subsequent frame to begin comparison of the patterns of interest. The selected frame is shifted at one-pixel increments and the similarity between the patterns is calculated using a correlation matrix. The location with the best match in the intensity pattern can be determined by finding which cell in the correlation matrix has the maximum value. The displacement of the particle is then calculated from the number of pixels being shifted to best align its images from two consecutive frames. Each tracking algorithm has its own set of advantages and disadvantages. For example, while centroid-based algorithms are computationally efficient, they are susceptible to background noise [13]. On the other hand, correlationbased methods are not as affected by noise but can be prone to erroneous matching of particles [9, 13]. More recent algorithms often combine elements from both approaches to exploit their respective strengths. Choice of tracking algorithm usually depends on the microscope, signal detector (camera or photomultiplier tube), and fluorescent probe being tracked since the resulting signal-to-noise ratio is one of the most important factors that needs to be considered. Figure 9.1 shows how trajectories of individual nonviral gene delivery vectors would appear in an intracellular setting when monitored through SPT. Readers are referred to reviews elsewhere for a more comprehensive discussion and comparison of commonly used tracking algorithms [8, 9, 13, 15].

9.3

ANALYSIS OF TRACKING DATA

The first transport data that is calculated in a particle tracking study is the mean squared displacement (MSD) of the tracked particles. In 2D particle tracking, the MSD of tracked particles can be calculated as follows: MSD( t ) = ( x( t ) − x ( t + Δτ ) + ( y( t ) − y( t + Δτ )) 2

2

(9.1)

The MSD can then be used to find the apparent rates of transport, or effective diffusivities (Deff), of individual particles: Deff = MSD ( t ) 4t

(9.2)

It is worth noting that the MSD described here is not a function of time, but that of time interval, t (also known as time scale). As a tracked particle follows a particular trajectory within a predetermined period of imaging time, multiple MSD values can be calculated from the same particle trajectory across different t, with t being dependent on the frame rate at which the particle is

c09.indd 163

7/30/2010 2:19:02 PM

164

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

Figure 9.1. Trajectories of nonviral gene vectors in a live cell tracked using SPT. The observed trajectories are highly heterogeneous, which suggests that different transport mechanisms may be involved. (Reproduced from Ref. 53 with permission.) (See color insert.)

monitored. Readers are referred to a review by Suh et al. [6] for a more detailed discussion of time scale and MSD calculation. While MSD itself mathematically describes the motion of the tracked particles, the changes of MSD, or Deff, across different time scales are indicative of the different transport modes, such as diffusive, subdiffusive, and active transport, which describe the motion of the particles. Various theoretical equations that relate MSD with t have been derived for the different transport modes [16]. Particles undergoing random diffusion are expected to exhibit a constant MSD across different time scales. Their motions can be described by MSD( t ) = 4 Do t

(9.3)

where Do is the diffusion coefficient independent of time scale. For particles experiencing subdiffusive transport, their impeded motion can be described by

c09.indd 164

7/30/2010 2:19:02 PM

ANALYSIS OF TRACKING DATA

165

MSD

Active: MSD = 4Dτ + ν2τ2

Diffusive: MSD = 4Dοτ Subdiffusive: MSD = Kτα

τ

Figure 9.2. Characteristic MSD curves for different transport modes.

MSD( t ) = Kt α

(9.4)

where K is a constant and a is an anomalous exponent <1. For particles undergoing active or convective transport, their motions can be described by MSD( t ) = 4 Dt + v2 t 2

(9.5)

where v represents the velocities of the particles. The v2t 2 term originally described convectional drift, but is commonly associated with active transport in an intracellular setting. At short t, the diffusive term dominates, and at long t, the velocity term dominates. As seen from the equations above, the MSD of tracked particles vary with t in distinct manners depending on the mode of transport involved. By plotting the MSD of individual particles against t (Figure 9.2), their mode of transport can be assessed graphically. For the case of simple diffusion, Do can be found from the slope of the line of best fit in a MSD versus t plot. Other coefficients such as D, K, α, and v can be determined by fitting the appropriate curve to the MSD data using equations that correspond to either subdiffusion or active transport. Suh et al. [17] developed a quantitative method to categorize tracked particles into their respective transport mode based on the relative change (RC) in their effective diffusivities. The diffusivities of purely diffusive particles remain constant at all time scales while particles undergoing active transport or subdiffusive motions display diffusivities that increase or decrease with time scale, respectively. Through the use of a standardized algorithm, a large MSD data set can be analyzed in a high-throughput manner. An RC value is calculated for each tracked particle according to the following equation:

c09.indd 165

7/30/2010 2:19:02 PM

166

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

0.5

2 1.5 1

Active Upper RC Bound RC Bound

Diffusive Hindered

0 0 0.5 1 1.5 2 Time Scale (s) 2

Hindered

4 5 6 7 8 9 10 11 12

Time Scale (s)

A D H A A A A D A D H H A H H

Active Diffusive

1

0.5

0.5 0

1

1.5

Diffusive Lower

Short Time Scale

Active

1.5

0.5

0 0 0.5 1 1.5 2 Time Scale (s)

Classification

Lhort Time Scale

Diffusive

RCShort Time Scale

1

Measured RC 2

RCLong Time Scale

RCLong Time Scale

RCShort Time Scale

Monte Carlo Simulations 2 Upper RC 1.5 Active Bound

% Active Fractions % Diffusive Fractions % Hindered Diffusive Fraction

Hindered

0

4 5 6 7 8 9 10 11 12

Time Scale (s)

Figure 9.3. Steps involved in the RC method to categorize quantitatively individual particles into different transport modes. (Adpated from Ref. 18.)

RC = Deff ( t comp ) Deff ( t ref )

(9.6)

where t comp is the comparison time scale and t ref is a reference time scale that is shorter than t comp. RC values of purely diffusive particles are normally distributed around 1; thus statistical RC value boundaries for diffusive transport are first established through either Monte Carlo simulation or particle tracking in purely homogeneous medium (e.g., glycerol). RC values are then calculated for the experimental particles of interest. Particles with RC values larger than the theoretical upper bound are classified to be undergoing active transport, while particles with RC values less than the theoretical lower bound are classified as subdiffusive. The analysis procedures are often performed at both short and long time scales to find possible changes in transport modes across different time scales. The overall mode of transport can be determined by taking into account the transport modes involved at short and long time scales. The steps involved in RC analysis and the categorization of overall transport mechanism is summarized in Figure 9.3. While mathematical criteria such as RC value have been set forth to sort out the dominant mode of transport of tracked particles, qualitative aspects of the analysis remain indispensable. For example, particles exhibiting “pearlon-a-string” trajectories that are not characteristic of simple diffusion can be misclassified as diffusive unless their trajectories are visually examined. The choice of time scale when using the RC method also requires a considerable amount of understanding and experience in particle tracking. For technical details on performing particle tracking experiments and its data analysis, readers are referred to a guide written by Lai et al. [18].

c09.indd 166

7/30/2010 2:19:02 PM

APPLICATIONS

9.4 9.4.1

167

APPLICATIONS Drug Carriers

Various drug carriers have been developed over the years to improve the efficacy of therapy and to minimize systemic toxicity through targeted delivery. Biodegradable polymeric nanoparticles (NPs) are increasingly popular drug carriers since they can achieve targeted delivery with a readily tunable release profile [19]. The intracellular fate of nanosized polymeric drug carriers has been studied by loading the NPs with fluorescent dyes and examining their subcellular localizations using fluorescence microscopy at fixed time points [20]. Unfortunately, SPT studies on NPs made of materials used in actual therapeutic applications are rare. Instead, model fluorescent probes are often used to explore how size and surface chemistry can affect the intracellular transport of NPs in real time. 9.4.1.1 Model Polymeric NPs Polymeric NPs, such as fluorescent polystyrene (PS) beads, have been used as model probes to investigate the intracellular trafficking behavior of nanosized polymeric drug/gene carriers. These model particles have high fluorescence output, allowing for easy tracking with fluorescence microscopy. They are also relatively monodisperse, thereby minimizing heterogeneity in transport properties due to inherent differences amongst the NPs. Using particle tracking, Suh et al. [17] quantitatively assessed the effect of surface modification with polyethylene glycol (PEG) on the cytoplasmic transport of PS beads in live cells. After cell entry through endocytosis, polymeric drug carriers may need to escape from endosomes to travel to their site of action, such as the nucleus, to exert their maximal therapeutic effect. The highly crowded cytoplasm can hinder the transport of drug carriers to their target organelles, therefore limiting their therapeutic efficacy. Surface modification with PEG, a hydrophilic polymer, was hypothesized to improve particle transport through the cytoplasm by reducing the nonspecific adhesion of particles to subcellular structures, such as cytoskeletal elements. PEGylated or unmodified NPs were microinjected into live cells and their transport properties were quantified by particle tracking. The fraction of PS particles that exhibit unhindered diffusive transport in the cytoplasm increases by 100% upon PEGylation, showing quantitatively how PEG can improve cytoplasmic transport of polymeric NPs. Model polymeric NPs have also been used to characterize new endocytotic pathways. Lai et al. [21] have shown that NPs can be sorted into different endocytotic pathways based on differences in particle size. Through inhibition of classical endocytosis pathways, it was found that 43-nm NPs are likely to undergo clathrin-mediated endocytosis while 23-nm NPs enter cells in a nonclathrin-, non-caveolae-dependent manner. Also, as opposed to 43-nm PS NPs, which are mostly sequestered within acidic vesicles (AVs) such as the

c09.indd 167

7/30/2010 2:19:02 PM

168

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

late endosomes or lysosomes, 23-nm PS NPs appear to be sorted into nonacidic vesicles (NAVs), a pathway that could potentially reduce the degradation of polymeric drug carriers. While particles of both sizes are found to accumulate in the perinuclear region within 2 hours, their transport properties are revealed to be significantly different. The ensemble average MSD of NPs in NAV is about four fold slower than that of NPs in AVs [21, 22]. While around 45% of AVs undergo microtubule-dependent active transport, only 15% of NAVs display active transport, accounting for their slower overall transport. The slower transport of particles within NAVs can potentially increase residence time of drug carriers in the perinuclear region. This may in turn allow for extended release near the perinuclear region and may potentially be beneficial for drug carriers that target organelles near the nucleus. 9.4.1.2 Quantum Dots Quantum dots (QDs) are fluorescent nanocrystals made of semiconductor materials. They are much brighter than most conventional fluorophores and are highly stable against photobleaching [23]. Moreover, their narrow, easily tunable emission spectra allow them to be used in multicolor experiments [24]. Together, these optical properties make QDs excellent probes for fluorescence microscopy. The intrinsically hydrophobic QDs are poorly soluble in aqueous solution, making them ill-suited for biological studies. Development of biocompatible, amphiphilic surface coatings [23, 25, 26] for QDs has led to an exponential increase in the use of QDs as probes for biological processes [27]. To facilitate the entry of QDs into living cells, ligands that induce receptor-mediated endocytosis, such as folate [28] and transferrin [23], are conjugated onto the surface of QDs and these functionalized QDs can be used to investigate the effects of different ligands on endocytotic events [29–31]. While functionalized QDs are now relatively established biological probes, their role as model drug carriers in intracellular transport studies is just beginning to be explored using SPT. A study performed by Tada et al. [32] has demonstrated the applicability of QDs as model drug carriers in SPT studies. Notably, this study also set a remarkable precedent for the application of SPT in live animals. In this study, PEG-coated QDs were conjugated with monoclonal anti-HER2 antibody and their movements from tumor blood vessels to the perinuclear region of tumor cells were tracked in real time after administration into live mice models. After cellular uptake, the functionalized QDs are found to exhibit two distinct stages of directed motion, possibly along two different types of cytoskeletal filaments, before undergoing Brownian motion in a confined area in the highly congested perinuclear region. The velocities of the QDs suggest active transport while the “stop-and-go” phenomenon is attributed by the authors to steric hindrance within the complex biological environment of the cytoplasm. While the intracellular trafficking properties of these model NPs may vary from actual drug carriers due to fundamental differences in the materials used, their usefulness has nonetheless been demonstrated in the characterization

c09.indd 168

7/30/2010 2:19:02 PM

APPLICATIONS

169

of a nonclassical endocytotic pathway and establishing the effects of physiochemical parameters, such as size and surface modification, on the intracellular routing of NPs. 9.4.2

Gene Carriers

Another area of research that benefits from particle tracking is the field of gene therapy. In gene therapy, therapeutic genes are introduced into cells to restore functionality or elicit new responses. Carriers are frequently used to package the therapeutic genes, facilitating their cellular uptake and protecting them from degradation within the cytoplasm. The ability for gene delivery vectors to reach specific organelles, in particular, the nucleus, directly affects the efficacy of gene therapy. Hence the intracellular routing and transport properties of gene vectors have been extensively studied [33, 34]. By monitoring the trajectories of gene delivery vectors in real time with SPT, researchers are able to quantitatively define their transport characteristics, gaining additional insights that could help guide the rational design of delivery vehicles toward better gene delivery efficiencies. 9.4.2.1 Viral Vectors The evolved abilities of many viruses to deliver genetic material into the nucleus of human cells have been harnessed for therapeutic applications. Viral vectors are highly efficient at delivering their genetic cargo and the observed efficacy is often attributed to the expedient transport of viral vectors toward the nucleus [35]. By tracking the journey of individual viral particles in live cells, the endocytotic pathways and cellular components involved in the intracellular trafficking of viral vectors can be precisely and quantitatively characterized. Such knowledge can be used to improve the design of synthetic gene delivery systems through biomimicry. To study the intracellular transport of viruses in live cells, they first need to be fluorescently labeled. The surface of viruses can be chemically labeled with low molecular weight fluorescent dyes, such as Cy5 [36] and Alexa Fluor [37]. While viruses need to be sufficiently labeled so that their motions can be captured with fluorescence microscopy, it is also important to ensure that the degree of labeling does not affect the infectivity of the viruses [36, 38]. Another common approach is to genetically fuse fluorescent proteins to viral capsid proteins [39–41]. However, since fluorescent proteins can be relatively large attachments to the viral capsid subunits, such fluorescent viruses need to be carefully characterized to ensure capsid integrity and infectivity before they can used for SPT studies. The work by Seisenberger et al. [36] is a landmark SPT study that was performed on a popular gene delivery vector, the adeno-associated virus (AAV). It was found that multiple modes of transport are involved in the intracellular transport of AAV. The majority of the tracked viruses either (1) exhibit diffusive behavior with two distinct diffusivities (depending on whether the viruses are vesicle-bound) or (2) hindered transport. A small

c09.indd 169

7/30/2010 2:19:02 PM

170

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

subset of viruses is also observed to be traveling at velocities that are in close agreement with microtubule-dependent active transport. Unexpected directed motions within the nucleus are also observed and it was postulated that AAV could be moving along tubular cytoplasmic structures that have extended into the nucleus. The influenza X-13 virus is another virus that has been studied extensively with SPT. By tracking the motions of individual endocytosed virus particles, Lakadamyali et al. [42] found that the trafficking of influenza viruses toward the nuclei can be divided into stages with distinct signature trajectories. Further quantitative analysis on these trajectories revealed characteristic velocities that are associated with transport along different cytoskeletal components. Similar multistaged trafficking patterns have also been observed for other viruses such as the simian virus 40 (SV40) [43], suggesting that this could be a prevalent feature among different viruses. Although the influenza virus is not a popular gene vector, the studies on influenza virus provide another clear demonstration of how SPT can be used to investigate and characterize viral infectious pathways. Intracellular trafficking pathways of other gene therapy related viruses, such as adenovirus [44, 45], herpes simplex virus (HSV) [46, 47], and human immunodeficiency virus (HIV) [41], have also been examined with SPT. A vast diversity of viruses exist in nature and there are considerable variations in how different viruses deliver their genomes into the cell nucleus. There are, however, some common features to the infectious pathways among different viruses, such as receptor-mediated endocytosis [38, 48, 49] and motor protein-guided active transport along cytoskeletal filaments [41, 42, 45, 50]. The intracellular transport of viruses can often be divided into distinct stages and different transport processes. SPT has been demonstrated to be an invaluable tool for discriminating and characterizing these different transport processes. Future investigations on how the infectivity of a virus particle is affected by its intracellular transport will continue to shape the design of advanced gene delivery vectors. 9.4.2.2 Nonviral Vectors Nonviral, synthetic vectors are attractive gene delivery systems since they are capable of delivering large genetic cargos with relatively little biosafety or immunogenicity concerns, especially when compared to viral vectors. However, the gene delivery efficiencies of synthetic vectors are generally considered to be lower than their viral counterparts. Inefficient intracellular transport is thought to be a critical barrier that lowers the effectiveness of synthetic vectors [2]. Particle tracking has been used to understand the intracellular transport properties of these vectors and to identify any rate-limiting steps involved. One particular nonviral carrier that has been studied extensively with SPT is polyethylene imine (PEI). PEI-based vectors are considered to be some of the most efficient synthetic vectors [51]. Being polycationic, PEI condenses negatively charged cargo genes into PEI/DNA nanocomplexes, protecting the genes and facilitating their intracellular transport. The intracellular trafficking

c09.indd 170

7/30/2010 2:19:02 PM

CONCLUSION

171

trajectory of these nanocomplexes can be tracked when either the PEI [44, 52] or DNA [53] component is fluorescently labeled. The intracellular trafficking of PEI/DNA nanocomplexes prior to nuclear entry has been quantitatively characterized using particle tracking. A hypothesis in the field was that nonviral gene vectors reach the cell nucleus via random diffusion, a slow and potentially rate-limiting step due to the highly crowded nature of the cytoplasm [2, 3]. However, Suh et al. [52, 54] observed that PEI/DNA nanocomplexes accumulate in the perinuclear region within 30 minutes of transfection. By analyzing the trajectories of individual nanocomplexes, it was found that a substantial population of PEI/DNA particles exhibit MSDs associated with active transport. When PEI/DNA nanocomplexes are applied to cells treated with nocodazole, a microtubule depolymerizing agent, active transport of nanocompelexes is inhibited, suggesting that the active transport is microtubule dependent. By tracking the movement of PEI/DNA nanocomplexes along fluorescently labeled cytoskeletal filaments, Bausinger et al. [53] further confirmed that vesicle-bound PEI/DNA nanocompelxes indeed travel along microtubules at velocities comparable to those involved in kinesin- and dynein-mediated transport. While most of the aforementioned studies have been conducted in readily available model cell lines, Suk et al. [44] have shown that similar results are also obtained in primary neurons. In addition, by comparing the intracellular trafficking between adenovirus and PEI/DNA, it was found that failure of synthetic vectors to escape late endosomes and lysosomes may account for the lower gene delivery efficiencies of nonviral vectors. Taken together, particle tracking studies have shown that the cytoplasmic transport of nonviral gene carriers toward the nucleus is not a limiting factor. Instead, design of synthetic vectors should focus on overcoming bottlenecks that emerge subsequent to perinuclear accumulation, such as endosomal escape and nuclear uptake. There is an apparent trade-off in efficiency and biosafety between viral and nonviral gene vectors. Studies suggest that viral vectors enjoy higher gene delivery efficiencies because they are more proficient at overcoming certain intracellular barriers, such as endosomal escape and nuclear translocation. Much research is focused on endowing virus-like features onto nonviral vectors in order to improve nonviral gene delivery efficiency. Further comparisons in the intracellular transport mechanisms between the two systems with SPT should help contribute to the development of highly efficient gene delivery vectors with limited biosafety concerns.

9.5

CONCLUSION

SPT is a powerful technique capable of tracking the movement of individual particles at high spatial–temporal resolution. Analysis of individual particle trajectories within the complex intracellular environment has revealed highly heterogeneous and unique transport properties that are likely overlooked by

c09.indd 171

7/30/2010 2:19:02 PM

172

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

ensemble average-based mobility assays. Continual advances in the development of imaging technologies and tracking algorithms should enhance the knowledge obtained from SPT studies. The expanding use of SPT to characterize intracellular trafficking pathways of different nanotherapeutics will continue to elucidate the link between intracellular transport properties and therapeutic efficacy, thereby guiding the future designs of nanoscale therapeutic devices. ACKNOWLEDGMENT This work was supported by the NSF Center for Biological and Environmental Nanotechnology (EEC-0647452).

REFERENCES 1. Farokhzad, O. C. and Langer, R. Nanomedicine: developing smarter therapeutic and diagnostic modalities. Adv. Drug Deliv. Rev. 58: 1456–1459 (2006). 2. Luby-Phelps, K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. Survey Cell Biol. 192: 189–221 (2000). 3. Tseng, Y., Kole, T. P., and Wirtz, D. Micromechanical mapping of live cells by multiple-particle-tracking microrheology. Biophys. J. 83: 3162–3176 (2002). 4. Miller, A. M. and Dean, D. A. Tissue-specific and transcription factor-mediated nuclear entry of DNA. Adv. Drug Deliv. Rev. 61: 603–613 (2009). 5. Schroder, T., et al. Peptoidic amino- and guanidinium-carrier systems: targeted drug delivery into the cell cytosol or the nucleus. J. Med. Chem. 51: 376–379 (2008). 6. Suh, J., Dawson, M., and Hanes, J. Real-time multiple-particle tracking: applications to drug and gene delivery. Adv. Drug Deliv. Rev. 57: 1551 (2005). 7. Ghosh, R. N. and Webb, W. W. Automated detection and tracking of individual and clustered cell-surface low-density-lipoprotein receptor molecules. Biophys. J. 66: 1301–1318 (1994). 8. Carter, B. C., Shubeita, G. T., and Gross, S. P. Tracking single particles: a userfriendly quantitative evaluation. Phys. Biol. 2: 60–72 (2005). 9. Levi, V. and Gratton, E. Exploring dynamics in living cells by tracking single particles. Cell Biochem. Biophys. 48: 1–15 (2007). 10. Anderson, C. M., et al. Tracking of cell-surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera—low-densitylipoprotein and influenza-virus receptor mobility at 4 °C. J. Cell Sci. 101: 415–425 (1992). 11. Schutz, G. J., Schindler, H., and Schmidt, T. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys. J. 73: 1073–1080 (1997). 12. Lee, G. M., Ishihara, A., and Jacobson, K. A. Direct observation of Brownianmotion of lipids in a membrane. Proc. Nat. Acad. Sci. U.S.A. 88: 6274–6278 (1991).

c09.indd 172

7/30/2010 2:19:02 PM

REFERENCES

173

13. Cheezum, M. K., Walker, W. F., and Guilford, W. H. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81: 2378–2388 (2001). 14. Gelles, J., Schnapp, B. J., and Sheetz, M. P. Tracking kinesin-driven movements with nanometre-scale precision. Nature 331: 450–453 (1988). 15. Jenkins, M. C. and Egelhaaf, S. U. Confocal microscopy of colloidal particles: towards reliable, optimum coordinates. Adv. Colloid Interface Sci. 136: 65–92 (2008). 16. Saxton, M. J. and Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26: 373–399 (1997). 17. Suh, J., et al. PEGylation of nanoparticles improves their cytoplasmic transport. Int. J. Nanomed. 2: 735–741 (2007). 18. Lai S. K., et al. Design and characterization of gene transfer vectors. In: Gene Therapy Protocols, Vol. 3. Humana Press, Totowa, NJ, 2008, pp. 81–97. 19. Panyam, J. and Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 55: 329–347 (2003). 20. Cartiera, M. S., et al. The uptake and intracellular fate of PLGA nanoparticles in epithelial cells. Biomaterials 30: 2790–2798 (2009). 21. Lai, S. K., et al. Privileged delivery of polymer nanoparticles to the perinuclear region of live cells via a non-clathrin, non-degradative pathway. Biomaterials 28: 2876–2884 (2007). 22. Lai, S. K., et al. Characterization of the intracellular dynamics of a non-degradative pathway accessed by polymer nanoparticles. J. Control. Release 125: 107–111 (2008). 23. Chan, W. C. W. and Nie, S. M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281: 2016–2018 (1998). 24. Jaiswal, J. K., et al. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 21: 47–51 (2003). 25. Bruchez, M., et al. Semiconductor nanocrystals as fluorescent biological labels. Science 281: 2013–2016 (1998). 26. Pellegrino, T., et al. Hydrophobic nanocrystals coated with an amphiphilic polymer shell: a general route to water soluble nanocrystals. Nano Lett. 4: 703–707 (2004). 27. Pinaud, F., et al. Advances in fluorescence imaging with quantum dot bio-probes. Biomaterials 27: 1679–1687 (2006). 28. Bharali, D. J., et al. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. J. Am. Chem. Soc. 127: 11364–11371 (2005). 29. Chen, B., et al. Transmembrane delivery of the cell-penetrating peptide conjugated semiconductor quantum dots. Langmuir 24: 11866–11871 (2008). 30. Delehanty, J. B., et al. Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. Bioconjug. Chem. 17: 920–927 (2006). 31. Medintz, I. L., et al. Intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides. Bioconjug. Chem. 19: 1785–1795 (2008). 32. Tada, H., et al. In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors of mice. Cancer Res. 67: 1138–1144 (2007).

c09.indd 173

7/30/2010 2:19:02 PM

174

REAL-TIME PARTICLE TRACKING FOR STUDYING INTRACELLULAR TRANSPORT

33. Lechardeur, D., Verkman, A. S., and Lukacs, G. L. Intracellular routing of plasmid DNA during non-viral gene transfer. Adv. Drug Deliv. Rev. 57: 755–767 (2005). 34. Payne, C. K. Imaging gene delivery with fluorescence microscopy. Nanomedicine 2: 847–860 (2007). 35. Whittaker, G. R. and Helenius, A. Nuclear import and export of viruses and virus genomes. Virology 246: 1–23 (1998). 36. Seisenberger, G., et al. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294: 1929–1932 (2001). 37. Ewers, H., et al. Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes. Proc. Nat. Acad. Sci. U.S.A. 102: 15110–15115 (2005). 38. Bartlett, J. S., Wilcher, R., and Samulski, R. J. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J. Virol. 74: 2777–2785 (2000). 39. Desai, P. and Person, S. Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid. J. Virol. 72: 7563–7568 (1998). 40. Lux, K., et al. Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking. J. Virol. 79: 11776–11787 (2005). 41. McDonald, D., et al. Visualization of the intracellular behavior of HIV in living cells. J. Cell Biol. 159: 441–452 (2002). 42. Lakadamyali, M., et al. Visualizing infection of individual influenza viruses. Proc. Nat. Acad. Sci. U.S.A. 100: 9280–9285 (2003). 43. Pelkmans, L., Kartenbeck, J., and Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 3: 473–483 (2001). 44. Suk, J. S., et al. Gene delivery to differentiated neurotypic cells with RGD and HIV Tat peptide functionalized polymeric nanoparticles. Biomaterials 27: 5143– 5150 (2006). 45. Suomalainen, M., et al. Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J. Cell Biol. 144: 657–672 (1999). 46. LaVail, J. H., et al. Retrograde axonal transport of Herpes simplex virus in squid axoplasm. Mol. Biol. Cell 10: 139a (1999). 47. Satpute-Krishnan, P., DeGiorgis, J. A., and Bearer, E. L. Fast anterograde transport of herpes simplex virus: role for the amyloid precursor protein of Alzheimer’s disease. Aging Cell 2: 305–318 (2003). 48. Daecke, J., et al. Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry. J. Virol. 79: 1581–1594 (2005). 49. Rust, M. J., et al. Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat. Struct. Mol. Biol. 11: 567–573 (2004). 50. Dohner, K., et al. Function of dynein and dynactin in herpes simplex virus capsid transport. Mol. Biol. Cell 13: 2795–2809 (2002). 51. Park, T. G., Jeong, J. H., and Kim, S. W. Current status of polymeric gene delivery systems. Adv. Drug Deliv. Rev. 58: 467–486 (2006).

c09.indd 174

7/30/2010 2:19:02 PM

REFERENCES

175

52. Suh, J., Wirtz, D., and Hanes, J. Efficient active transport of gene nanocarriers to the cell nucleus. Proc. Nat. Acad. Sci. U.S.A. 100: 3878–3882 (2003). 53. Bausinger, R., et al. The transport of nanosized gene carriers unraveled by live-cell imaging. Angew. Chem. Int. Ed. 45: 1568–1572 (2006). 54. Suh, J. H., Wirtz, D., and Hanes, J. Real-time intracellular transport of gene nanocarriers studied by multiple particle tracking. Biotechnol. Prog. 20: 598–602 (2004).

c09.indd 175

7/30/2010 2:19:02 PM

c09.indd 176

7/30/2010 2:19:02 PM

CHAPTER 10

Tracking Intracellular Polymer Localization Via Fluorescence Microscopy SIMON C. W. RICHARDSON

10.1 AN INTRODUCTION TO THE STUDY OF THE ENDOCYTIC AND SECRETORY PATHWAYS USING FLUORESCENT MICROSCOPY Organelles integral to the sorting and movement of material through the endocytic and secretory pathways (Figure 10.1) are critical to cellular homeostatic balance and perform a diverse array of physiological functions [1, 2]. Examples of the diversity of function performed by the two aforementioned pathways include communicating with and responding to the external environment [3], the secretion of structural material and enzymes (i.e., to remodel the cell’s environment) [4], the attenuation of external signals (i.e., regulating cell growth) [5], and the feeding of the cell through the internalization of essential nutrients such as iron [6] and albumin [7]. All of these functions require the sorting and compartmentalization of material into discrete membrane delimited structures and the regulated movement of material between these structures [1]. As this regulated movement requires not only the anterograde (forward) movement of material but also the retrograde (backward) recycling of cellular machinery involved in this regulation, there exists the possibility of exploiting not only the endocytic pathway and lysosomotrophic targeting for drug delivery [8], but also the transit of material between the secretory and endocytic pathways [9–11]. This occurs in nature and has been documented during the characterization of several bacterial and plant toxins [11, 12]. Over the last 35 years much interest has been shown in the use of synthetic, polymeric drug delivery systems that have demonstrated lysosomotrophic targeting in many types of mammalian cell (in vitro), in vivo and in the clinic Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

177

c10.indd 177

7/30/2010 2:19:04 PM

178

TRACKING INTRACELLULAR POLYMER LOCALIZATION

Secretory pathway

Endocytic pathway

Secretory Vesicles

Recycling Endosomes (TfR /RAB11a)

trans-Golgi (TGN46/38 or Ceramide)

Early Sorting Endosome (EEA1 /Rab5a)

MedialCis-Golgi (GM130) ER (BiP)

Cytosol

Early Endocytic Structures

Late Endosome (LAMP1-3 / M6PR / Rab7)

Hybrid Organelle

Lysosome (LAMP1-3)

Late Endocytic Structures

Nucleus

Figure 10.1. Generic intracellular trafficking pathways.

[8]. However, if investigators wish to optimize the intracellular movement, or kinetics of drug delivery at the intracellular level, they are obliged to investigate the intracellular fate of the delivery system [11]. As organelle intracellular localization and morphology are very diverse, it is often useful to identify subcellular compartments using physiological, immunological markers that have their roots in a fundamental understanding of organelle physiology and function [13]. Consequently, if the appropriate subcellular markers are employed, it is possible to effectively monitor the subcellular fate of nanoscale drug delivery systems in a temporal and qualitative manor [11]. It has been reported that it is possible (though extremely difficult) to use fluorescent microscopy to approximate the concentration of fluorescent material within a subcellular compartment. This difficulty is primarily due to the effects of fluorophore concentration-dependent quenching and possible pH-driven fluorescent phenomena in live cells. However, due to its inherent difficulty and complexity, this topic will not be considered further. The qualitative localization of synthetic biocompatible drug delivery systems is far from simple. One commonly used method capable of untangling

c10.indd 178

7/30/2010 2:19:04 PM

AN INTRODUCTION TO THE STUDY OF THE ENDOCYTIC AND SECRETORY PATHWAYS

179

the vast network of intracellular organelles is immunofluorescent microscopy. Typically, immunofluorescent microscopy requires the use of specific immunoreactive proteins, documented to mediate organelle-specific function. As these proteins define intracellular compartments, they can act as “landmarks” or markers. Often proteins that are responsible for regulating fusion to an organelle are chosen as these have the potential to represent “lock and key” type specificity through the very nature of their intracellular function. That said, the models used are often not perfect as there may be promiscuity associated with a particular protein’s function, that is, certain molecules may catalyze more than one fusion event (e.g., Syntaxin 6 [14, 15]), or cycle between more than one compartment (e.g., transferrin receptor [16]). Equivalently, there may be a great deal of primary protein sequence similarity between members of a particular family (i.e., Syntaxin 7 and Syntaxin 13), mediating fusion between the late endosome and early endosome, respectively [17], or the Rab family of GTPases [18, 19], which makes the immunological identification of specific family members difficult. Typical strategies used during the fluorescent identification of a subcellular compartment may be either (1) direct (i.e., by detecting a fluorescent physiological probe or fluorophore conjugated antibody) or (2) indirect (i.e., by immunolabeling the sample with a primary antibody specific to a protein enriched either within or upon the target compartment or organelle). The primary antibody is then identified using a fluorophore labeled secondary antibody (specific to the primary antibody) [16, 20, 21]. However, with respect to the subcellular localization of synthetic macromolecules, a paradox becomes apparent. Antibodies are macromolecules and are unable to penetrate cellular membranes. As the compartmentalization of macromolecules is one of the primary functions of the cellular membrane, it is obvious that, with the exception of antigens on the exofacial leaflet of the plasma membrane, an extracellular antibody applied by the researcher will not be able to assimilate its target unless some sort of membrane extraction or permeabilization is undertaken. Yet, it is also critical that intracellular macromolecules remain in place and are thus representative of the cell “in life.” Antibody intracellular penetration is usually achieved by lipid extraction using either solvents or detergents [20]. However, both forms of extraction potentially allow the escape (or movement) of poorly fixed (i.e., immobilized) synthetic material such as a soluble polymer forming part of a drug delivery system during sample preparation. This specific quandary set the basic tenet for this chapter, which will discuss ways of localizing soluble, nonfixed material to specific organelles or intracellular compartments (Section 10.4). But, before embarking on this discussion, it is critical to understand the aforementioned intracellular markers that will serve as counterstains, landmarks, and reference points, allowing the localization of a material with an unknown intracellular fate into a specific organelle or compartment over time. The following is not an exhaustive list, which would be extensive, but a list of tools that the author is familiar with and has used successfully [11].

c10.indd 179

7/30/2010 2:19:04 PM

180

TRACKING INTRACELLULAR POLYMER LOCALIZATION

10.2 ENDOCYTIC AND SECRETORY ORGANELLES: FUNCTION AND MARKERS Figure 10.1 depicts both the secretory and endocytic membrane trafficking pathways. It is of note that this is a general depiction of cellular function as several cell types have evolved and differentiated to utilize these marker proteins to catalyze fusion between not one subset but many different organelles. An example of this is seen in the murine melanoma cell line B16 F10. B16 F10 cells localize Syntaxin 6 (normally found in the trans-Golgi network (TGN) and on early sorting endosomes) to lysosome-associated membrane glycoprotein (LAMP) positive structures, typically discreet and separate from the TGN and early endosome [15]. However, within the context of this work, Figure 10.1 represents a useful rule of thumb. Table 10.1 describes most of the reagents discussed herein and documents suppliers and catalog numbers. 10.2.1 Plasma Membrane At the cellular level, the first barrier encountered by a scientist wishing to enhance the bioavailability of a molecule with an intracellular target is the

TABLE 10.1

Commonly Used Reagents

Antigen/Reagent

Clone/Cat #

Source

EEA1 Syn6 TfR LAMP1 LAMP2 CD63 TGN38 TGN46 Texas Red anti-rabbit Texas Red anti-mouse Alexa Fluor 488 anti-rabbit Alexa Fluor 488 anti-mouse Cy5 anti-rabbit Cy5.5 anti-mouse Texas Red-NHS Alexa Fluor 488-NHS Lysotracker (Red) Lysotracker (Green) Texas Red-WGA Alexa Fluor 488-WGA Lysine fixable Dextran

(#E41120) (#S5542) (H68.4) (H4A3) (H4B4) (RFAC4) (T69020) (AHP500) (#T-2767) (#T-862) (#A11008) (#A11001) (611 110122) (610 713124) (T-6134) (A-20000) (L-7528) (L-7526) (W21405) (W112161) (D-1864)

Transduction Labs Transduction Labs Zymed DSHB* DSHB* Biodesign Int. Transduction Labs ABd SeroTech Invitrogen Invitrogen Invitrogen Invitrogen Rockland Inc. Rockland Inc. Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen

*DSHB (Development studies hybridoma bank at the University of Iowa.

c10.indd 180

7/30/2010 2:19:04 PM

ENDOCYTIC AND SECRETORY ORGANELLES: FUNCTION AND MARKERS

181

plasma membrane. This may be the exofacial leaflet of an endothelial cell forming the lumen of a microcapillary in the lung [22], or of the endothelial cells forming part of the blood–brain barrier [23], or that of the target cell. Imaging the plasma membrane is useful when asking questions such as: “Is the ‘cargo’ or drug to be delivered inside the cell?” Even if the exact nature of the compartment occupied by the drug is not positively identified, data demonstrating that a substance has been internalized may still be informative. An excellent example of a directly labeled marker that can be used to label the plasma membrane is Texas Red-labeled Wheat Germ Agglutinin (TxR-WGA). If a fixed cell has not been subject to permeabilization (using either detergent of solvent), WGA can be used to label sialic acid and N-acetylglucosaminyl residues extending from the exofacial leaflet of the plasma membrane [24], the WGA being unable to penetrate this structure. It is also possible to image and delineate the plasma membrane living cells using WGA by keeping the cells at 0 °C. It is important to note that as WGA binds to sugar moieties on the cell surface, it will be internalized by live cells at 37 °C and, at this point, is no longer at the cell surface. 10.2.2 Early Endosome Following endocytic internalization, the first intracellular sorting station reached by exogenous material is the early endosome (EE), also called the sorting endosome. This will typically house material 5–10 min after internalization and is characterized by the presence of the small GTPase Rab5 [25], Rab5 effectors such as early endosomal antigen 1 (EEA1) [26] as well as a host of other proteins such as Syntaxin 13 [27]. In this acidic (pH typically about 6.5) organelle, material is sorted into a putative default catabolic (lysosomal) pathway or recycling pathways (Figure 10.1) [1, 28, 29]. This organelle also contains much of the sorting machinery required for the ubiquitin-mediated entry into multivesicular bodies that ultimately results in the degradation of trans-membrane proteins [29, 30]. Furthermore, the segregation of soluble material from receptors and membranes is a function of decreased pH and manipulating surface area to volume ratio, sorting the membrane into long reticular tubes [1]. EEA1 is a tethering protein that forms homo-oligomers and is responsible for bringing prospective fusion partners into proximal contact, that is, a donor vesicle and a recipient compartment, prior to soluble N-ethylmaleimidesensitive factor (NSF) attachment protein receptors (SNARE) pair formation [27]. SNARE proteins, such as the Syntaxin and vesicle associated membrane protein (VAMP) family of integral membrane proteins, are commonly held to be the proteins responsible for driving vesicular fusion [31]. Fusion is thought to be achieved by overcoming the entropy associated with pushing together two hydrated phospholipid bilayers, squeezing out water from between the two prospective fusion partners [32]. EEA1 can be

c10.indd 181

7/30/2010 2:19:04 PM

182

TRACKING INTRACELLULAR POLYMER LOCALIZATION

immunolabeled using commercial monoclonal antibodies, which require an aldehyde rather than a cold solvent fix (Section 10.3.2). Unfortunately, this makes counterstaining samples fixed in aldehyde with a second antibody that require a cold solvent fix (i.e., LAMP-specific antibodies), difficult [16]. As fixation and permeabilization of the cell is required, this antibody is not usually a good choice for labeling live cells. If it is necessary to image the EEs of a live cell then the overexpression of Rab5a fused in frame to enhanced green fluorescent protein (eGFP) may be used [25]. Constitutively active Rab5 mutants (Rab5:Q79L-GFP) [33] are well characterized and should be used with care as their expression can cause the redistribution of intracellular markers (e.g., LAMP1) [34]. 10.2.3 Recycling Endosomes The recycling endosome (RE) is a Rab11 positive, reticular structure that has been documented to contain recycled material (i.e., apo-transferrin) 30 min after internalization [35]. The transferrin receptor (TfR) is an often-used marker for this organelle. The trafficking of TfR is markedly different from that of some other cellular receptors such as the epidermal growth factor receptor (EGFR), which is subject to ubiquitinylation and sorting into the lysosomal catabolic pathway following its activation [5]. From the early endosome, the TfR will follow either a kinetically fast or kinetically slow path back to the cell surface, the latter transiting through the Rab11 positive recycling endosome [18]. A consequence of this is that if TfR is used as an endocytic marker, there will be significant overlap between the Rab5 positive recycling endosome and the Rab11 positive recycling endosome [16]. TfR may be visualized indirectly by either immunolabeling using a commercially available antibody (in aldehyde fixed cells) or by using fluorescently labeled transferrin, which will be internalized and transit through the sorting and recycling endosomes in cells expressing TfR. Similar to the overexpression of Rab5a-GFP, Rab11a-fluorescent protein overexpression is well characterized and may be used to label recycling endosomes in living cells [36]. 10.2.4 Late Endosomes (LEs) LEs may be defined as LAMP positive, mannose-6-phosphate receptor (M6PR) positive structures. They are devoid of Rab5 and Rab11 and positive for Rab7, which regulates both homotypic and heterotypic fusion to LEs [34]. Morphologically, these structures have been documented by electron microscopy to contain internal membrane and are consequently also referred to as multivesicular endosomes. Material subject to endocytic capture may be documented in LEs after 60 min. There is some controversy as to the genesis of LEs with some reports documenting the transit of material from early sorting endosomes to LEs in vesicles (i.e., endocytic carrier vesicles [37]). However,

c10.indd 182

7/30/2010 2:19:04 PM

ENDOCYTIC AND SECRETORY ORGANELLES: FUNCTION AND MARKERS

183

there is also evidence documenting the maturation of early sorting endosomes into LEs [34]. LAMPs are highly glycosylated integral membrane proteins that contain a C-terminal cytosolic sorting signal that directs these proteins to late endocytic structures [38]. The luminal portion of these molecules is highly glycosylated, a physiological adaptation that is thought to help prevent the autolysis of the LE/Lys limiting membrane following lysosomal heterotypic fusion [39]. Furthermore, the primary protein structure of the LAMP family of proteins is highly species specific in terms of immunoreactivity. In primates and humans LAMP1 and LAMP2 specific antibodies will not cross-react with their rat orthologous (lysososmal glycoprotein (Lgp110 and Lgp120)). Commercially available antibodies to the above lysosomal proteins are readily available and work well after sample fixation using cold methanol. As previously mentioned, the LE is also defined by the presence of the M6PR [40]. The M6PR may be divided into two families, one being cation dependent (CD) and the other being cation independent (CI). CD-MP6R is a type 1 trans-membrane protein (having its C-terminus in the cytosol) that is responsible for transporting biogenic material destined for the lysosome from the Golgi body [41]. A well-characterized example of one such MP6R cargo would be the lysosomal protease cathepsin D. LAMP2 has also recently been reported to be involved in the process of lysosomal biogenesis, transporting molecules from the Golgi to the LE [42]. 10.2.5 Lysosomes (Lys) The lysosome is typically the terminal compartment of the endocytic system and may be thought of not as a “suicide bag” but as an enzyme storage depot. During endocytosis and the entry of material into the late endocytic compartment, material is sorted to the LE, which then fuses with Lys. The Lys then disgorge their contents into the lumen of the LE, creating a LE/Lys hybrid organelle where digestion occurs [43]. Although there is little literature describing the events after this fusion event, Lys are thought to reform from this LE/Lys hybrid organelle [44]. BSA–colloidal gold has been documented within a LAMP positive compartment after 60 min and aggregation of BSA– colloidal gold continues over the next 20 h, indicating that digestion of the BSA is occurring [45]. It is worthy of note that the catabolic nature of lysosomes makes their study difficult. However, tools exist that can help localize potentially labile material to late endocytic structures (such as enzyme inhibitors). Leupeptin (200 μM) fed to cells during and after a pulse of proteinaceous fluorescent labeled fluid phase or adsorptive marker (such as Texas Red-labeled Dextran, BSA, or WGA) can maintain the integrity of fluorophore-labeled material for up to 72 h. If the leupeptin is omitted there will be much nonspecific labeling of the cell and very little will be lysosomal (data not shown). Fluorophore-labeled WGA, BSA, and Dextran will yield almost complete colocalization with late

c10.indd 183

7/30/2010 2:19:04 PM

184

TRACKING INTRACELLULAR POLYMER LOCALIZATION

endocytic markers following a 4–18-h incubation period (denoted the chase time) in the presence of leupeptin [11, 16]. This time is extended beyond the initial 60 min required for the detection of material within late endocytic structures to allow the clearing of earlier endocytic structures [16]. Although some cells express BSA-specific receptors [7], adsorptive endocytosis has been reported to drive the endocytosis of BSA (in specific instances). However, in the vast majority of cells and at high BSA concentration, the majority of BSA uptake appears to be nonspecific and concentration dependent (i.e., by fluid phase endocytic capture). This is a consequence of the relatively high concentrations of fluorophore-labeled BSA that are necessary to obtain an acceptable signal-to-noise ratio. Typically, this will be between 5 and 10 mg/mL BSA in PBS. This has been used to successfully delineate late endocytic (LAMP positive) structures in a range of developmentally different cell types derived from both fibroblasts and epithelia [11, 16, 46]. Commercially available lysine fixable Texas Red-labeled Dextran contains lysine residues. The lysine residues are intended to make this molecule susceptible to aldehyde fixation (the epsilon amine being able to react with an aldehyde). However, the charge associated with the lysine makes this molecule (in the author’s hands) very nonspecifically “sticky.” A result of this is a very high background, nonspecific, fluorescent signal. Although this molecule is less susceptible to degradation than BSA (internal signal persisting up to 72 h with or without leupeptin), it is of note that this advantage is often offset by the high background signal, lowering the signal-to-noise ratio obtained. After solubilization in PBS at 10 mg/mL, this reagent benefits from centrifugation at 100,000 g at 4 °C for 60 min. The supernatant may then be used without very bright nonsoluble aggregates interfering with the process of image acquisition. Similar to fluorophore-labeled BSA, Texas Red-labeled WGA can be allowed to bind to cell surface sialic acid and N-acetylglucosaminyl moieties and then be internalized. As this binding is absorptive (i.e., to membrane associated sugar residues, which are in abundance) [11], relatively low concentrations of WGA can be used to generate an acceptable signal-to-noise ratio. After a 1-h incubation with the fluorophore-labeled WGA in cell culture media (a pulse of WGA) at 37 °C and, following rigorous washing and a further 4-h incubation time (the chase period, also at 37 °C), WGA can be detected in LAMP positive structures (although these structures are larger than native LAMP positive structures). This phenomenon may be attributed to WGA having multiple sugar-binding sights and causing the crosslinking of membrane constituents [24]. Whether this is due to the increased trafficking of material to LEs or a reduction in the efficiency of LE reformation from the LE/Lys hybrid is not known. LysoTracker® is an aminic fluorophore that, at a neutral pH, freely permeates cell membranes. Once this reagent enters an acidic compartment, protonation impairs the molecule’s ability to cross membranes, leading to its selective

c10.indd 184

7/30/2010 2:19:04 PM

ENDOCYTIC AND SECRETORY ORGANELLES: FUNCTION AND MARKERS

185

accumulation in acidic membrane-bound intracellular structures. Consequently, in live cells with active membrane proton pumps, this reagent can be very successfully used to label acidic membrane-bound structures (i.e. EE, RE, LE, and Lys). At a 10-nM concentration, the photobleaching of the LysoTracker reagent has been an issue for the author [11]. This can be countered by increasing the concentration of the lysotracker in the cell media to 100 nM. This action also increases background noise and may interfere with membrane fusion events, which have been documented to be sensitive to vesicular luminal pH [47]. This is an issue as the accumulation of LysoTracker molecules within an acidic vesicle has been documented to cause an alkalizing effect [48]. 10.2.6 The Golgi Apparatus The Golgi body may be thought of as acting in opposition to the EE. As the EE mediates the sorting of material (and membrane components) entering the cell through the endocytic pathway (i.e., from the outside), the Golgi body sorts material that has been synthesized by the cell, directing it either to another intracellular location or into the secretory pathway. The Golgi may be divided into four subcompartments—the trans-Golgi network (TGN), the medial-Golgi (or Golgi ribbon), the cis-Golgi, and the endoplasmic reticulum to Golgi intermediate compartment (ERGIC) [49]. The TGN may be labeled using a variety of reagents in live or fixed cells. Antibodies specific to Syntaxin 6 are well-characterized Golgi markers. However, due the promiscuity of Syntaxin 6, it is not always the best choice when labeling the TGN [14, 15]. Additionally, varying expression levels of Syntaxin 6 may also result in poor signal-to-noise ratio. Monoclonal antibodies specific for TGN38 and TGN46 proteins may also be used to decorate the TGN. The species-specific expression of these proteins means they should be approached with care [50], for example, TGN38 is not expressed in Vero cells (whereas TGN46 is). Fluorescently labeled WGA may also be used to label the TGN in fixed, permeabilized cells. Ribosome inactivating proteins (RIPs) may also be chased into the Golgi and visualized by indirect immunofluorescence using polyclonal antibodies or following direct labeling [11]. If the TGN of live cells is to be imaged, then there are a variety of fluorescently labeled materials that have worked well for the author. Fluorescently labeled ceramide may be used to label the Golgi body in live cells and is suitable for use over a time course [11]. If the cis- or medial-Golgi is to be imaged, then monoclonal antibodies specific for the protein GM130 may be useful [11]. Like EEA1, GM130 is a tethering protein that helps regulate endoplasmic reticulum (ER) to Golgi transport. GM310 exists functionally within a complex of several other proteins, such as (though not exclusively) P115 and Giantin [51]. This protein may be labeled using commercially available antibodies after an aldehyde fix.

c10.indd 185

7/30/2010 2:19:04 PM

186

TRACKING INTRACELLULAR POLYMER LOCALIZATION

10.2.7 The Endoplasmic Reticulum The ER is the site of lipid synthesis (smooth ER) and acts as part of the cell’s quality control mechanism, ensuring integral and secreted proteins are folded properly prior to sending these proteins to the Golgi for distribution around the cell or for secretion [52]. In order to facilitate this process, luminal proteins called “chaperones” are employed such as heat-shock protein 70 (HSP70). Monoclonal anti-immunoglobulin heavy chain binding protein (BiP) antibodies may be used to visualize the ER. BiP is an ER chaperone protein [53] and can readily be detected with a commercially available antibody. To draw this section to a close, it is of note that the researcher’s choice of fluorophore will impact on the success of imaging a given structure. If a probe is to be directly labeled and then subject to endocytic capture, Texas Red or Alexa Fluor 488 are good candidates. When chasing fluorophore-labeled BSA into cells, the author, despite exhausting Molecular Probe’s (now Invitrogen) comprehensive catalog (and obtaining consistent labeling efficiencies), was unable to image intracellular structures using a “blue” fluorophore. However, Marina Blue-labeled WGA was regularly and successfully used to label late endocytic structures.

10.3

FIXATION

Fixation is performed to ensure that the spatial location of material is not altered during the preparation of samples for microscopy. Commonly, one of two methods are employed, utilizing either solvents or aldehyde. If the immunodetection of specific markers is to be employed after fixation, a blocking step is necessary prior to antibody hybridization. This is typically performed using animal serum diluted to 2% (v/v) in phosphate buffered saline (PBS) and prevents the nonspecific adsorption of antibodies onto the sample [20]. It is important to note that it is critical not to use a blocking agent that will be recognized by the secondary or primary antibodies. For example, if the secondary antibodies have been raised in a donkey, donkey serum would be an excellent choice as a blocking agent. However, if the intended secondary antibodies were specific to rabbit IgGs, rabbit serum would not be a good choice as a blocking agent. Occasionally, unanticipated artifacts are encountered using the more standard blocking agents and if this is an issue Teleostean gelatine may be used in place of mammalian serum. If the researcher is in any doubt with regard to secondary antibody specificity, omitting the primary antibody from a negative control, processed in exactly the same way as the samples, will usually document which structures are being specifically labeled and which are not, the difference denoting specific labeling. Similarly, if a researcher using a confocal microscope is worried about bleed from one fluorophore’s channel to another, simply turning off the relevant laser and imaging as before will act as a negative control. If signal persists in the channel with

c10.indd 186

7/30/2010 2:19:04 PM

FIXATION

187

Scheme A Formaldehyde Methylene Paraformaldehyde Glycol O H OH H (In water) HO O n OH H H H n=2-8 Scheme B Protein X R

O N N

R′

H N R′

Methylene Glycol H

+ NH2 H O

OH+ OH

Protein Y O

R″ N N

R″″

H N

O R″′

O

R′ HN

Fixed protein aggregate R″″ R″ O H N N R″′ O HN

NH

R″

O R

Figure 10.2. Aldehyde fixation.

the extinguished laser, another laser is exciting the fluorophore in question, which may be emitting light at several wavelengths, triggering a photodetector (not necessarily the intended one). Turning down any other active laser may solve this problem. 10.3.1 Aldehyde Fixation Formaldehyde is a gas that is soluble in aqueous media (the solution then being formalin) and can be used to crosslink proteins, resulting in the fixation of proteinaceous tissue [20]. Once in solution, formaldehyde exists in equilibrium with methylene glycol (Figure 10.2) [20]. As a starting material, paraformaldehyde is commonly used and is the polymeric form of formaldehyde, shown diagrammatically in Figure 10.2. However, to be used as a fixative, paraformaldehyde needs to be reduced into its monomeric components [20]. Formaldehyde solutions are typically prepared on the day of use [11, 16, 54]. Usually, for cells grown on a cover-slip, a 20-min fixation time using 2% (w/v) formaldehyde solution is adequate. If a 4% (w/v) formaldehyde solution is employed, the fixation time may be reduced to 2 min. Both fixation times are representative of fixation being performed at room temperature [11, 16, 54]. Following fixation, washing with PBS and quenching with 50-mM glycine removes or neutralizes excess unreacted aldehyde. It is common practice to perform the detergent extraction and quenching step simultaneously by adding 0.02% (v/v) Triton X-100 to the glycine solution. This will disorder cell membranes, allowing the penetration of macromolecules, such as antibodies, into the interior of the cell. Typically, a quenching and permeabilization time of 20 min at room temperature is not uncommon.

c10.indd 187

7/30/2010 2:19:04 PM

188

TRACKING INTRACELLULAR POLYMER LOCALIZATION

10.3.2 Solvent Fixation Methanol cooled to −20 °C can be used to effect fixation, as this treatment will precipitate proteins and extract lipid from the cell. A variety of other solvents may be used and will also negate the need for a detergent extraction step as the lipidic portion of cell membranes will be dissolved and effectively discarded. Typically 5 min at −20 °C is adequate to achieve fixation. As methanol is volatile, it is important not to allow the samples to dry out. Consequently, it is a good idea to add PBS to the cold methanol after fixation and to act as soon as possible once the samples have been removed from the freezer. Rapid washing with an additional three charges of PBS will normally remove residual methanol [11, 16, 54].

10.3.3 Pre-fixation Methodologies Immunoreactive cytosolic pools of peripheral membrane proteins can mask a membrane-bound pool, obscuring the labeling of a target compartment. In this instance, cytosolic extraction may be desirable, in order to remove the immunoreactive pool of protein prior to fixation. There are several ways of achieving this end. Extraction with saponin prior to fixation removes membrane-bound cholesterol, effectively leaving pores in the cell membrane through which ions and proteins may escape or be washed out [11]. If this step is to be used “prealdehyde fixation,” it is vital to reduce the time of the subsequent detergent extraction step (author uses 5 min). The use of this agent prefixation may (like leupeptin) slightly alter vesicular morphology, giving an expanded “doughnutlike” appearance [11]. Streptolysin O (SLO) is a bacterial toxin that also can be used to generate pores in the plasma membrane. Typically, it is introduced to the cell at 0 °C in the presence of calcium ions. Once the calcium ions are chelated using ethylene glycol tetraacetic acid (EGTA), the SLO will form pores large enough for proteins to escape the intracellular environment while keeping large intracellular supramolecular assemblies inside the cell [55]. The use of some lipidic transfection reagents can, at times of up to 48 h post-transfection, significantly alter the distribution of intracellular markers such as the LAMP proteins, independently of the transgene being expressed. Further cellular morphology can also be altered, revealing a highly vacuolated phenotype. Care should be taken when using transient transfection and lipidic vectors that any observed effects are not attributable to the transfection vehicle. With the careful selection of transfection vehicle and the application of positive and negative controls, it is possible to control for any transfection vector induced artifacts. As has been previously mentioned [11, 16], leupeptin is a useful and necessary tool when using fluorophore-labeled biodegradable markers for temporally dissecting the endocytic pathway. However, like saponin, leu-

c10.indd 188

7/30/2010 2:19:04 PM

REFERENCES

189

peptin may also induce artifacts in the form of expanded vesicular structures [11, 16].

10.4

SAMPLE PREPARATION FOR POORLY FIXABLE MATERIAL

One solution to the “washing out” of a poorly fixed, soluble, drug delivery system (i.e., Oregon Green-labeled poly(ethylene glycol), Dextrin, or N-(2 hydroxypropyl methacrylamide) has recently been published by the author. Central to this methodology is the use and characterization of internalized markers such as fluorophore-labeled BSA (i.e., TxR-BSA) or TxR-WGA as previously discussed (Section 10.2.5). The kinetics of fluorophore-labeled BSA (fBSA) movement through early and late endocytic structures can be delineated and documented using standard fixation methodologies in conjunction with immunolabeling and may be viewed as being relatively constant under similar growth conditions. Once the temporal location of the fBSA is known, fBSA may be coincubated with a soluble, synthetic drug delivery system (DDS) (labeled with a second, nonspectrally overlapping fluorophore). By virtue of any colocalization between the DDS (an unknown quantity), and the precharacterized endocytic marker (a known quantity), the intracellular kinetics and localization of the DDS may be identified. Furthermore, this may be achieved without the need for a permeabilization step, negating the opportunities for poorly fixed or nonfixed material (i.e., the DDS) to move, distorting the data set obtained. As antibodies have already been used to characterize the localization of fBSA over time, they can be dispensed with during the characterization of the DDS. Consequently, it is also possible to utilize this methodology to document DDS localization in live or fixed cells relative to fBSA, which has already been characterized using immunofluorescent subcellular markers such as EEA1, LAMP, and GM130 [11]. This technique assumes that the unknown quantity will not alter the distribution of the marker (fBSA) and this assumption needs to be controlled for. Particular care needs to be taken regarding the osmolarity of the buffers used. PBS, in the hands of the author, has worked well.

REFERENCES 1. Gruenberg, J. The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2(10): 721–730 (2001). 2. Wickner, W. and Schekman, R. Membrane fusion. Nat. Struct. Mol. Biol. 15(7): 658–664 (2008). 3. Caswell, P. and Norman, J. Endocytic transport of integrins during cell migration and invasion. Trends Cell Biol. 18(6): 257–263 (2008). 4. Benes, P., Vetvicka, V., and Fusek, M. Cathepsin D—many functions of one aspartic protease. Crit. Rev. Oncol. Hematol. 68(1): 12–28 (2008).

c10.indd 189

7/30/2010 2:19:04 PM

190

TRACKING INTRACELLULAR POLYMER LOCALIZATION

5. Sorkin, A. and Goh, L. K. Endocytosis and intracellular trafficking of ErbBs. Exp. Cell Res. 315(4): 683–696 (2009). 6. Subramaniam, V. N., Summerville, L., and Wallace, D. F. Molecular and cellular characterization of transferrin receptor 2. Cell Biochem. Biophys. 36(2–3): 235–239 (2002). 7. John, T., Stephen, T. A., Vogel, M., Tiruppathi, C., Malik, A. B., and Minshall, R. D. Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer. Am. J. Physiol. Lung Cell Mol. Physiol. 284: L187–L196 (2003). 8. Duncan, R. Polymer–drug conjugates as anticancer nanomedicines. Nat. Rev. Drug Discov. 6: 688–701 (2006). 9. Tarragó-Trani, M. T. and Storrie, B. Alternate routes for drug delivery to the cell interior: pathways to the Golgi apparatus and endoplasmic reticulum. Adv. Drug Deliv. Rev. 59(8): 782–797 (2007). 10. Medina-Kauwe, L. K. “Alternative” endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery? Adv. Drug. Deliv. Rev. 59(8): 798–809 (2007). 11. Richardson, S. C., Wallom, K. L., Ferguson, E. L., Deacon, S. P., Davies, M. W., Powell, A. J., Piper, R.C., and Duncan, R. The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery. J Control. Release 127(1): 1–11 (2008). 12. Sandvig, K. and van Deurs, B. Delivery into cells: lessons learned from plant and bacterial toxins. Gene Ther. 12(11): 865–872 (2005). 13. Sorkin, A. The endocytosis machinery. J. Cell Sci. 113(24): 4375–4376 (2000). 14. Wendler, F. and Tooze, S. Syntaxin 6: the promiscuous behaviour of a SNARE protein. Traffic 2: 606–611 (2001). 15. Wade, N., Bryant, N. J., Connolly, L. M., Simpson, R. J., Luzio, J. P., Piper, R. C., and James, D. E. Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP) 8, and VAMP7 in b16 melanoma cells. J. Biol. Chem. 276(23): 19820–19827 (2001). 16. Richardson, S. C., Winistorfer, S. C., Poupon, V., Luzio, J. P., and Piper, R. C. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol. Biol. Cell 15(3): 1197–1210 (2004). 17. Mullock, B. M., Smith, C. W., Ihrke, G., Bright, N. A., Lindsay, M., Parkinson, E. J., Brooks, D. A., Parton, R. G., James, D. E., Luzio, J. P., and Piper, R. C. Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and is required for late endosome-lysosome fusion. Mol. Biol. Cell 11(9): 3137–3153 (2000). 18. Zerial, M. and McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2: 107–119 (2001). 19. Pfeffer, S. and Aivazian, D. Targeting Rab GTPases to distinct membrane compartments. Nat. Rev. Mol. Cell Biol. 5(11): 886–896 (2004). 20. Melan, M. A. Overview of cell fixation and permeabilisation. Methods Mol. Biol. 34: 55–66 (1994).

c10.indd 190

7/30/2010 2:19:04 PM

REFERENCES

191

21. Ramos-Vara, J. A. Technical aspects of immunohistochemistry. Vet. Pathol. 42(4): 405–426 (2005). 22. Forbes, B. and Ehrhardt, C. Human respiratory epithelial cell culture for drug delivery applications. Eur. J. Pharm. Biopharm. 60(2): 193–205 (2005). 23. Abbott, N. J. Dynamics of CNS barriers: evolution, differentiation, and modulation. Cell Mol. Neurobiol. 25(1): 5–23 (2005). 24. Wright, C. S. Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II. J. Mol. Biol. 178(1): 91–104 (1984). 25. Roberts, R. L., Barbieri, K. M., Pryse, K. M., Chua, M., Morisaki, J. H., and Stahl, P. D. Endosome fusion in living cells overexpressing GFP-rab5. J. Cell Sci. 112: 3667–3675 (1999). 26. Mills, I. G., Urbe, S., and Clague, M. J. Relationships between EEA1 binding partners and their role in endosome fusion. J. Cell Sci. 114: 1959–1965 (2001). 27. McBride, H. M., Rybin, V., Murphy, C., Giner, A., Teasdale, R., and Zerial, M. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and Syn 13. Cell 98: 377–386 (1999). 28. Bishop, N. E. Dynamics of endosomal sorting. Int Rev Cytol. 232: 1–57 (2003). 29. Luzio, J. P., Bright, N. A., and Pryor, P. R. The role of calcium and other ions in sorting and delivery in the late endocytic pathway. Biochem. Soc. Trans. 35(5): 1088–1091 (2007). 30. Bonifacino, J. S. and Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72: 395–447 (2003). 31. Sollner, T. H. Regulated exocytosis and SNARE function. Mol. Membr. Biol. 20: 209–220 (2003). 32. Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T. H., and Rothman, J. E. SNAREpins: minimal machinery for membrane fusion. Cell 92(6): 759–772 (1998). 33. Barbieri, M. A., Li, G., Mayorga, L. S., and Stahl, P. D. Characterization of Rab5:Q79L-stimulated endosome fusion. Arch. Biochem. Biophys. 326(1): 64–72 (1996). 34. Rink, J., Ghigo, E., Kalaidzidis, Y., and Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122(5): 735–749 (2005). 35. Macedo, M. F. and de Sousa, M. Transferrin and the transferrin receptor: of magic bullets and other concerns. Inflamm. Allergy Drug Targets 7(1): 41–52 (2008). 36. Savina, A., Fader, C. M., Damiani, M. T., and Colombo, M. I. Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner. Traffic 6(2): 131–143 (2005). 37. Gu, F. and Gruenberg, J. Biogenesis of transport intermediates in the endocytic pathway. FEBS Lett. 452(1–2): 61–66 (1999). 38. Guarnieri, F. G., Arterburn, L. M., Penno, M. B., Cha, Y., and August, J. T. The motif Tyr-X-X-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1. J. Biol. Chem. 268(3): 1941–1946 (1993). 39. Eskelinen, E. L. Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol. Aspects Med. 27(5–6): 495–502 (2006). 40. Pryor, P. R. and Luzio, J. P. Delivery of endocytosed membrane proteins to the lysosome. Biochim. Biophys. Acta. 1793(4): 615–624 (2009).

c10.indd 191

7/30/2010 2:19:04 PM

192

TRACKING INTRACELLULAR POLYMER LOCALIZATION

41. Gary-Bobo, M., Nirdé, P., Jeanjean, A., Morère, A., and Garcia, M. Mannose 6-phosphate receptor targeting and its applications in human diseases. Curr. Med. Chem. 14(28): 2945–2953 (2007). 42. Reczek, D., Schwake, M., Schroder, J., Hughes, H., Blanz, J., Jin, X., Brondyk, W., Van Patten, S., Edmunds, T., and Saftig, P. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of β-glucocerebrosidase. Cell 131(4): 770–783 (2007). 43. Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R., and Luzio, J. P. Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent. J. Cell Biol. 140(3): 591–601 (1998). 44. Piper, R. C. and Luzio, J. P. CUPpling calcium to lysosomal biogenesis. Trends Cell Biol. 14(9): 471–473 (2004). 45. Bright, N. A., Reaves, B. J., Mullock, B. M., and Luzio, J. P. Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles. J. Cell Sci. 110: 2027–2040 (1997). 46. Chu, Y., Piper, R., Richardson, S., Watanabe, Y., Patel, P., and Heistad, D. D. Endocytosis of extracellular superoxide dismutase into endothelial cells: role of the heparin-binding domain. Arterioscler. Thromb. Vasc. Biol. 26(9): 1985–1990 (2006). 47. Marshansky, V. The V-ATPase α2-subunit as a putative endosomal pH-sensor. Biochem. Soc. Trans. 35(5): 1092–1099 (2007). 48. Invitrogen product sheet. LysoTracker® and LysoSensor™ Probes. Available at http://probes.invitrogen.com/media/pis/mp07525.pdf. 49. Jackson, C. L. Mechanisms of transport through the Golgi complex. J. Cell Sci. 122(4): 443–452 (2009). 50. Banting, G. and Ponnambalam S. TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology. Biochim. Biophys. Acta. 1355 (3): 209–217 (1997). 51. Rosing, M., Ossendorf, E., Rak, A., and Barnekow, A. Giantin interacts with both the small GTPase Rab6 and Rab1. Exp. Cell Res. 313(11): 2318–2325 (2007). 52. Roth, J., Yam, G. H., Fan, J., Hirano, K., Gaplovska-Kysela, K., Le Fourn, V., Guhl, B., Santimaria, R., Torossi, T., Ziak, M., and Zuber, C. Protein quality control: the who’s who, the where’s and therapeutic escapes. Histochem. Cell Biol. 129(2): 163–177 (2008). 53. Ni, M. and Lee, A. S. ER chaperones in mammalian development and human diseases. FEBS Lett. 581(19): 3641–3651 (2007). 54. Pryor, P. R., Mullock, B. M., Bright, N. A., Lindsay, M. R., Gray, S. R., Richardson, S. C. W., Stewart, A., James, D. E., Piper, R. C., and Luzio, J. P. Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events. EMBO Rep. 5(6): 590–595 (2004). 55. Campbell, M. A., Kessler, P. D., and Fambrough, D. M. The alternative carboxyl termini of avian cardiac and brain: sarcoplasmic reticulum/endoplasmic reticulum Ca2+-ATPases are on opposite sides of the membrane. J. Biol. Chem. 267(13): 9321–9325 (1992).

c10.indd 192

7/30/2010 2:19:04 PM

CHAPTER 11

Can QSAR Models Describing SmallMolecule Xenobiotics Give Useful Tips for Predicting Uptake and Localization of Nanoparticles in Living Cells? And If Not, Why Not? RICHARD W. HOROBIN Division of Integrated Biology, FBLS, The University of Glasgow, Glasgow, Scotland, United Kingdom

11.1

QUESTIONS AND THEIR CONTEXT

Nanoparticles come with a wide variety of composition and form, and possess a wide variety of surface features. When considering their biomedical applications, cellular uptake and localization is almost always of significance, whether as drug delivery agents or as biosensors. Consequently, being able to predict these behaviors from a knowledge of the physicochemical properties of nanoparticles would be very advantageous. And yet, apart from the finetuning of endocytosis (for some recent work see Hild et al. [1], Rejman et al. [2], and Sánchez-Martín et al. [3]), this is not deemed possible—and moreover what is known is rarely spelled out in any summary and explicit way. But have we been too pessimistic? The question of whether intracellular localization of nanoparticles can be predicted is worth asking again, since intracellular uptake and localization in many organelles can now be predicted for small-molecule species. This has been achieved using both first-principles and quantitative structure–activity Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

193

c11.indd 193

7/30/2010 2:19:07 PM

194

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

(QSAR) models. While, as discussed below, it was unlikely that such models would apply in detail to nanoparticles, nevertheless can such approaches at least give us tips for predicting the localization of such species?

11.2

SOME BACKGROUND: THE MEANING OF “NANOPARTICLES”

There are many radically different types of nanoparticles—including aggregates of hydrophobic organics such as hypericin or porphyrins; liposomes; modified proteins (e.g., albumin and gelatin); metal crystallites such as iron and gold; crystallites of iron oxide, silica, or titania; quantum dots; polymers; and dendrimers. The notion that such particles have features in common, other that is than size, is perhaps odd but is nevertheless a widely implicit assumption of “nanoscience.” In any event, nanoparticles certainly do differ radically from “small molecules.” Thus sheer size plus surface (i.e., not overall) properties may be controlling factors in the interactions of nanoparticles with living systems. Perhaps nanoparticles should not be regarded as one end of a spectrum that has small molecules at the other end; rather, they are on a different distribution altogether. Another, unavoidable, issue raised by asking “What are nanoparticles?” is that of nomenclature and definition. Defining a nanoparticle is somewhat arbitrary, one widely held position merely being that it is a particle falling between 10 and 100 nm in “diameter.” But this lacks precision and significance, both physicochemical and biological. Indeed, such a definition should be seen more as a staking out of academic territory rather than as entailing boundaries emerging from the biological and physical worlds. So while “diatom nanotechnology” [4] exceeds such a range, certain “nanocrystal lumiphores” [5] fall below it. And do we have to ignore possible data from “colloid chemistry” or indeed from “micro” particles? Indeed we do not, since the operational conclusion of researchers as shown by journal terminology is clearly less restrictive, with illuminating comparisons emerging, for instance, from comparisons of particles ranging from less than 10 nm up to more than 1000 nm. Also, beyond mere size, the effects of the amazing variety of entities we are dealing with, noted above, is significant—and yet all these variations are on occasion shoehorned into the “nano” category.

11.3 PREDICTING UPTAKE AND LOCALIZATION OF SMALLMOLECULE XENOBIOTICS USING QSAR MODELS For small-molecule xenobiotics (typically < 1000 daltons) it has been found that rules can be specified connecting the physicochemical character of the xenobiotics with their uptake and localization in live cells of many types. Two quite different and workable strategies have been investigated. First is a

c11.indd 194

7/30/2010 2:19:07 PM

PREDICTING UPTAKE AND LOCALIZATION OF SMALL-MOLECULE XENOBIOTICS

195

physicochemically first-principles approach, where interaction of small xenobiotics with a simplified model cell is described by utilizing the Henderson– Hasselbach and Nerst–Planck equations, Fick’s law of diffusion, and a hydrophil–lipophil partition constant [6, 7]. Although this approach is currently limited in the number of organelles that can be modeled, it has being applied successfully in several contexts of biological significance, for example, soil–water [8] and water–fish [9] partitioning of ionizable organic chemicals. The other strategy involves correlational QSAR models, formally expressed as dichotomous decision rules. The limits of this approach are different, namely, having the correct set of exemplar xenobiotics to accurately specify the models. Again, this has achieved application over a wide range of biological systems, from microorganisms to whole plants. However, neither of these approaches is universally applicable at this time, as shown in a recent comparative study [10]. Intriguingly, both strategies are based on overall molecular properties, suggesting that the widely adopted “tag” or “motif” approach is not in actuality the only game in town. In this chapter it is the second strategy, based on QSAR decision rules, that is utilized. The relevant QSAR decision rules relate the uptake of xenobiotics (e.g., drugs and fluorescent probes) into living cells to the structure of the xenobiotics as described numerically. These correlations were established following direct microscopic observation of the interaction of a wide range of fluorescent compounds with live cells. The strategy requires the numerical description of the structure of xenobiotics in terms of a few key physicochemical properties. Thus electric charge (Z) is specified directly, and acid/base strength described in terms of pKa values. Overall size is modeled using the relative molecular mass (i.e., molecular weight, MW). Overall size of the conjugated, usually substantially aromatic, system is modeled using the conjugated bond number (CBN) parameter and that of the largest planar conjugated region by the largest conjugated fragment (LCF) parameter. Lipophilicity/hydrophilicity is modeled using the logarithm of the octanol–water partition coefficient (log P), and amphilicity by the amphiphilicity index (AI). For information concerning the derivation, application, and limits of these parameters see Horobin [11] and Christensen et al. [12]. QSAR decision rule models are now available to predict the occurrence, or nonoccurrence, of a wide variety of processes: namely, membrane permeability, adhesion to the cell surface, exclusion from a cell’s interior, and passage through internal cellular barriers. Most of these processes can occur by more than one mechanism, each of which requires a separate model, as discussed in Horobin [11] and Stockert et al. [13]. A summary of the available QSAR decision rule models is provided in Figure 11.1. QSAR decision rule models are also available to predict the accumulation of xenobiotics in a variety of cellular regions and organelles. To date these include generic biomembranes [11] as well as those of the endoplasmic reticulum [14], Golgi apparatus [15], and plasma membrane [11]; the cytosol [16];

c11.indd 195

7/30/2010 2:19:07 PM

196 Endocytosis

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

Membrane permeability, of plasmalemma & others

Fluid phase (pinocytosis)

Adsorptive

Lysosome or other acidic organelle Passage through gap junction or plasmadesmata Phagosome

Phagocytosis

nucleus No binding to, or passage through, plasma membrane Passage through nuclear pore

Figure 11.1. Relationships between chemical properties of small-molecule xenobiotics (e.g., drugs or fluorescent probes) and their cellular uptake and intracellular transfer behavior in live cells. Structural requirements for occurrence of the arrowed processes are summarized below; see text for explanation of terms. Adsorptive endocytosis requires AI or log P > 8 or CBN > 40. Fluid-phase endocytosis requires that if CBN < 40 then log P < 0 or if CBN > 40, then log P < −10. Membrane permeability, plasmalemma, and others require that 8 > AI or log P > 0; CBN < 40. No binding to cells requires that if pKa << 7 or >>7 and CBN < 40, then log P < 0; or if pKa << 7 or >>7 and CBN > 40, then log P < −10; or if pKa ∼ 7 and CBN < 40, then log P (most ionized species) <−5. Note that uptake into cells is still possible if pinocytosis occurs. Passage through gap junctions or plasmadesmata requires that MW < 1000 (gap junction) or MW < 700 (plasmadesmata). Passage through nuclear pores requires that MW < 5000. Phagocytosis requires particles with a “diameter” greater than ∼500 nm.

lysosomes and other acidic structures [17]; mitochondria [18, 19]; nuclear chromatin [11, 20]; and nucleoli and ribosomes (Rashid-Doubell, Stockert, and Horobin, unpublished findings). Again, there are often multiple accumulation mechanisms for each organelle, each with a separate QSAR model. A summary of the available decision rules is given in Figure 11.2. There are various limitations of such models. For instance, some needed structure parameters may be unavailable. Examples of such problems are the lack of log P fragment values for key moieties, and the lack of micro pKa values for certain polybasic compounds. Moreover, these QSAR models, being based on simple physicochemical effects, will not cope with the effects of sophisticated targeting systems, for example, the involvement of antibody–antigen, enzyme–substrate, or peptide–receptor interactions, or of nucleic acid complementarity. This is a radical limitation, given that such targeting strategies are often used with nanoparticle delivery pharmaceuticals.

c11.indd 196

7/30/2010 2:19:07 PM

PREDICTING UPTAKE AND LOCALIZATION OF SMALL-MOLECULE XENOBIOTICS

197

Membrane bound

Lipid domains Protein domains

Plasma membrane

Precipitation trapping of weak acids

Potential driven &/or cardiolipin complexation

Ion-trapping of weak bases

Ion-trapping of weak acids

Mitochondria

Lysosomes & acidic organelles

Fluid lipid domains, e.g., apoptotic cells

Outer, fluid, membrane

Phagosome Endoplasmic reticulum membrane

Generic biomembranes Ribosomal RNA

Golgi membrane

Nucleolus, nucleic acids Fat droplet

Cytosol Histones

Nuclear chromatin DNA

Figure 11.2. Relationships between chemical properties of small-molecule xenobiotics (e.g., drugs or dyes) and their patterns of localization in live cells. Structural requirements for localization in the named organelles and other sites are summarized below; see text for explanation of terms. Cytosol requires AI < 3.5; CBN < 40; 5 > log P > 0; Z = 0. Endoplasmic reticulum membranes require that 6 > AI > 3.5; 6 > log P > 0; Z > 0. Fat droplets require that AI < 3.5; HGS < ∼20; log P > 5.0; pKa (if relevant) < 6. Generic biomembranes, for reversible uptake, require that 8 > AI or log P > 5. Golgi membranes require that 5 > log P > 3. Lysosomes/acidic organelles, weak base ion-trapping requires that 0 > log P (cation) > −5; 10 > pKa > 6; Z > 0. Lysosomes/acidic organelles, weak acid precipitation trapping requires that log P (free acid) > 0; pKa = 7 ± 3; Z(most ionized species) < 0. Lysosomes and endosomes, membranes, lipid domains require that AI or log P > 8. Lysosomes and endosomes, membranes, protein domains require that CBN > 40. Mitochrondria, potential driven and/or cardiolipid complexation require that 5 > log P > 0; pKa > 12; Z > 0. Mitochrondria, ion trapping of weak acids requires that 5 > log P (least ionized species) > 0; pKa = 7 ± 3; Z(most ionized species) < 0. Mitochondria, outer membrane, require that 6 > AI > 3.5; log P > 0; Z > 0. Nuclear DNA, chromatin requires that LCF > 24; −2 > log P(cation) > 0; pKa > 10; Z(ionized species) > 0. Nuclear histones require that if Z(most ionized species) < 0, and CBN > 40, then log P > −10; or if Z < 0 and CBN > 16, then 8 > log P(anion) > 0. Nucleoli, nucleic acids require that LCF > 17; 8 > log P(least ionized species) > 0; pKa > 10; Z > 0. Phagosomes require particles with a “diameter” greater than ∼500 nm. Plasma membrane uptake, fluid lipid domains (e.g., in apoptotic cells) require that ∼5.5 > AI > 3.5; log P < 5.0; HGS > 400. Plasma membrane uptake, lipid domains require that AI or log P > 8 or AI > 5; HGS > 400. Plasma membrane uptake, protein domains require that log P < −10; CBN > 40. Ribosomal RNA requires that LCF > 17; 8 > log P(least ionized species) > 0; pKa > 10; Z > 0.

c11.indd 197

7/30/2010 2:19:08 PM

198

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

11.4 MORE BACKGROUND: WHAT FACTORS CONTROL UPTAKE AND INTRACELLULAR LOCALIZATION OF NANOPARTICLES? First, a disclaimer: this chapter, as acknowledged above, does not discuss the major approaches to the targeting of nanoparticles adopted to date. Most investigators have been focused on the development and application of “smart targeting” mediated by various entities. Thus a recent review of the cellular and intracellular targeting of quantum dots gave considerable attention to work involving targeting by antibodies, by ligands for cell-surface receptors such as folate, and by peptide ligands for various tumor cell markers, with some attention also directed at ligands binding to receptors for angiotensin and neurotransmitters [1]. Other interactions of nanoparticles and cells were discussed under the heading of “nonspecific particle uptake,” which is precisely the type of phenomenon we will be considering in the present chapter. Given the focus on smart targeting, it is perhaps not surprising that there is a paucity of rigorous investigations into such “nonspecific” mechanisms of uptake and localization of nanoparticles within living cells, not only because of the issues noted above, but also because preparation of novel nanoparticles is within the technical capabilities of many chemical and related laboratories. Indeed, a wait-and-see approach to such a complex problem as nanoparticle uptake and targeting may be a valid, if not the only, way to successfully solve pressing problems of drug delivery and toxicology. However, we will now briefly review some of the available rigorous studies, first concerning uptake into cells, and then the post-uptake localization within cells. It is immediately apparent, and of course no surprise, that the most dramatic particle-associated variable influencing cellular uptake is the size of the nanoparticle. Of course, nanoparticles are (depending on definitions, see above) very large objects to pass through the plasma membrane in the same manner that is easily possible for small molecules entering a cell by passive diffusion. Instead by far the most common mode of entry is by endocytosis, whether the particles are lipoplexes [21], liposomes [22], quantum dots [23], or some other variety. At the extreme, it was observed as long ago as the early twentieth century—when India ink was used as the probe—that particles exceeding approximately 0.5 μm in diameter were extensively taken up by phagocytes [24]; for more modern accounts see Besterman and Low [25] and Haas [26]. However, for “true” nanoparticles, a discriminating and critical investigation has been carried out more recently by Hoekstra’s group [2]. This involved exposing nonphagocytic eukaryotic cells to fluorescent latex beads ranging in size from 50 to 1000 nm. All except the largest (1000-nm) particles were taken up, by energy-dependent processes. In the size range up to 200 nm this involved a clathrin-mediated process, with the particles finally arriving in late endosomal/lysosomal compartments. In the larger size range, above 200 nm to below 1000 nm, uptake involved a caveolae-mediated process, with the final destination not being lysosomal. This latter process was distinct from

c11.indd 198

7/30/2010 2:19:08 PM

MORE BACKGROUND: WHAT FACTORS CONTROL UPTAKE

199

macropinocytosis, which can also favor larger particles [27]. These size criteria have been found applicable to a variety of cell types (e.g., epithelial [28] and fibroblast [29]) and to diverse types of particle (e.g., lipoplexes [30] and porphyrin aggregates [31]. Endocytic uptake, however, is modulated by other factors. One such is a particle’s surface electric charge. Thus endocytotic uptake of nanoparticles of very widely varying types is often facilitated by a positive surface charge, as reviewed, for instance, by Blau et al. [32]. This is generally considered to be due to an initial adsorption to surface negative charges, usually on the plasma membrane. Indeed, this does often result in an observed increase (short or long term) of the surface adhesion of the nanoparticles at that stage [33]. However, a positive surface charge does not always result in entry into the cell, since if a cell has an external coating of an anionic glygosaminoglycan, for instance, then a cationic nanoparticle can be trapped in that layer [34]. Another surface property dramatically modifying endocytic uptake is the lipophilicity or hydrophilicity of the particle’s surface. In vivo, it was observed many years ago that after intravenous administration nanoparticles were rapidly removed from the circulation as a result of being phagocytosed by macrophages, especially in bone marrow, liver, lungs, and spleen [35]. It was shown that this was largely due to adsorption of proteins onto the particle surface resulting in opsonization [36]. In keeping with this, occluding the hydrophobic surface with hydrophilic moieties reduces protein binding and consequently phagocytic endocytosis, as demonstrated quantitatively by Chang’s group [37]. Hydrophilic materials as varied as polyethylene glycol [38] and polysaccharides [39] have been used in this way. Of course, this reduction of competition from macrophages enhances uptake into other cell types by other endocytic mechanisms. Before moving on to consider the intracellular localization of nanoparticles, it should be noted that their entry into cells is not entirely restricted to endocytosis. For instance, membrane fusion can also enable internalization, albeit largely that of liposomes. However, even for those structures it is not the most common mechanism (although it has given rise to some wonderful electron micrographs; e.g., see Dini et al. [40], Figure 5d). Crucially, membrane fusion can result in direct delivery of hydrophilic materials into the cytosol [41]. In addition, some nanoparticles do cross the plasma membrane. Least surprising is to see this occuring with small (∼1 nm) fullerene derivatives, which include lipophilic species [42, 43]. More surprisingly, it has also been reported for particles with diameters above 10 nm. Thus Jablonski et al. [44] have developed a system involving coating the particle with a cationic species with a subsequent application of a hydrophobic counterion, pyrenebutyrate, in the published example. This is not a permeabilization process, and the negative surface charge of the particle means that the plasma membrane potential strongly influences uptake. Another process involves coating quantum dots with amphiphilic polymers [45].

c11.indd 199

7/30/2010 2:19:08 PM

200

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

Post-uptake intracellular localization of nanoparticles will now be considered. As described above, particle size strongly influences which particular endocytotic mechanism is involved. Consequently, particle size substantially controls whether the nanoparticle will end up in a lysosome or an acidified late endosome, or instead in a nonacidified vesicle. Once again, however, a particle’s electric charge modulates this: for instance, in directing down which intracellular pathway the nanoparticles travel. A recently reported example involves the application of various anionic and cationic derivatives of poly(ethylene glycol)-d,l-polylactide nanoparticles, all with diameters between 50 and 100 nm, to cultured epithelial cells. Most were taken up via the clathrin pathway. However, whereas many anionic particles transited through the lysosomal route, cationic particles typically did not but instead diverted to the lateral plasma membrane [46]. Ionization can also play a role in escape from endosomes and related vesicles. For instance, weak bases such as protonsponge dendrimers can become ionized within acidified vesicles, thus dramatically raising the local osmotic pressure, favoring vesicle rupture [47]. Amphilicity, and the molecular features influencing this property, also has an impact on membrane rupture and so on escape of particles from endosomes. For instance, nanoparticles will escape more readily if their surfaces carry amphiphils with surfactant properties. In particular, this effect is influencial in lipoplexes, where chain length effects have been discussed using the structure parameter AI [48]. A related, but distinct, property influencing membrane rupture and fusion is lipid shape. In this case it is membrane curvature that is the key property. This is controlled both by the relative sizes of the head group and hydrocarbon tail, and by the cross-sectional area of the head group; for an accessible account of which Israelachvili’s [49] formulation is still to be recommended.

11.5 SO CAN SMALL-MOLECULE QSAR MODELS TELL US ANYTHING ABOUT NANOPARTICLE UPTAKE AND LOCALIZATION? As discussed above, the factors found to be of significance do include those considered in the small-molecule models—namely, amphilicity, electric charge, head group size, lipophilicity, and pKa. However, it is hard to see how the small-molecule models can be applied directly to such vast structures as nanoparticles. So is that all? Are there are no regularities to which the smallmolecule QSAR models can be applied, or even any useful tips? It does seem that there are two distinct ways in which numerical structure–localization rules may be usefully applied to nanoparticle uptake into cells. First, consider nanostructures at the lower end of the size range, such as C60 fullerene derivatives. Such, if lipophilic, do penetrate the plasma membrane directly rather than by endocytosis. In one study, a dicarboxylic C60 fullerene was found to localize in biomembranes including the plasma mem-

c11.indd 200

7/30/2010 2:19:08 PM

SIMPLE NUMERICAL SUMMARIES OF NANOPARTICLE UPTAKE

201

brane and, most markedly, in mitochondria [43]. This fullerene is small enough for structure parameters to be estimated, and it was found to be a lipophilic weak acid, with some species being extremely lipophilic. Plugging the parameters into the relevant QSAR models, accumulation of the weak acid by iontrapping in mitochondria, and more general partitioning of the strongly lipophilic species into membranes, with superlipophilic species favoring the plasma membrane, matched observation. However, when a hexacarboxylic acid C60 fullerene was investigated it was observed to still accumulate in mitochondria, with cytoskeletal keratin fiber binding also occuring [42]. Again, estimation of structure parameters and application of the QSAR models gave congruent predictions, again of lipophilic weak acid ion trapping and, in this case, protein binding by the hydrophilic carboxylate anions. Morover, some larger nanoparticles do behave somewhat as suggested by the small-molecule models. Examples are provided by mitochondrial targeting of nanoparticles with lipophilic and cationic surfaces, noting that small molecules with lipophilic and cationic character are typically mitochondrially targeted. One such instance involved DQAsomes derived from dequalinium and its derivatives in intact cultured cells [16]. Another involved linking triphenylphosphonium to hydroxypropylmethacrylamide polymers [50], which nanoparticles appeared to show localization to mitochondria, but only with the smallest (<5 kDa) species, as larger particles were restricted to the lysosomal compartment. Such observations suggest that with the smallest nanoparticles available QSAR small-molecule models do have some direct applicability; and even with larger particles the models, although not rigorously applicable, can give useful tips. Perhaps further experimental studies of nanosystems of this type would have predictive value. In particular, it would be useful to know whether the loss of predictability with increase in size is merely a methodological limit of the modeling system or also involves physicochemical limits.

11.6 SIMPLE NUMERICAL SUMMARIES OF NANOPARTICLE UPTAKE AND INTRACELLULAR LOCALIZATION Second, note the striking fact that authors discussing the various physicochemical influences on nanoparticle uptake and localization have been curiously reluctant to provide simple summaries of factors involved. This seems unduely reticent: whatever its limitations such a summary would certainly have some utility. Consequently, in Figure 11.3, in a more optimistic spirit, I offer a graphical summary of the data mentioned in this chapter. One final complication must, however, be pointed out at this point. Very dramatic cell-line effects can occur when nanoparticles interact with live cells— there are indeed “smart cells” as well as “smart targeting” of nanoparticles [51, 52].

c11.indd 201

7/30/2010 2:19:08 PM

202

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

Particle size effects

« 50nm

phagocytosis

200-500nm Macropinocytosis

Mit

caveolinmediated

Pha Lys

Ma

CyPr

50-200 nm BioM clathrin-mediated endocytosis

Uptake & localization show log P, pKa, Z effects

500-1000nm

Particle charge effects

End

Al & pKa dependent

Coat with cationic species

Coat with various hydrophils

Coat with cationic species, then a hydrophobic anion OR with an amphiphilic block copolymer

Tactics to enhance different types of cellular uptake

Figure 11.3. Graphical summary of physicochemical factors influencing cellular uptake and intracellular localization of nanoparticles. Targeted organelles abbreviated as follows: BioM, generic biological membranes; CyPr, cytoskeletal proteins; End, endosomes: Lys, lysosome; Ma, macropinosome; Mit, mitochondrium; Pha, phagosome.

ACKNOWLEDGMENT I wish to acknowledge Prof. I. McGrath, Division of Integrated Biology, FBLS, The University of Glasgow, Scotland, for provision of facilities.

REFERENCES 1. Hild, W. A., Breunig, M., and Goepferich, A. Quantum dots—nano-sized probes for the exploration of cellular and intracellular targeting. Eur. J. Pharm. Biopharm. 68: 152–168 (2008).

c11.indd 202

7/30/2010 2:19:08 PM

REFERENCES

203

2. Rejman, J., Oberle, V., Zuhorn, I. S., and Hoekstra, D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem. J. 377: 159–169 (2004). 3. Sánchez-Martín, R. M., Alexander, L., and Bradley, M. Multifunctialized biocompatible microspheres for sensing. Ann. N.Y. Acad. Sci. 1130: 207–217 (2008). 4. Gordon, R., Losic, D., Tiffany, M. A., Nagy, S. S., and Sterrenburg, F. A. The glass menagerie: diatoms for novel applications in nanotechnology. Trends Biotechnol. 27: 116–127 (2009). 5. Ma, N., Sargent, E. H., and Kelley, S. O. One-step, DNA-programmed growth of luminescent and biofunctionalized nanocrystals. Nat. Nanotechnol. 4: 121–125 (2009). 6. Trapp, S. and Horobin, R. W. A predictive model for the selective accumulation of chemicals in tumor cells. Eur. Biophys. J. 34: 959–966 (2005). 7. Trapp, S., Rosania, G., Horobin, R. W., and Kornhuber, J. Quantitative modeling of selective lysosomal targeting for drug design. Eur. J. Biol. 7: 1317–1328 (2008). 8. Franco, A. and Trapp, S. Estimation of soil–water partition coefficient normalized to organic carbon for ionizable organic chemicals. Environ. Toxicol. Chem. 27: 1995–2004 (2008). 9. Fu, W., Franco, A., and Trapp, S. Methods for estimating the bioconcentration factor of ionizable organic chemicals. Environ. Toxicol. Chem. 28(7): 1372–1379 (2009). 10. Horobin, R. W., Trapp, S., and Weissig, V. Mitochondriotropics: a review of their mode of action, and their applications for drug and DNA delivery to mammalian mitochondria. J. Control. Release 121: 125–136 (2007). 11. Horobin, R. W. Uptake, distribution, and accumulation of dyes and fluorescent probes within living cells. A structure–activity modelling approach. Adv. Colour Sci. Technol. 4: 101–107 (2001). 12. Christensen, H., Garton, N. J., Horobin, R. W., Minnikin, D. E., and Barer, M. R. Lipid domains of mycobacteria studied with fluorescent molecular probes. Mol. Microbiol. 31: 1561–1572 (1999). 13. Stockert, J. C., Can~ete, M., Juarranz, A., Villaneuva, A., Horobin, R. W., Borrell, J. I., Texidó, J., and Nonell, S. Porphycenes: facts and prospects in photodynamic therapy of cancer. Curr. Med. Chem. 14: 997–1026 (2007). 14. Colston, J., Horobin, R. W., Rashid-Doubell, F., Pediani, J., and Johal, K. K. Fluorescent probes for endoplasmic reticulum—Why are they selective? An experimental and qsar-modelling study. Biotechnic. Histochem. 78: 323–333 (2003). 15. Rashid-Doubell, F. and Horobin, R. W. Selection of fluorescent Golgi complex probes using structure–activity relationship models. In: P. Bach, C. H. Reynolds, J. M. Clark, J. Mottley, and P. L. Poole, eds. Biotechnology Applications of Microinjection, Microscopic Imaging and Fluorescence. Plenum Press, New York, 1993, pp. 73–78. 16. D’Souza, G. G. M., Cheng, S. M., Boddapati, S. V., Horobin, R. W., and Weissig, V. Nanocarrier–assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel. J. Drug Target. 6: 578–585 (2008).

c11.indd 203

7/30/2010 2:19:08 PM

204

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

17. Rashid, F., Horobin, R. W., and Williams, M. A. Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure–activity models. Histochem. J. 23: 450–459 (1991). 18. Rashid, F. and Horobin, R. W. Interaction of molecular probes with living cells and tissues. 2. A structure–activity analysis of mitochondrial staining by cationic probes, and a discussion of the synergistic nature of image-based and biochemical approaches. Histochemistry 94: 303–308 (1990). 19. Rashid, F. and Horobin, R. W. Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations, a proposed mechanism and some implications. J. Microsc. 163: 233–241 (1991). 20. Horobin, R. W., Stockert, J., and Rashid-Doubell, F. Fluorescent cationic probes for nuclei of living cells: Why are they selective? A quantitative structure–activity relations analysis. Histochem. Cell Biol. 126: 165–175 (2006). 21. Furuhata, M., Izumisawa, T., Kawakami, H., Toma, K., Hottori, Y., and Maitani, Y. Decaarginine-PEG-liposome enhanced transfection efficiency and function of arginine length and PEG. Int. J. Pharm. 371: 40–46 (2009). 22. Huang, S. K., Lee, K. D., Hong, K., Friend, D. S., and Papahadjopoulos, D. Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice. Cancer Res. 52: 5135–5143 (1992). 23. Luccardini, C., Yakovlev, A., Gaillard, S., van’t Hoff, M., Alberola, A. P., Mallet, J. M., Parak, W. J., Feltz, A., and Oheim, M. Getting across the plasma membrane and beyond: intracellular uses of colloidal semiconductor nanocrystals. J. Biomed. Biotechnol. 2007: 68963 (2007). 24. Foot, N. C. Studies on endothelial reactions. J. Exp. Med. 32: 513–546 (1920). 25. Besterman, J. M. and Low, R. B. Endocytosis: a review of mechanisms and plasma memrbrane dynamics. Biochem. J. 210: 1–13 (1983). 26. Haas, A. The phagosome: compartment with a license to kill. Traffic 8: 311–330 (2007). 27. Schmidt, S. M., Moran, K. A., Tweed Kent, A. M., Slosar, J. L., Webber, M. J., McCready, M. J., Deering, C., Veranth, J. M., and Ostafin, A. Uptake of calcium phosphate nanoshells by osteoblasts and their effect on growth and differentiation. J. Biomed. Mater. Res. A 87: 418–428 (2008). 28. Innes, N. P. T. and Ogden, G. R. A technique for the study of endocytosis in human oral epithelial cells. Arch. Oral Biol. 44: 519–523 (1999). 29. Soenen, S. J. H., Vercauteren, D., Braeckmans, K., Noppe, W., De Smedt, S., and De Cuyper, M. Stable long-term intracellular labelling with fluorescently tagged cationic magnetoliposomes. ChemBioChem 10: 257–267 (2009). 30. Zuhorn, I. S., Kalicharan, R., and Hoekstra, D. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J. Biol. Chem. 277: 18021–18028 (2002). 31. Hu, Z., Pan, Y., Wang, J., Chen, J., Li, J., and Ren, L. Meso-tetra(carboxyphenyl) porphyrin (TCPP) nanoparticles were internalized by SW480 cells by a clathrinmediated endocytosis pathway to induce high phototoxicity. Biomed. Pharmacother. 63: 155–164 (2009). 32. Blau, S., Jubeh, T. T., Haupt, S. M., and Rubinstein, A. Drug targeting by surface cationization. Crit. Rev. Ther. Drug Carrier Syst. 17: 425–465 (2000).

c11.indd 204

7/30/2010 2:19:08 PM

REFERENCES

205

33. Nam, H. Y., Kwon, S. M., Chung, H., Lee, S.-Y., Kwon, S.-H., Jeon, H., Kim, Y., Park, J. H., Kim, J., Her, S., Oh, Y.-K., Kwon, I. C., Kim, K., and Jeong, S. Y. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J. Control. Release 135: 259–267 (2009). 34. Behrens, I., Pena, A. I., Alonso, M. J., and Kissel, T. Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: the effect of mucus on particle adsorption and transport. Pharm. Res. 19: 1185–1143 (2002). 35. Schroit, A. J., Madsen, J., and Nayar, R. Liposome–cell interactions: in vivo discrimination of uptake mechanism and in vivo targeting strategies to mononuclear phagocytes. Chem. Phys. Lipids 40: 373–393 (1986). 36. Armstrong, T. I., Davies, M. C., and Illum, L. Human serum albumin as a probe for protein adsorption to nanoparticles: relevance to biodistribution. J. Drug Target. 4: 389–398 (1997). 37. Chang, E., Wu, W. W., Colvin, V. L., and Drezek, R. Quantifying the influence of surface coatings on quantum dot uptake in cells. J. Biomed. Nanotechnol. 1: 397–401 (2005). 38. Woodle, M. C. and Lasic, D. D. Sterically stabilized liposomes. Biochim. Biophys. Acta 1113: 171–199 (1992). 39. Mailänder, V., Lorenz, M. R., Holzapfel, V., Musyanovych, A., Fuchs, K., Wiesneth, M., Walther, P., Landfester, K., and Schrezenmeier, H. Carboxylated superparamagnetic iron oxide particles label cells intracellularly without transfection agents. Mol. Imaging Biol. 10: 138–146 (2008). 40. Dini, L., Falasca, L., Ruzzittu, M. T., Mossa, G., Finazzi-Agrò, A., and Di Giulio, A. Interaction between isolated and purified liver cells and small unilamellar liposomes. Liver 18: 229–238 (1998). 41. Trigiante, G. and Huestis, W. H. Selective virus-mediated intracellular delivery of membrane-impermeant compounds by means of plasma membrane vesicles. Antiviral Res. 45: 211–221 (2000). 42. Chirico, F., Fumelli, C., Marconi, A., Tinari, A., Straface, E., Malorni, W., Pellicciari, R., and Pincelli, C. Carboxyfullerenes localize within mitochondria and prevent the UVB-induced intrinsic apoptotic pathway. Exp. Dermatol. 16: 429–436 (2007). 43. Foley, S., Crowley, C., Smaihi, M., Bonfils, C., Erianger, B. F., Seta, P., and Larroque, C. Cellular localization of a water-soluble fullerene derivative. Biochem. Biophys. Res. Commun. 294: 116–119 (2002). 44. Jablonski, A. E., Humphries, W. H., and Payne, C. K. Pyrenebutyrate-mediated delivery of quantum dots across the plasma membrane of living cells. J. Phys. Chem. B 113: 405–408 (2009). 45. Yildiz, I., McCaughan, B., Cruikshank, S. F., Callan, J. F., and Raymo, S. M. Biocompatible CdSe-ZnS core–shell quantum dots coated with hydrophilic polythiols. Langmuir 25: 7090–7096. 46. Harush-Frenkel, O., Rozentur, E., Benita, S., and Altschulter, Y. Surface charge on nanoparticles determines their endocytic and transcytotic pathway in polarized cells. Biomacromolecules 9: 435–443 (2008).

c11.indd 205

7/30/2010 2:19:08 PM

206

CAN QSAR MODELS FOR SMALL-MOLECULE XENOBIOTICS GIVE USEFUL TIPS?

47. Behr, J. P. Overcoming barriers to nonviral gene delivery. In: G. Gregoriadis and B. McCormack, eds. Targetting of Drugs IOS Press, Amsterdam, 2000, pp. 76–80. 48. Horobin, R. W. and Weissig, V. A QSAR-modeling perspective on cationic transfection lipids. 1. Predicting efficiency and understanding mechanisms. J. Gene Med. 7: 1023–1034 (2005). 49. Israelachvili, J. N. Thermodynamic and geometric aspects of amphiphile aggregation into micelles, vesicles and bilayers, and the interactions between them. In: D. Giorgio and M. Corti, eds. Physics of Amphiphiles: Micelles, Vesicles, and Microemulsions. North Holland, Amsterdam, 1985, pp. 24–58. 50. Callahan, J. and Kopecek, J. Semitelechelic, HPMA copolymers functionalized with triphenylphosphonium as drug carries for membrane transduction and mitochondrial localization. Biomacromolecules 7: 2347–2356 (2006). 51. Barua, S. and Rege, K. Cancer-cell phenotype-dependent differential intracellular trafficking of unconjgated quantum dots. Small 5: 370–376 (2009). 52. Peachman, K. K., Rao, M., Alving, C. R., Palmer, D. R., Sun, W., and Rothwell, S. W. Human dendritic cells and macrophages exhibit different intracellular processing pathways for soluble and liposome-encapsulated antigens. Immunobiology 210: 321–333 (2005).

c11.indd 206

7/30/2010 2:19:08 PM

CHAPTER 12

Self-Unpacking Gene Delivery Scaffolds MILLICENT O. SULLIVAN Department of Chemical Engineering, University of Delaware, Newark, Delaware

12.1

INTRODUCTION

Gene therapy can be defined as the introduction of genetic material into specific cells of an individual for the purpose of treating disease [1]. Gene therapy has the potential to treat a broad variety of human diseases, including those that involve heritable genetic disorders such as cystic fibrosis, hemophilia, muscular dystrophy, and familial hypercholesterolemia that could be treated by the stable expression of one or a few mutant genes within a target tissue. More broadly, gene therapy has been explored for the treatment of multiple acquired diseases, including cancer, infectious diseases, cardiovascular disease, wound healing, Parkinson’s disease, and Alzheimer’s disease. In these cases, the goals of the therapy are more diverse and might entail either stable or transient gene expression within the target cell population. For example, cancer gene therapies under exploration include those in which the goal is to permanently replace the function of a missing or altered gene, such as the retinoblastoma susceptibility gene, whose inactivation has been linked to the neoplastic phenotype in human retinoblastoma and osteosarcoma cells [2]. Other cancer gene therapies under development aim to promote the transient expression or overexpression of genes that stimulate natural defense mechanisms, act as “suicide genes” to promote tumor cell apoptosis, enhance the response to drug therapies, or block the development of tumor-induced angiogenesis. Despite broad interest in the potential applications for gene therapy, its success rate has been very low. The first clinical trial for gene therapy was

Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

207

c12.indd 207

7/30/2010 2:19:10 PM

208

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

initiated in 1990 and was directed at the treatment of severe combined immunodeficiency (SCID) [3]. The first clinical successes did not occur until 10 years later, in trials involving SCID, hemophilia, and squamous cell carcinoma of the head and neck [4–6]. Since 1990, 1537 clinical trials have been approved with a disappointing overall success rate [7]. Significant hurdles remain to effective gene therapy, including the identification of therapeutic target genes, the identification and functional elucidation of target therapeutic nucleic acids, the elucidation of methods for inducing safe chromosomal integration when desired, and the development of controllable, efficient, and safe methods for gene delivery. It is clear that, in most cases, tight regulation of the levels and duration of gene expression will be critical [8]. Gene delivery has been called the Achilles heel of gene therapy [9] and, in general, is regarded as the most significant hurdle to its success [10].

12.2 METHODS OF GENE PACKAGING Two major classes of gene delivery vehicles have been widely pursued: recombinant viruses (viral) and synthetic materials (nonviral). Viral vehicles are created from recombinant viruses in which a portion of the viral genome has been replaced with a therapeutic gene of interest. Synthetic vehicles are typically comprised of polycationic lipids or polymers, which self-assemble with DNA to form lipo- or polyplexes, respectively (Figure 12.1). 12.2.1 Viral Gene Delivery Vehicles Viruses have evolved for the purpose of highly efficient gene delivery, and viral vehicles are the more widely explored class of delivery vehicles reported

Figure 12.1. Polyplex self-assembly. Plasmid DNA (grey, left) and polycationic polymers (black, left) or lipids (not shown) electrostatically self-assemble to form multicomponent complexes (right) containing multiple plasmids and thousands of polymers. Complex formation is entropically driven by the release of bound water molecules from the polyions.

c12.indd 208

7/30/2010 2:19:10 PM

METHODS OF GENE PACKAGING

209

in the literature and in current clinical trials [7]. Viral vehicles have been created from viruses, including retrovirus, lentivirus, adenovirus, adenoassociated virus, vaccinia virus, and herpes simplex virus, and are designed to be replication incompetent by replacement of appropriate viral genes with the therapeutic transgene. Unfortunately, viral vehicles have faced numerous real and perceived obstacles in clinical development including their inherent toxicity and immunogenicity and concerns that they may revert to an infective form. Viruses are also limited in terms of their transgene carrying capacity by the size of the viral capsid and, in many cases, must have their native cell-specificity reprogrammed for the appropriate therapeutic target cell or tissue. 12.2.2 Nonviral Gene Delivery Vehicles Synthetic vehicles have received significant interest for their potential as safer and more controllable gene carriers than their viral cousins. These vehicles are formed by electrostatic interactions between cationic residues on the lipid or polymer carrier and negatively charged phosphate anions along the backbone of DNA (Figure 12.1). Complexation occurs spontaneously, and is an entropically driven phenomenon: interactions between the cations and anions release bound water molecules into the surrounding medium [11]. If sufficient polycation is added, the hydrophobicity of the partially neutralized DNA becomes so high that it loses its extended conformation and is forced to collapse [12]. The process compacts DNA and results in the formation of kinetically trapped spherical or toroidal complexes of roughly ten to several hundred nanometers in diameter [13, 14]. The formed complexes tend to be relatively polydisperse, with sizes and morphologies that depend on the conditions under which they are formed (i.e., mixing, order of addition) [15]. Lipoplexes and polyplexes typically contain several plasmids (or several thousand small RNA molecules) and several thousand lipids or polymers [16]. DNA complexation serves several purposes with respect to gene delivery. The DNA is condensed and compacted, which protects it from shear-induced degradation and sterically prohibits nuclease access to the DNA. The condensation process also imparts a level of control on the overall sizes of the complexes formed. The average size of these complexes depends on factors including the solution ionic strength and the formulation charge ratio [15, 17, 18] (for amine-containing polycations, this is defined as the ratio of amine nitrogens, N, to DNA phosphates, P) and can be tuned, to some extent, to promote more efficient extra- and intracellular transport. Furthermore, because complexes are typically formulated with an excess of the polycation (i.e., at N : P > 1), complexation enhances cellular internalization of DNA by promoting electrostatic interactions between the positively charged complexes and negatively charged glycosaminoglycan residues displayed on the extracellular surfaces of cells. Finally, the condensing polymers or lipids provide a scaffold onto which a variety of ligands can be attached, with the goal of enhancing the in vivo properties or appropriate trafficking of the complexes.

c12.indd 209

7/30/2010 2:19:10 PM

210

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

For example, cell-binding and other targeting ligands have been grafted to free amine residues within the vehicle to stimulate vehicle uptake and appropriate subcellular trafficking by target cells.

12.3

VEHICLE TRANSPORT BARRIERS

Despite promising developments in vitro, nonviral vehicles have had limited success in vivo and have rarely reached clinical application. The relatively poor in vivo gene transfer efficiency of this class of vehicles has been attributed to a variety of factors, including their inadequate tissue and cellular targeting and poorly controlled intracellular trafficking and processing. One significant and understudied issue is the lack of control over the timing and location of DNA release. Polyplexes and lipoplexes can be prematurely unpackaged by exposure to extracellular components such as heparan sulfate proteoglycans (HSPGs), leading to DNA degradation by serum nucleases [19–25]. Inefficient unpackaging is also problematic, as condensed DNA has a limited ability to interact with the transcriptional machinery [26]. The following subsections will highlight major barriers in the extra- and intracellular gene delivery pathways and will briefly review material design strategies that address these barriers. The subsequent section will include a detailed discussion of the factors that determine polyplex/lipoplex stability and unpackaging, as well as new material-based strategies for programming site-targeted DNA release and activation. 12.3.1 Serum Stability and Extracellular Transport The gene delivery pathway presents formidable challenges to the delivery vehicle immediately following the introduction of the vehicle into the extracellular environment (Figure 12.2A). For in vivo gene transfer via systemic administration, the initial challenge to the vehicle is survival in the bloodstream. In this environment, the half-life of a vehicle depends on its ability to avoid aggregation and protein adsorption following contact with serum components. Charged particles like polyplexes are destabilized by electrostatic screening in the 150 mM physiological saline environment, which can cause their rapid aggregation, reduced activity, and significant adverse consequences to the host such as particle embolization in the lung [8]. Interactions between polyplexes and lipoplexes and serum proteins have been shown to initiate rapid vehicle elimination by phagocytic cells and the reticuloendothelial system [22, 27, 28]. To avoid these issues, poly(ethylene glycol) (PEG) and other hydrophilic and uncharged polymers, proteins, or oligosaccharides are frequently grafted as a brush onto free amine residues within the condensing polycation to provide a steric barrier against salt-induced aggregation [22, 27, 29–31]. These molecules can also prevent protein- or complement-induced inactivation [29, 32–34].

c12.indd 210

7/30/2010 2:19:10 PM

VEHICLE TRANSPORT BARRIERS

211

(A)

(B)

Figure 12.2. Barriers to DNA transport. (A) Extracellular barriers. Poly- or lipoplex vehicles (black circles) travel through the host vasculature following administration, and must then extravasate (1) from the vasculature within the target tissue. Following extravasation, the vehicles must traverse the ECM containing collagen, proteoglycans, GAGs, and other ECM proteins to reach the target cell (2a). In some cases, interactions between the vehicle and the ECM can cause vehicle unpackaging and lead to the subsequent degradation of the DNA (2b). Inset image shown in (B). (B) Intracellular barriers. Vehicles enter cells by receptor-mediated endocytosis (3a) or macropinocytosis (3b). Subsequently, the early endocytic vesicles containing the vehicles are either recycled (4a) or mature into late endosomes (4b). Vehicles trapped within late endosomes might escape (5a) or be retained during endolysosomal maturation (5b); retention typically leads to DNA degradation. Released vehicles must travel through the cytosol to the vicinity of the nucleus (5a). Vehicles might enter the nucleus intact (6a) and subsequently unpackage (7a); alternatively, the vehicles might unpackage in the cytosol (6b), and the unpackaged DNA might enter the nucleus (7b) or be degraded by cytosolic nucleases (7c). (Author thanks John D. Larsen for assistance with figure preparation.)

c12.indd 211

7/30/2010 2:19:10 PM

212

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

Provided the vehicle maintains sufficient serum stability, it must then begin the process of navigation to its target cell, which for nonhematopoietic and nonendothelial targets requires vascular extravasation and transport through the interstitial extracellular matrix (ECM) and/or lymphatics. As a general rule, methods to predictably control or promote vascular extravasation are not well established, given the low capillary permeability in most organs. For select tissues and organs, passive targeting strategies have been developed that take advantage of the unique physical properties of the target tissue. For example, nanoparticles such as polyplexes and lipoplexes tend to passively accumulate in the liver due to phagocytic clearance by the reticuloendothelial system [35–38]. A similar passive accumulation effect has been observed as a result of the aberrant architecture and increased vascular permeability and hydraulic conductivity of tumor endothelium [39–42]. This occurrence of tumor-specific accumulation, termed the enhanced permeability and retention (EPR) effect [43], is strongly size and molecular weight dependent. The average vascular pore sizes in various human and rodent tumors have been estimated as ∼400–500 nm in diameter [40], and macromolecules with sufficient serum stability and molecular weights greater than ∼50 kDa accumulate in tumor tissues. Transport of polyplexes and lipoplexes through dense ECM is mediated by a combination of convection and diffusion and depends on both their size and surface chemistry [44–46]. For example, in vitro studies employing hydrogels and multicellular spheroids have estimated the influence of nanoparticle characteristics on the efficiency of transport through simplified ECM. These studies suggest that particles with diameters of ∼100 nm or less can penetrate model matrices, but that penetration was relatively limited even for relatively small particles (∼20 nm) [47–49]. The surface chemistry of the nanoparticles was also critical. For example, PEGylated nanoparticles were found to move more rapidly through model ECM than unPEGylated nanoparticles, an effect attributed to reduced interactions between the PEGylated vehicles and fibrin networks within the ECM [50]. In another study, liposomes with neutral or low surface charges were found to penetrate to the central regions of spheroids, whereas charged liposomes were restricted [51]. A related issue with respect to the penetration of dense ECM by polyplexes and lipoplexes is the innate stability of the polyplexes and lipoplexes, and their tendency to unpackage and release DNA following contact with ECM components. This issue is covered in more detail in the following section on vehicle unpackaging. 12.3.2 Cellular Internalization and Intracellular Transport Upon arrival at their target cell, lipoplexes and polyplexes must navigate cellular entry and subcellular trafficking, ideally resulting in the delivery of intact cargo DNA to the nucleus (Figure 12.2B). Gene delivery vehicles typically enter the cell by either nonspecific macropinocytosis or receptor-mediated endocytosis, depending on their surface functionalization and size. Positively

c12.indd 212

7/30/2010 2:19:11 PM

VEHICLE TRANSPORT BARRIERS

213

charged lipoplexes and polyplexes are generally internalized via macropinocytosis as a result of their nonspecific electrostatic interactions with the cell surface, as noted previously. Neutral or negatively charged complexes containing cell-binding ligands typically traffic through the relevant receptor-mediated pathway. The size of a lipoplex or polyplex is an important determinant of its internalization rate, and the optimal size for endocytosis has been investigated for various receptor-mediated pathways. As a general rule, previous experimental and computational studies have suggested that particles with diameters <50 nm are optimal for receptor-mediated endocytosis [52–57]. The mechanism of cellular entry largely determines the trafficking pathway employed by gene delivery vehicles. The majority of vehicles are thought to traffic into late endosomes, which acidify and can mature into acidic (pH ∼4–5) lysosomes containing a variety of lysosomal proteases and degradative enzymes [8]. Thus the endosomal membrane represents an important gene delivery barrier, and endosomal release prior to the acidification process is considered to be critical for effective gene delivery. A variety of strategies have been explored to mediate escape from the endosome into the cytoplasm, including the use of “proton-sponge” polymers such as poly(ethylenimine) (PEI) as DNA condensing agents, and the incorporation of pH-sensitive membranolytic polymers and peptides onto the delivery vehicle. Branched PEI contains a large number of secondary and tertiary amines, whose ability to buffer the endosomal acidification process has been proposed to result in osmotic swelling and rupture of the endosome [58–61]. pH-sensitive agents have been shown to enhance endosomal escape as a result of their structural transitions as acidic pH; for example, peptides that adopt an amphiphilic alpha-helical conformation at acidic pH can disrupt the endosomal membrane and promote the subsequent escape of encapsulated nanoparticles into the cytosol [62]. Once released into the cytosol, gene delivery vehicles must be transported to and into the nucleus for processing. Cytosolic transport represents a recently recognized barrier for the gene delivery process: cytosolic mobility is highly size dependent, restricting the free diffusion of DNA > 2000 bp and that of nanoscale particles [63–65]. Microtubule-dependent active transport of PEI polyplexes has been reported for a fraction of the internalized polyplexes (∼17%) [64]. Recently, anionic plasmid carriers formed from poly(lactic-coglycolic acid) and from 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes were conjugated to actin comet tails and shown to move through cytoplasmic extracts much more rapidly than their unconjugated counterparts [66]. Polyplex redistribution has also been proposed as a result of the mixing that occurs during mitosis [8]. The final intracellular transport barrier for gene delivery is the nuclear membrane. Transport into the nucleus is mediated by the nuclear pore complex, a multiprotein transmembrane assembly that permits the passage of small molecules but prohibits the free diffusive transport of macromolecules larger than 10–20 kDa [8]. The upper limit for active intranuclear transfer of proteins has been estimated as ∼50 kDa [67, 68], making it nearly prohibitive

c12.indd 213

7/30/2010 2:19:11 PM

214

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

for DNA and nanoscale particles. Thus the majority of gene delivery vehicles are thought to accumulate in the nucleus during cell division [69], a theory that is supported by the findings that transfection of nondividing cells occurs at only very low levels, and that transfection of dividing cells is 30- to 500-fold more efficient when transfection is performed immediately prior to cell division as opposed to at the beginning of the cell cycle [8, 70].

12.4

VEHICLE UNPACKAGING

The final critical step in the gene delivery pathway for lipoplexes and polyplexes is the release of DNA from the complex, given that condensed DNA does not allow efficient access of the transcriptional machinery to the underlying DNA sequences [26]. This self-disassembly, or self-unpackaging, presents an important challenge in materials design, in that the same beneficial characteristics (i.e., a high density of positive charge [71–73]) that promote tight association of a polycation with DNA during gene transport eventually inhibit the activity of the DNA within the nucleus. A change in complex packaging state requires a change in the underlying structure or charge state of the condensing polycation. Unfortunately, there is a lack of obvious intracellular triggers (i.e., ionic strength, temperature, or pH) to stimulate this transition, given that the local pH drop in the endosome does not deprotonate the cationic amine residues that constitute the basis for many condensing agents. Indeed, the stability of various polyplexes to intracellular dissociation has been noted [26, 64, 74]. Inefficient polyplex disassembly has been identified as a critical rate-limiting barrier for gene delivery [26, 64, 74–78]. While unpackaging is a clear requirement for efficient transcription, the desired time and place for DNA release are less clear [71]. For example, premature unpackaging can be as problematic as inefficient unpackaging: interactions between lipoplexes or polyplexes and various extracellular components have been shown to cause destabilization, DNA release, and DNA degradation by serum nucleases. Intracellular components have also been shown to mediate lipo- or polyplex destabilization and DNA release, although in many cases, higher levels of intracellular release are correlated with improved transfection, despite the presence of cytoplasmic nucleases. Thus DNA packaging materials face opposing requirements, as they must protect DNA by tight packaging during transport, and release DNA prior to transcription. Materials designed with mechanisms to initiate the site-specific release of DNA in the vicinity of the cell’s nucleus would be expected to significantly enhance the efficient utilization of administered DNA. It is worth note that despite the development of numerous nonviral methods for achieving high levels of in vitro cell transfection, including those that employ lipoplexes and polyplexes, no method exists that could truly be considered efficient. For example, for a typical in vitro Lipofectamine-mediated transfection (Lipofectamine is a product of Invitrogen Corporation, Carlsbad, CA), millions of copies of

c12.indd 214

7/30/2010 2:19:11 PM

VEHICLE UNPACKAGING

215

plasmid DNA must be administered per cell (this assumes that 10 μg of plasmid DNA would be used to transfect 106 cells). Given that the accumulation of uncomplexed intranuclear DNA appears to very minimal, even when the levels of overall intracellular lipo- or polyplex accumulation are high [26, 64], it is clear that intracellular trafficking and unpackaging remain critical and unsolved barriers with respect to gene delivery. In this section, the parameters determining the innate stability of lipo- and polyplexes will be addressed, and known mechanisms that promote the unpackaging of these complexes in the extra- and intracellular environments will be explored. Subsequently, several classes of new materials that have been designed for the explicit purpose of site-specific unpackaging and DNA release within the cell will be reviewed, with emphasis on identifying the most promising new directions for further development. 12.4.1 Complex Structure and Stability Both lipids and polymers have demonstrated the capacity to compact and deliver DNA payloads in vitro. Polycation structure plays an important role in the efficiency of DNA binding and condensation. For example, branched PEI has been shown to more efficiently compact DNA then linear PEI [79], perhaps as a result of the higher density of primary amine groups on branched PEI relative to linear PEI. In general, primary amine groups within cationic lipids and polymers have been found to compact DNA more efficiently than higher order amines within corresponding molecules, presumably due to the reduced steric interference around the primary amine groups [80, 81]. The number and density of cationic groups on the polycation have also been shown to affect condensation efficiency. With polypeptide structures, several groups have found that a minimum of six to eight charged residues per polypeptide were necessary for tight polyplex packaging (i.e., packaging at a level that would reduce ethidium bromide staining of the complexed DNA) [26, 82, 83]. Different structural requirements for packaging were identified by these same groups, where Plank and co-workers found that the inclusion of tryptophan residues did not affect DNA binding affinity but reduced DNA compaction [82], and Wadhwa and co-workers found that the inclusion of tryptophan increased DNA binding efficiency [83]. Differences in the structures (branched vs. linear) and charge density of the studied polypeptides could underlie these different findings. Correlations between polycation length and packaging efficiency have also been identified for chitosan-based carriers [84, 85]. Furthermore, other work to systematically address the impact of various characteristics of polycationic polymers on DNA binding and condensation suggested that polymers with a low density of cationic residues were unable to efficiently compact DNA [86]. Random copolymers formed from neutral monomers and monomers containing a single quaternary amine residue bound to DNA inefficiently when the ratio of neutral monomer was 50% or greater [86].

c12.indd 215

7/30/2010 2:19:11 PM

216

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

The functionalization of lipoplexes and polyplexes with stabilizing or targeting groups has also been shown to influence the efficiency of DNA compaction. For example, hydrophilic polymers such as PEG are often grafted to DNA condensing agents via reaction with primary amine groups along the backbone of the polycation [29, 31, 32, 87]. When a polycation is functionalized with PEG or another hydrophilic polymer prior to complexation, the charge density of the agent is reduced and DNA compaction is adversely affected [28, 88–91]. Furthermore, despite the fact that this surface grafting is typically performed for the purpose of reducing polyplex self-interactions and polyplex interactions with serum components [92], efficiently compacted polyplexes grafted with hydrophilic polymers have been found to be less stable to competing ions in blood than their ungrafted counterparts [19, 24, 32, 93, 94]. This effect has been attributed to thermodynamic destabilization caused by hydrophilic chains dispersed within the polyplex [24, 32]. However, even when grafting is performed on surface-exposed free amines postcomplexation, grafted polyplexes are often less stable to competitive ions [19, 23], and PEGylated polyplexes formed from lower molecular weight polycations can be destabilized by the addition of salt [28]. 12.4.2 Extracellular Unpackaging Depending on the innate stability of a lipo- or polyplex, a variety of components within the extracellular environment can drive premature unpackaging prior to cellular entry. Immediately following introduction into the bloodstream, PEG–PEI polyplexes have been shown to dissociate significantly [32, 93]. Polypeptide–DNA complexes have also been shown to dissociate within minutes upon introduction into the blood, allowing the degradation of DNA by serum endonucleases; when these same peptide-based polyplexes were crosslinked with glutaraldehyde prior to in vivo administration, their in vivo half-life was improved significantly [95]. Serum-induced destabilization may result from the enhanced electrostatic screening of DNA–polycation interactions at physiological salt levels. Alternatively, charged serum components such as albumin may destabilize these polyplexes by competitive DNA or polycation displacement. However, despite the fact that negatively charged model polyanions have been shown to displace DNA from polyplexes, albumin has been shown to bind to N-(2-hydroxypropyl)-methacrylamide/ 2-(trimethylammonio)ethyl methacrylate polyplexes without significant polyplex destabilization and only minor increases in the overall polyplex size [24]. Thus albumin-mediated polyplex clearance has been proposed to result from enhanced interactions between albumin-bound polyplexes and serum opsonins, as opposed to polyplex dissociation [24]. Following vascular extravasation, interactions with ECM proteins can also cause rapid dissociation of lipo- and polyplexes and the subsequent degradation of the DNA. For example, extracellular glycosaminoglycans (GAGs) such as heparin have been shown to cause polyplex destabilization/unpackaging,

c12.indd 216

7/30/2010 2:19:11 PM

VEHICLE UNPACKAGING

217

resulting in the degradation of the freed DNA by extracellular nucleases [19–21, 96, 97]. Burke and Pun demonstrated that both soluble and insoluble ECM GAGs and proteoglycans facilitated polyplex unpackaging in a fashion that correlated with the density of negatively charged residues, supporting the hypothesis of DNA displacement by competing anions in the ECM [19]. 12.4.3 Intracellular Unpackaging Interactions between lipo- and polyplexes and intracellular anions have also been proposed to stimulate polyplex unpackaging. For example, several groups have demonstrated the release of plasmid DNA from DNA-condensing lipids and polymers by cytosolic components [98–102]. In the case of lipoplexes, anionic HSPGs on the cell surface have been shown to interact with polycationic lipids within the lipoplex to initiate receptor-mediated endocytosis [103–106]. These interactions have been proposed to destabilize the endosomal membrane, cause the membrane lipids to flip to the cytoplasmic face of the membrane, and displace the DNA from the lipoplex into the cytoplasm [100, 102]. Polyplexes have been proposed to unpackage in the cytoplasm as a result of displacing interactions with other cytoplasmic polyanions. For example, Okuda et al. [99] demonstrated that the cytoplasmic component causing the release of plasmid DNA from dendritic poly(l-lysine) (PLL) and jetPEI was protease sensitive, as proteinase K-treated cytosolic fractions were inactive at DNA release. This effect was only observed at low PEI : DNA ratios, prompting speculation that other factors such as cytoplasmic RNA might be involved in cytosolic DNA displacement at high polycation : DNA ratios [101]. Huth et al. [101] demonstrated that the release of plasmid DNA from linear and branched PEI and PLL in cytoplasmic extracts was RNA-dependent, as RNase-treated cytoplasmic fractions did not release DNA. Furthermore, these authors demonstrated that in the absence of cytoplasmic extracts, the addition of RNA at levels similar to intracellular levels of RNA also resulted in DNA release from polyplexes formed at high polycation : DNA ratios. Chromosomal DNA has also been suggested to play a role in the displacement and release of DNA from lipo- and polyplexes [26, 107]. For example, fluorescence imaging analyses with PLL–DNA polyplexes demonstrated that a fraction of the polyplexes reached the nucleus, and that the amount of intranuclear polyplex dissociation was correlated with the length of the PLL chain [26]. Whereas long PLL chains (180 residues) remained closely associated with plasmid DNA, shorter PLL chains (19 and 36 residues) were found to be completely separated from the plasmid within the nucleus. In subsequent experiments, Schaffer et al. [26] demonstrated that double-stranded DNA could more easily dissociate the shorter PLLs from the plasmid than the longer PLL, a result consistent with cation exchange to the surrounding chromatin. Other investigators have identified a more minor role for chromosomal DNA in unpackaging. For example, Zabner et al. [103] demonstrated that lipoplexes

c12.indd 217

7/30/2010 2:19:11 PM

218

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

injected into the nucleus showed low transfection efficiency, implying that chromosomal DNA plays only a small role in unpackaging [102]. Together, these experiments suggest that polycation structure and the innate stability of the lipo- or polyplex are important determinants of the site, mechanism, and extent of unpackaging, and support roles for multiple components in the unpackaging process. Although lipo- and polyplex materials capable of intracellular DNA release often have high in vitro gene transfer activities in comparison with their more stably packaged counterparts [26, 58, 84, 98, 108–112], cytoplasmic release has also been suggested to correlate with lower gene transfer activity [113]. For example, Oupicky et al. [113] demonstrated that cytoplasmic microinjection of free DNA resulted in lower levels of transfection than cytoplasmic microinjection of polyplexes, whereas nuclear microinjection of the same samples had the opposite effect. These findings were attributed in part to the enhanced resistance of the polyplex-contained DNA to cytoplasmic nucleases, although the authors also noted that the polyplexes might have improved cytoplasmic mobility and transport to the nucleus in comparison with the free DNA. In general, it is probable that cytoplasmic degradation of at least a portion of the released DNA occurs even with materials that exhibit high in vitro transfection efficiencies. Presumably, only a portion of the released DNA is sacrificed, and the remaining intact free DNA is utilized relatively efficiently.

12.5

SELF-UNPACKAGING MATERIALS

Several strategies have been proposed with respect to the design of materials capable of packaging and releasing DNA for efficient in vivo gene delivery. For example, the utilization of materials with an intermediate level of packaging has been suggested; ideally, these materials would be sensitive to the existing intracellular release mechanisms but still stable in serum and during contact with the ECM. However, given the similar structures and charge densities of the extracellular and intracellular GAGs and other anions that have been shown to initiate complex unpackaging, it would seem difficult to strike such a balance. Thus a variety of materials containing active DNA-releasing strategies have recently been explored. These include materials that release DNA by carrier degradation, carrier charge reduction, and crosslinker degradation (Figure 12.3). These major categories of active materials will be explored in the following subsections. 12.5.1 Release by Carrier Degradation Given that a correlation between the length of a polycationic carrier and its tendency to dissociate from DNA has been identified by several investigators [26, 82–85], as discussed above, one strategy that has been pursued for targeted unpackaging involves the use of high molecular weight degradable polycations

c12.indd 218

7/30/2010 2:19:11 PM

SELF-UNPACKAGING MATERIALS

219

(A)

(B)

(C)

Figure 12.3. Strategies for DNA release by active self-unpackaging. DNA within lipoor polyplexes can be released by hydrolytic or reductive degradation of the cationic carrier (A), charge reversal of the carrier (B), or degradation of surface crosslinkers on the carrier (C). Strategy (C) is often combined with carriers that degrade as in (A).

(Figure 12.3A). These species are typically either hydrolytically [109, 114–120] or reductively [75, 77, 110, 121] degradable, and thus are designed to gradually release DNA as they transition into low molecular weight (and fast-dissociating) species following exposure to aqueous media or reducing environments such as the endosome [108]. For example, Langer and co-workers have synthesized libraries of poly(β-aminoesters) and related biodegradable materials that condense DNA at physiological pH, but are hydrolytically degradable into nontoxic fragments [114, 115, 119, 120]. These investigators identified several candidate polymers with high in vitro gene transfection efficiencies. Hydrolytically degradable PEI derivatives have also been created and shown to have DNA-binding properties similar to high molecular weight (25-kDa) PEI with comparably low cytotoxicity and high gene transfer efficiency [109]. Forrest et al. [109] speculated that enhanced DNA release might be one determinant of the enhanced in vitro transfection efficiency of these materials. Jain and co-workers have synthesized pH-sensitive linear poly(amido amines)

c12.indd 219

7/30/2010 2:19:11 PM

220

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

whose hydrolysis rate is significantly enhanced at pH values correspondent to the lysosomal pH [116]. Because of their capacity to selectively degrade in the intracellular environment, as opposed to in a sustained fashion, these new materials would be expected to have desirable properties for targeted DNA release. With a similar goal of targeted intracellular degradation and DNA release, reductively degradable DNA-packaging materials have also been synthesized. For example, Pichon et al. [77] synthesized high molecular weight PLL polymers for gene delivery by the reductive crosslinking of low molecular weight PLL via disulfide linkages. In a similar strategy, Gosselin et al. [75] created crosslinked PEI polymers from low molecular weight PEI linked by disulfide bonds. These reductively linked polymers had high in vitro gene transfer efficiencies relative to the corresponding uncrosslinked low molecular weight species, and were shown to release DNA upon incubation with reducing agents such as glutathione [75, 77]. More recently, reductively degradable poly(amido amines) and poly(amido ethylenimines) were synthesized and shown to exhibit high levels of in vitro gene transfection in the presence of serum, even when serum incubation times were extended for several hours [110, 121]. These results suggest that the intracellular targeted release mechanism employed by these materials had good potential for in vivo activity. 12.5.2 Release by Carrier Charge Reduction Other strategies for the targeted release of DNA have also been pursued, including the use of polycationic carriers designed to undergo controlled reductions in their net charge when exposed to physiologically relevant environments [71, 108, 111, 112, 122–125] (Figure 12.3B). For example, Hennink and co-workers have created charge-reducing polycations from poly(methacrylates) whose amine-containing side chains are attached to the polymer backbone via hydrolyzable carbonate bonds; thus hydrolytic cleavage of these polymers results in the gradual loss of cationic amine groups [123–125]. These polymers were shown to release DNA in aqueous media in a fashion correlated with the expected time-dependent changes in electrostatic interactions, and mediated high levels of in vitro cell transfection. Others have also created materials whose positive character is reduced by hydrolysis. For example, Lynn and co-workers have created charge-shifting cationic polymers whose hydrolysis results not only in the loss of cationic groups, but also in the introduction of anionic residues [71, 108, 112]. The DNA-releasing capacities of these materials have been demonstrated for surface-mediated [25] and polyplex-mediated [71, 108] delivery. Prata et al. [111] have designed chargereversal amphiphiles that transition from positively to negatively charged within cells. These amphiphiles bind to DNA to form supramolecular lipoplex complexes, but are cleaved by endosomal esterases to become anionic and release DNA. The supramolecular lipoplexes had high in vitro activity that was dependent upon esterase-mediated lipid cleavage.

c12.indd 220

7/30/2010 2:19:11 PM

REFERENCES

221

12.5.3 Release by Crosslinker Degradation Although the above approaches for targeted release are promising, the lack of evaluation (to date) of these systems in vivo combined with the increasing concerns about the stability of lipo- and polyplexes during prolonged exposure to the extracellular environment have led to recent interest in new methods to reversibly stabilize polyplexes. For example, several approaches have focused on the development of methods to covalently crosslink the surfaces of polyplexes with degradable linkages [76, 126] (Figure 12.3C). Oupicky et al. [76] explored the potential of polyplexes formed from reducible polycations and stabilized by surface crosslinking with hydrophilic polymers. Surface coating was shown to suppress the unpackaging of the polyplexes by poly(acrylic acid)-mediated displacement. The stabilized particles were capable of moderate levels of cellular transfection when cell-binding ligands were conjugated to their surfaces. In another approach, PEI–DNA polyplexes were crosslinked with disulfide bonds [126]. These crosslinked polyplexes were stable against dissociation by polyanions and high ionic strength and had improved biocompatibility against albumin and erythrocyte interactions in comparison with uncrosslinked PEI–DNA complexes, suggesting the potential applicability for systemic application. 12.6

CONCLUSIONS

Gene delivery represents the most significant hurdle to the realization of nucleic acid-based therapies, and despite years of effort directed at the design of new nonviral delivery materials, successes have been limited. Many pervasive problems with these materials are related to the lack of correlation between their in vitro and in vivo performance. With respect to lipo- and polyplex-mediated delivery, the ability to protect DNA within the extracellular environment but release it within the cell has presented a particular challenge. Many new materials are now being developed with promising potential for the targeted intracellular release of DNA. These materials offer possible solutions to a significant problem in gene delivery, and improved understanding in this area may enable the synthesis of nonviral materials with efficacies that rival those of viral delivery systems, without the related concerns of toxicity and immunogenicity. The demonstration of these new releasing materials in vivo will improve understanding of the mechanisms that control gene delivery and will pave the way for their clinical translation. REFERENCES 1. Mulligan, R. C. The basic science of gene therapy. Science 260: 926–932 (1993). 2. Huang, H., Yee, J., Shew, J., Chen, P., Bookstein, R., Friedmann, T., Lee, E., and Lee, W. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242: 1563–1566 (1988).

c12.indd 221

7/30/2010 2:19:11 PM

222

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

3. Blaese, R. M., Culver, K. W., Miller, A. D., Carter, C. S., Fleisher, T., Clerici, M., Shearer, G., Chang, L., Chiang, Y., Tolstoshev, P., Greenblatt, J. J., Rosenberg, S. A., Klein, H., Berger, M., Mullen, C. A., Ramsey, W. J., Muul, L., Morgan, R. A., and Anderson, W. F. T lymphocyte-directed gene therapy for ADA–SCID: initial trial results after 4 years. Science 270(5235): 475–480 (1995). 4. Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J.-L., Bousso, P., Le Deist, F., and Fischer, A. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288(5466): 669–672 (2000). 5. Kay, M. A., Manno, C. S., Ragni, M. V., Larson, P. J., Couto, L. B., McClelland, A., Glader, B., Chew, A. J., Tai, S. J., Herzog, R. W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A. W., and High, K. A. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24: 257–261 (2000). 6. Khuri, F. R., Nemunaitis, J., Ganly, I., Arseneau, J., Tannock, I. F., Romel, L., Gore, M., Ironside, J., MacDougall, R. H., Heise, C., Randlev, B., Gillenwater, A. M., Bruso, P., Kaye, S. B., Hong, W. K., and Kirn, D. H. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with displatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat. Med. 6: 879–885 (2000). 7. Gene Therapy Clinical Trials Worldwide [online]. March 2009. Available at: http:// www.wiley.co.uk/genetherapy/clinical/ (accessed July 2009). 8. Pack, D. W., Hoffman, A. S., Pun, S., and Stayton, P. S. Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 4(7): 581–593 (2005). 9. Verma, I. M., Naldini, L., Kafri, T., Miyoshi, H., Takahashi, M., Blömer, U., Somia, N., Wang, L., and Gage, F. H. Gene therapy: promises, problems, and prospects. In: V. Boulyjenkov, K. Berg, and Y. Christen, eds. Genes and Resistance to Disease. Springer, New York, 2000. 10. Verma, I. M. and Somia, N. Gene therapy—promises, problems and prospects. Nature 389: 239–242 (1997). 11. Bakeev, K. N., Izumrudov, V. A., Kuchanov, S. I., Zezin, A. B., and Kabanov, V. A. Kinetics and mechanism of interpolyelectrolyte exchange and addition-reactions. Macromolecules 25: 4249–4254 (1992). 12. Manning, G. S. Limiting laws and counterion condensation in polyelectrolyte solutions. I. Colligative properties. J. Chem. Phys. 51: 924–938 (1969). 13. Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M. L. Transferrin–polycation– DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. PNAS 88: 4255–4259 (1991). 14. Hansma, H. G., Golan, R., Hsieh, W., Lollo, C. P., Mullen-Ley, P., and Kwoh, D. DNA condensation for gene therapy as monitored by atomic force microscopy. Nucleic Acids Res. 26(10): 2481–2487 (1998). 15. Lai, E. and van Zanten, J. H. Monitoring DNA/poly-l-lysine polyplex formation with time-resolved multiangle laser light scattering. Biophys. J. 80: 864–873 (2001). 16. Bartlett, D. W. and Davis, M. E. Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles. Bioconjug. Chem. 18: 456–468 (2007).

c12.indd 222

7/30/2010 2:19:11 PM

REFERENCES

223

17. Bloomfield, V. A. DNA condensation by multivalent cations. Biopolymers 44: 269–282 (1997). 18. Kabanov, A. V., Astafyeva, I. V., Chikindas, M. L., Rosenblat, G. F., Kiselev, V. I., Severin, E. S., and Kabanov, V. A. DNA interpolyelectrolyte complexes as a tool for efficient cell transformation. Biopolymers 31(12): 1437–1443 (1991). 19. Burke, R. S. and Pun, S. H. Extracellular barriers to in vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver. Bioconjug. Chem. 19(3): 693– 704 (2008). 20. Ruponen, M., Ronkko, S., Honkakoski, P., Pelkonen, J., Tammi, M., and Urtti, A. Extracellular glycosaminoglycans modify cellular trafficking of lipoplexes and polyplexes. J. Biol. Chem. 276(36): 33875–33880 (2001). 21. Ruponen, M., Ylä-Herttuala, S., and Urtti, A. Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies. Biochim. Biophys. Acta Biomembranes 1415(2): 331–341 (1999). 22. Dash, P., Read, M., Barrett, L., Wolfert, M., and Seymour, L. Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Ther. 6: 643–650 (1999). 23. Oupicky, D., Howard, K. A., Konak, C., Dash, P. R., Ulbrich, K., and Seymour, L. W. Steric stabilization of poly-l-lysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2-Hydroxypropyl)methacrylamide]. Bioconjug. Chem. 11(4): 492–501 (2000). 24. Oupicky, D., Konak, C., Dash, P. R., Seymour, L. W., and Ulbrich, K. Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block. Bioconjug. Chem. 10(5): 764–772 (1999). 25. Eliyahu, H., Joseph, A., Schillemans, J. P., Azzam, T., Domb, A. J., and Barenholz, Y. Characterization and in vivo performance of dextran-spermine polyplexes and DOTAP/cholesterol lipoplexes administered locally and systemically. Biomaterials 28: 2339–2349 (2007). 26. Schaffer, D. V., Fidelman, N. A., Dan, N., and Lauffenburger, D. A. Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol. Bioeng. 67(5): 598–606 (2000). 27. Nishikawa, M., Takemura, S., Takakura, Y., and Hashida, M. Targeted delivery of plasmid DNA to hepatocytes in vivo: optimization of the pharmacokinetics of plasmid DNA/galactosylated poly(l-lysine) complexes by controlling their physicochemical properties. J. Pharm. Exp. Ther. 287(1): 408–415 (1998). 28. Pun, S. H. and Davis, M. E. Development of a nonviral gene delivery vehicle for systemic application. Bioconjug. Chem. 13: 630–639 (2002). 29. Ogris, M., Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. PEGylated DNA/ transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6(4): 595–605 (1999). 30. Toncheva, V., Wolfert, M. A., Dash, P. R., Oupicky, D., Ulbrich, K., Seymour, L. W., and Schacht, E. H. Novel vectors for gene delivery formed by self-assembly of DNA with poly(l-lysine) grafted with hydrophilic polymers. Biochim. Biophys. Acta 1380(3): 354–368 (1998).

c12.indd 223

7/30/2010 2:19:11 PM

224

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

31. Kircheis, R., Blessing, T., Brunner, S., Wightman, L., and Wagner, E. Tumor targeting with surface-shielded ligand–polycation DNA complexes. J. Control. Release 72(1–3): 165–170 (2001). 32. Merdan, T., Kunath, K., Petersen, H., Bakowsky, U., Voigt, K. H., Kopecek, J., and Kissel, T. PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice. Bioconjug. Chem. 16(4): 785–792 (2005). 33. Ogris, M., Steinlein, P., Carotta, S., Brunner, S., and Wagner, E. DNA/ polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression. AAPS PharmSci. 3(3): E21 (2001). 34. Petersen, H., Fechner, P. M., Martin, A. L., Kunath, K., Stolnik, S., Roberts, C. J., Fischer, D., Davies, M. C., and Kissel, T. Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system. Bioconjug. Chem. 13(4): 845–854 (2002). 35. Illum, L., Davis, S. S., Muller, R. H., Mak, E., and West, P. The organ distribution and circulation time of intravenously injected colloidal carriers sterically stabilized with a block copolymer—Poloxamine 908. Life Sci. 40: 367–374 (1987). 36. Bazile, D., Robert, C., Huve, P., Verrecchia, T., Marlard, M., Frydman, A., Veillard, M., and Spenlehauer, G. Body distribution of fully biodegradable 14C-poly(lactic acid) nanoparticles coated with albumin after parenteral administration to rats. Biomaterials 13: 1093–1102 (1992). 37. Allemann, E., Gurny, R., Doelker, E., Skinner, F. S., and Schutz, H. Distribution, kinetics and elimination of radioactivity after intravenous and intramuscular injection of savoxepine loaded poly(d,l-lactic acid) nanospheres to rats. J. Control. Release 29: 97–104 (1994). 38. Kreuter, J., Tauber, U., and Illi, V. Distribution and elimination of poly(methyl2–14C-methacrylate) nanoparticles radioactivity after injection in rats and mice. J. Pharm. Sci. 68: 1443–1447 (1979). 39. Gerlowski, L. E. and Jain, R. K. Microvascular permeability of normal and neoplastic tissues. Microvasc. Res. 31(3): 288–305 (1986). 40. Yuan, F., Dellian, M., Fukumura, D., Leunig, M., Berk, D. A., Torchilin, V. P., and Jain, R. K. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res. 55(17): 3752–3756 (1995). 41. Yuan, F., Leunig, M., Berk, D. A., and Jain, R. K. Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice. Microvasc. Res. 45(3): 269–289 (1993). 42. Yuan, F., Leunig, M., Huang, S. K., Berk, D. A., Papahadjopoulos, D., and Jain, R. K. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Res. 54(13): 3352–3356 (1994). 43. Maeda, H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv. Enzyme Regul. 41: 189–207 (2001).

c12.indd 224

7/30/2010 2:19:11 PM

REFERENCES

225

44. Goodman, T. T., Ng, C. P., and Pun, S. H. 3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. Bioconjug. Chem. 19(10): 1951–1959 (2008). 45. Netti, P. A., Berk, D. A., Swartz, M. A., Grodzinsky, A. J., and Jain, R. K. Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res. 60(9): 2497–2503 (2000). 46. Pluen, A., Boucher, Y., Ramanujan, S., McKee, T. D., Gohongi, T., di Tomaso, E., Brown, E. B., Izumi, Y., Campbell, R. B., Berk, D. A., and Jain, R. K. Role of tumor–host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. PNAS 98(8): 4628–4633 (2001). 47. Goodman, T. T., Olive, P. L., and Pun, S. H. Increased nanoparticle penetration in collagenase-treated multicellular spheroids. Int. J. Nanomed. 2(2): 265–274 (2007). 48. Kuhn, S., Hallahan, D., and Giorgio, T. Characterization of superparamagnetic nanoparticle interactions with extracellular matrix in an in vitro system. Ann. Biomed. Eng. 34(1): 51–58 (2006). 49. Enger, P. Ø., Thorsen, F., Lønning, P. E., Bjerkvig, R., and Hoover, F. Adenoassociated viral vectors penetrate human solid tumor tissue in vivo more effectively than adenoviral vectors. Hum. Gene Ther. 13(9): 1115–1125 (2002). 50. Valentine, M. T., Perlman, Z. E., Gardel, M. L., Shin, J. H., Matsudaira, P., Mitchison, T. J., and Weitz, D. A. Colloid surface chemistry critically affects multiple particle tracking measurements of biomaterials. Biophys. J. 86: 4004–4014 (2004). 51. Saga, T., Neumann, R. D., Heya, T., Sato, J., Kinuya, S., Le, N., Paik, C. H., and Weinstein, J. N. Targeting cancer micrometastases with monoclonal antibodies: a binding-site barrier. PNAS 92: 8999–9003 (1995). 52. Gao, H., Shi, W., and Freund, L. B. Mechanics of receptor-mediated endocytosis. PNAS 102(27): 9469–9474 (2005). 53. Aoyama, Y., Kanamori, T., Nakai, T., Sasaki, T., Horiuchi, S., Sando, S., and Niidome, T. Artificial viruses and their application to gene delivery. Size-controlled gene coating with glycocluster nanoparticles. J. A. Chem. Soc. 125(12): 3455–3457 (2003). 54. Nakai, T., Kanamori, T., Sando, S., and Aoyama, Y. Remarkably size-regulated cell invasion by artificial viruses. Saccharide-dependent self-aggregation of glycoviruses and its consequences in glycoviral gene delivery. J. Am. Chem. Soc. 125(28): 8465–8475 (2003). 55. Osaki, F., Kanamori, T., Sando, S., Sera, T., and Aoyama, Y. A quantum dot conjugated sugar ball and its cellular uptake. On the size effects of endocytosis in the subviral region. PNAS 126(21): 6520–6521 (2004). 56. Desai, M. P., Labhasetwar, V., Walter, E., Levy, R. J., and Amidon, G. L. The mechanism of uptake of biodegradable microparticles in Caco-2 cells is size dependent. Pharm. Res. 14(11): 1568–1573 (1997). 57. Prabha, S., Zhou, W.-Z., Panyam, J., and Labhasetwar, V. Size-dependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles. Int. J. Pharm. 244(1–2): 105–115 (2002). 58. Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. PNAS 92(16): 7297–7301 (1995).

c12.indd 225

7/30/2010 2:19:11 PM

226

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

59. Akinc, A., Thomas, M., Klibanov, A. M., and Langer, R. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J. Gene Med. 7(5): 657–663 (2005). 60. Behr, J. P. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 51: 34–36 (1997). 61. Sonawane, N. D., Szoka, F. C. Jr., and Verkman, A. S. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine–DNA polyplexes. J. Biol. Chem. 278(45): 44826–44831 (2003). 62. Wagner, E. Application of membrane-active peptides for nonviral gene delivery. Adv. Drug Deliv. Rev. 38(3): 279–289 (1999). 63. Luby-Phelps, K., Castle, P. E., Taylor, D. L., and Lanni, F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. PNAS 84: 4910–4913 (1987). 64. Suh, J., Wirtz, D., and Hanes, J. Efficient active transport of gene nanocarriers to the cell nucleus. PNAS 100: 3878–3882 (2003). 65. Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275(3): 1625–1629 (2000). 66. Ng, C. P., Goodman, T. T., Park, I.-K., and Pun, S. H. Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility. Biomaterials 30(5): 951–958 (2009). 67. Görlich, D. and Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15: 607–660 (1999). 68. Macara, I. G. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65: 570–594 (2001). 69. Subramanian, A., Ranganathan, P., and Diamond, S. L. Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat. Biotechnol. 17: 873–877 (1999). 70. Brunner, S., Sauer, T., Carotta, S., Cotten, M., Saltik, M., and Wagner, E. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7: 401–407 (2000). 71. Liu, X., Yang, J. W., Miller, A. D., Nack, E. A., and Lynn, D. M. Charge-shifting cationic polymers that promote self-assembly and self-disassembly with DNA. Macromolecules 38: 7907–7914 (2005). 72. Kircheis, R., Wightman, L., and Wagner, E. Design and gene delivery activity of modified polyethylenimines. Adv. Drug Deliv. Rev. 53(3): 341–358 (2001). 73. Wagner, E. Strategies to improve DNA polyplexes for in vivo gene transfer: Will “artificial viruses” be the answer? Pharm. Res. 21(1): 8–14 (2004). 74. Godbey, W. T., Wu, K. K., and Mikos, A. G. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. PNAS 96: 5177–5181 (1999). 75. Gosselin, M. A., Guo, W. and Lee, R. J. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjug. Chem. 12(6): 989–994 (2001). 76. Oupicky, D., Parker, A. L., and Seymour, L. W. Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors. J. Am. Chem. Soc. 124(1): 8–9 (2002).

c12.indd 226

7/30/2010 2:19:11 PM

REFERENCES

227

77. Pichon, C., LeCam, E., Guerin, B., Coulaud, D., Delain, E., and Midoux, P. Poly[Lys(AEDTP)]: a cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection. Bioconjug. Chem. 13(1): 76–82 (2002). 78. Forrest, M. L., Meister, G., Koerber, J., and Pack, D., Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm. Res. 21(2): 365–371 (2004). 79. Dunlap, D. D., Maggi, A., Soria, M. R., and Monaco, L. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res. 25(15): 3095–3101 (1997). 80. Savvaa, M., Aljaberia, A., Feiga, J., and Stolzb, D. B. Correlation of the physicochemical properties of symmetric 1,3-dialkoylamidopropane-based cationic lipids containing single primary and tertiary amine polar head groups with in vitro transfection activity. Colloids Surf. B Biointerfaces 43(1): 43–56 (2005). 81. Wu, D. C., Liu, Y., Jiang, X., He, C. B., Goh, S. H., and Leong, K. W. Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery. Biomacromolecules 7(6): 1879–1883 (2006). 82. Plank, C., Tang, M. X., Wolfe, A. R., and Szoka, F. C. Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum. Gene Ther. 10(2): 319–332 (1999). 83. Wadhwa, M. S., Collard, W. T., Adami, R. C., McKenzie, D. L., and Rice, K. G. Peptide-mediated gene delivery: influence of peptide structure on gene expression. Bioconjug. Chem. 8(1): 81–88 (1997). 84. Koping-Hoggard, M., Varum, K. M., Issa, M., Danielsen, S., Christensen, B. E., Stokke, B. T., and Artursson, P. Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers. Gene Ther. 11(19): 1441–1452 (2004). 85. MacLaughlin, F. C., Mumper, R. J., Wang, J., Tagliaferri, J. M., Gill, I., Hinchcliffe, M., and Rolland, A. P. Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J. Control. Release 56(1–3): 259–272 (1998). 86. Wolfert, M. A., Dash, P. R., Nazarova, O., Oupicky, D., Seymour, L. W., Smart, S., Strohalm, J., and Ulbrich, K. Polyelectrolyte vectors for gene delivery: influence of cationic polymer on biophysical properties of complexes formed with DNA. Bioconjug. Chem. 10(6): 993–1004 (1999). 87. Mishra, S., Webster, P., and Davis, M. E. PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles. Eur. J. Cell Biol. 83(3): 97–111 (2004). 88. Garrett, S. W., Davies, O. R., Milroy, D. A., Wood, P. J., Pouton, C. W., and Threadgill, M. D. Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery. Bioorg. Med. Chem. 8(7): 1779–1797 (2000). 89. Kaminskas, L. M., Karellas, P., Krippner, G. Y., Lessene, R., Kelly, B., and Porter, C. J. The impact of molecular weight and PEG chain length on the systemic pharmacokinetics of PEGylated poly l-lysine dendrimers. Mol. Pharm. 5: 449–463 (2008). 90. Rimann, M., Luhmann, T., Textor, M., Guerino, B., Ogier, J., and Hall, H. Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA. Bioconjug. Chem. 19: 548–557 (2008).

c12.indd 227

7/30/2010 2:19:11 PM

228

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

91. Choi, Y., Liu, F., Kim, J.-S., Choi, Y., Park, J., and Kim, S. Poly(ethylene glycol)grafted poly-l-lysine as polymeric gene carrier. J. Control. Release 54: 39–48 (1998). 92. Moghimi, S. M. Mechanisms regulating body distribution of nanospheres conditioned with pluronic and tetronic block co-polymers. Adv. Drug Deliv. Rev. 16 (2–3): 183–193 (1995). 93. Fischer, D., Osburg, B., Petersen, H., Kissel, T., and Bickel, U. Effect of poly(ethylene imine) molecular weight and pegylation on organ distrubition and pharmacokinetics of polyplexes with oligodeoxynucleotides in mice. Drug Metab. Dispos. 32: 983–992 (2002). 94. Mullen, P. M., Lollo, C. P., Phan, Q.-C., Amini, A., Banaszczyk, M. G., Fabrycki, J. M., Wu, D., Carlo, A. T., Pezzoli, P., Coffin, C. C., and Carlo, D. J. Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo. Biochim. Biophys. Acta Gen. Subjects 1523(1): 103–110 (2000). 95. Collard, W. T., Yang, Y., Kwok, K. Y., Park, Y., and Rice, K. G. Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates. J. Pharm. Sci. 89(4): 499–512 (2000). 96. Moret, I., Esteban Peris, J., Guillem, V. M., Benet, M., Revert, F., Dasí, F., Crespo, A., and Aliño, S. F. Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum. J. Control. Release 76(1–2): 169–181 (2001). 97. Ruponen, M., Honkakoski, P., Rönkkö, S., Pelkonen, J., Tammi, M., and Urtti, A. Extracellular and intracellular barriers in non-viral gene delivery. J. Control. Release 93(2): 213–217 (2003). 98. Itaka, K., Harada, A., Yamasaki, Y., Nakamura, K., Kawaguchi, H., and Kataoka, K. In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J. Gene Med. 6(1): 76–84 (2004). 99. Okuda, T., Niidome, T., and Aoyagi, H. Cytosolic soluble proteins induce DNA release from DNA-gene carrier complexes. J. Control. Release 98(2): 325–332 (2004). 100. Bhattacharya, S. and Mandal, S. S. Evidence of interlipidic ion-pairing in anioninduced DNA release from cationic amphiphile; DNA complexes. Mechanistic implications in transfection. Biochemistry 37(21): 7764–7777 (1998). 101. Huth, S., Hoffmann, F., Gersdorff, K.V., Laner, A., Reinhardt, D., Rosenecker, J., and Rudolph, C. Interaction of polyamine gene vectors with RNA leads to the dissociation of plasmid DNA-carrier complexes. J. Gene Med. 8(12): 1416–1424 (2006). 102. Xu, Y. and Szoka, F. C. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35(18): 5616–5623 (1996). 103. Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A., and Welsh, M. J. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270(32): 18997–19007 (1995). 104. Zhou, X. and Huang, L. DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim. Biophys. Acta Biomembranes 1189(2): 195–203 (1994).

c12.indd 228

7/30/2010 2:19:11 PM

REFERENCES

229

105. Legendre, J.-Y. and Szoka, F. C. Jr. Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes. Pharm. Res. 9(10): 1235–1242 (1992). 106. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. PNAS 84: 7413–7417 (1987). 107. Remy, J. S., Kichler, A., Mordvinov, V., Schuber, F., and Behr, J. P. Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses. PNAS 92: 1744–1748 (1995). 108. Liu, X., Yang, J. W., and Lynn, D. M. Addition of “charge-shifting” side chains to linear poly(ethyleneimine) enhances cell transfection efficiency. Biomacromolecules 9: 2063–2071 (2008). 109. Forrest, M. L., Koerber, J. T., and Pack, D. W. A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjug. Chem. 14(5): 934–940 (2003). 110. Lin, C., Zhong, Z., Lok, M. C., Jiang, X., Hennink, W. E., Feijen, J., and Engbersen, J. F. J. Novel bioreducible poly(amido amine)s for highly efficient gene delivery. Bioconjug. Chem. 18(1): 138–145 (2007). 111. Prata, C. A. H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D., McIntosh, T. J., Lee, S. J., and Grinstaff, M. W. Charge-reversal amphiphiles for gene delivery. J. Am. Chem. Soc. 126(39): 12196–12197 (2004). 112. Zhang, J. and Lynn, D. M. Ultrathin multilayered films assembled from chargeshifting cationic polymers: extended, long-term release of plasmid DNA from surfaces. Adv. Mater. 19(23): 4218–4223 (2007). 113. Oupicky, D., Konák, C., Ulbrich, K., Wolfert, M. A., and Seymour, L. W. DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers. J. Control. Release 65(1–2): 149–171 (2000). 114. Akinc, A., Lynn, D. M., Anderson, D. G., and Langer, R. Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery. J. Am. Chem. Soc. 125(18): 5316–5323 (2003). 115. Anderson, D. G., Lynn, D. M., and Langer, R. Semi-automated synthesis and screening of a large library of degradable cationic polymers for gene delivery. Angew. Chem. Int. Ed. 42(27): 3153–3158 (2003). 116. Jain, R., Standley, S. M., and Frechet, J. M. J. Synthesis and degradation of pHsensitive linear poly(amidoamine)s. Macromolecules 40(3): 452–457 (2007). 117. Lim, Y.-B., Kim, S.-M., Lee, Y., Lee, W.-K., Yang, T.-G., Lee, M.-J., Suh, H., and Park, J.-S. Cationic hyperbranched poly(amino ester): a novel class of DNA condensing molecule with cationic surface, biodegradable three-dimensional structure, and tertiary amine groups in the interior. J. Am. Chem. Soc. 123(10): 2460–2461 (2001). 118. Lim, Y.-B., Kim, S.-M., Suh, H., and Park, J.-S. Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjug. Chem. 13(5): 952–957 (2002). 119. Lynn, D. M., Anderson, D. G., Putnam, D., and Langer, R. Accelerated discovery of synthetic transfection vectors: parallel synthesis and screening of a degradable polymer library. J. Am Chem. Soc. 123(33): 8155–8156 (2001).

c12.indd 229

7/30/2010 2:19:11 PM

230

SELF-UNPACKING GENE DELIVERY SCAFFOLDS

120. Lynn, D. M. and Langer, R. Degradable poly(beta-amino esters): synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 122(44): 10761–10768 (2000). 121. Christensen, L. V., Chang, C.-W., Kim, W. J., Kim, S. W., Zhong, Z., Lin, C., Engbersen, J. F. J., and Feijen, J. Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery. Bioconjug. Chem. 17(5): 1233–1240 (2006). 122. Shim, M. S. and Kwon, Y. J. Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine. Biomacromolecules 9(2): 444–455 (2008). 123. Funhoff, A., van Nostrum, C., Janssen, A., Fens, M., Crommelin, D., and Hennink, W. Polymer side-chain degradation as a tool to control the destabilization of polyplexes. Pharm. Res. 21(1): 170–176 (2004). 124. Jiang, X., Lok, M. C., and Hennink, W. E. Degradable-brushed pHEMApDMAEMA synthesized via ATRP and click chemistry for gene delivery. Bioconjug. Chem. 18(6): 2077–2084 (2007). 125. Luten, J., Akeroyd, N., Funhoff, A., Lok, M. C., Talsma, H., and Hennink, W. E. Methacrylamide polymers with hydrolysis-sensitive cationic side groups as degradable gene carriers. Bioconjug. Chem. 17(4): 1077–1084 (2006). 126. Neu, M., Sitterberg, J., Bakowsky, U., and Kissel, T. Stabilized nanocarriers for plasmids based upon cross-linked poly(ethylene imine). Biomacromolecules 7(12): 3428–3438 (2006).

c12.indd 230

7/30/2010 2:19:11 PM

CHAPTER 13

Cellular Trafficking of Dendrimers YUNUS EMRE KURTOGLU and RANGARAMANUJAM M. KANNAN Department of Chemical Engineering and Materials Science, NICHD Perinatology Research Branch, Wayne State University, Detroit, Michigan

13.1

DENDRITIC ARCHITECTURE

Dendrimers are polymeric molecules made of multiple monomers branching radially from a central core. The monomers used determine the dendrimer subfamilies, while the number of branching points between the core and the surface of dendrimers determine their generation number. The monomer used for the synthesis, generation number, and surface groups determines the chemical, physical, and biological characteristics. For biological applications various dendrimer structures have been synthesized, which include polyamidoamines [1], poly(aryl ethers) [2], polyamines [3], polyesters [4, 5], nucleic acids [6, 7], polypeptides [8], and carbohydrates [9]. These dendrimers have shown great promise in diverse biological applications such as gene and drug delivery, magnetic resonance imaging (MRI), antivirals, and antibacterials, as well as tissue scaffolds [10]. In gene and drug delivery applications, usually the aim is a combination of drug solubility enhancement, tissue targeting, increasing blood circulation time, reduction in drug/gene metabolism rate, or overcoming biological barriers. Gene delivery strategies often involve complexation of nucleic acids to positively charged dendrimers, whereas most small drug delivery approaches involve covalent attachment of active molecules to dendrimer surface groups. Alternatively, encapsulation of drug molecules to the interior space of a dendrimer has also been investigated. In general, dendrimers have lower polydispersity indices compared to linear polymers due to their stepwise synthesis schemes and highly symmetrical branching patterns. The low polydispersity index is reflected by welldefined number of surface functional groups as compared to hyperbranched Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

231

c13.indd 231

7/30/2010 2:19:13 PM

232

CELLULAR TRAFFICKING OF DENDRIMERS

and linear polymers synthesized by statistically controlled polymerization reactions. As a result of the branching, higher generation dendrimers have more surface end groups and higher molecular weights. On the other hand, the surface groups get more densely packed as the generation numbers go up, which may increase the charge density on the surface depending on the end groups. Most dendrimers are not synthesized over generation 10 (G10), since the surface crowding does not leave any room for further branching and typical sizes for dendrimers vary between 2 and 10 nm. The lower generation dendrimers (up to G4) are generally ellipsoidal, whereas higher generations are more spherical in shape. Well-defined globular structure and the multivalency at their surfaces give dendritic structures some advantages over linear and hyperbranched polymers for drug delivery applications. While the well-defined structure can provide more uniform biodistribution and pharmacokinetics, the functional surface groups enable the attachment of multiple drugs, targeting ligands, and imaging agents. Due to their globular structures and surface charge densities the biological characteristics of dendrimers differ from linear polymers. This macromolecular architecture of dendrimers creates opportunities for cellular targeting and fine-tuning their uptake mechanisms and rates. The specific aim of this chapter is to discuss how the surface properties of these threedimensional (3D) globular polymers govern their cellular transport characteristics. Polyamidoamine (PAMAM) dendrimers have been most widely investigated among the other dendritic molecules perhaps due to their commercial availability and the various sizes and surface functional groups readily available. Therefore, in order to understand how the globular 3D structure and surface functionality affects the cellular uptake of dendritic structures, it is imperative to discuss how various types of PAMAM dendrimers and other dendrimers behave in a comparative manner. Mechanism and rate of cellular uptake on particular cell types and their fate after internalization are important considerations for drug delivery applications and these properties are closely related to surface characteristics. Cellular uptake of dendrimers is mainly affected by their size, the type and charge of surface groups, and the attachment of drugs or other moieties to their functional groups on the periphery. The mechanisms for transport of molecules through biological barriers include endocytosis, passive diffusion, and carrier-mediated and paracellular transport. Like other polymeric macromolecules, endocytotic mechanisms are responsible for cellular uptake of most dendrimers even though smaller and hydrophobic dendrimers may be able to enter the cells by diffusion through the cellular membrane [11]. Endocytosis is an active cellular uptake mechanism to internalize small and large macromolecules and particles. Two types of endocytotic processes have been defined: phagocytosis and pinocytosis. Phagocytosis involves the uptake of large particles and is a characteristic mechanism of white blood cells, whereas pinocytosis follows clathrin-coated pits, caveolae, or clathrin- and

c13.indd 232

7/30/2010 2:19:13 PM

DENDRITIC VERSUS LINEAR AND HYPERBRANCHED POLYMERS

233

caveolae-independent pathways. The pathway by which a macromolecule is taken up can vary with the cell type and the proteins involved in the intracellular vesicles and size of these vesicles, as well as the intracellular fate of the macromolecule. Typically, after internalization polymers are transported to early endosomes followed by late endosomes and later in catalytic environment of lysosomes. The cellular uptake rates and residence times in intracellular compartments are important for designing dendrimer-based therapeutics since they often rely on release of covalently linked drugs intracellularly.

13.2 DENDRITIC VERSUS LINEAR AND HYPERBRANCHED POLYMERS The rate and mechanism of cellular uptake of G2, G3, and G4 amine terminated (cationic) PAMAM dendrimers were investigated in comparison to linear and branched polyethylene imine (PEI) polymers, which are also cationic with amine functional groups [12]. The polymers were labeled by Oregon green (through conjugation), and their cell membrane binding, endocytosis, and exocytosis characteristics were studied on B16F10 melanoma cells. While the cellular uptake mechanism of all the polymers was determined to be endocytosis, the hyperbranched PEI and the PAMAM dendrimers had cholesteroldependent pathways, whereas linear PEI uptake was cholesterol and clathrin independent. The rate of uptake was the highest for G4 PAMAM followed by branched PEI, linear PEI, and G3 and G2 PAMAM. The globular structure and the cationic surface charge density of G4 PAMAM is believed to enhance the cellular uptake by adsorptive endocytosis compared to lower generation dendrimers and PEI polymers. Particularly, the surface functional groups of the dendrimer can produce a highly localized charge density, which can have a significant influence on its interactions with the negatively charged proteoglycans in the cell membrane. All the polymers showed significant membrane binding, which suggests the cationic charge may give rise to nonspecific absorption. This type of adsorptive endocytosis usually follows curvilinear cell uptake kinetics due to saturation of the membrane binding sites. Cellular entry dynamics of amine-terminated G4 and G3 PAMAM dendrimers and hydroxyl-terminated PAMAM dendrimers were studied with human lung epithelial carcinoma cells (A549) in comparison to hyperbranched polyol polymer (Figure 13.1) [13]. Additionally, the amine-terminated G3 PAMAM dendrimer surface was modified with poly(ethylene glycol) PEG to investigate the PEG attachment effects on cellular uptake kinetics. Approximately 90% of amine-terminated PAMAM dendrimers (G3 and G4) entered the cells within 1 hour while the initial rate of cell entry of G4-NH2 was faster compared to that of G3-NH2 (Figure 13.2). In comparison, approximately 55% of G3 PEG and 75% of polyol were inside the cells in 1 hour. G4 PAMAM dendrimers were taken up by cells more rapidly when compared to G3 dendrimers with or without PEG modification and polyol. The higher cell

c13.indd 233

7/30/2010 2:19:13 PM

c13.indd 234

234 H2N H2N H2N

H2N

NH O NH O N

OH

H2N NH2

NH O

NH2 NH NH2 O NH NH2 O NH NH2 N O O NH NH NH2 O NH N NH NH O O N O N NH O O NH NH NH OO N N HN NH O O N

NH H2N O NH NH H2N O N O N NH N HO O N NH H2N N O NH O H2N HO N N O N NO H H NH N NO O H N NHO O NHNH2 N H2N O NH O O O O O H2N NH N O N NH N NH N H N NH N N H N NH O NHO O O NH OO O NH NH2 NH NH NH H2N O NH2 N N O N N O N NH H2N H O N O NH O NH NH O O NH O N H2N NH NH NH O NH H2N NH NH 2 N OO O 2 N NH O NH NH2 NH O NH H2N NH2 NH H2N NH2 H2N

PAMAM Dendrimer

HO O

O

HO OH HO

OH O

O

O

O

O

O

O O

O

OH OH

OH

HO OH HO

O

O

O O

OH OH O O O OH

O O O

HO

O

HO HO

O

O

O O

HO O O HO O HO HO

O

O

O O O

O

O O

O

O O

O

O

O O

O

O

O

O O O

OO O

O O

OO

O

OH HO

OH

O

O

OH OH O

OH OH OH

O O

O HO OH O OH HO HO OH

O

HO

OH O

O

Hyperbranched polyol

Figure 13.1. Schematic representation of the PAMAM dendrimer (left) and the polyol hyperbranched polymer (right). The “imperfect” branching pattern in the hyperbranched polymer can be seen [13]. 7/30/2010 2:19:13 PM

DENDRITIC VERSUS LINEAR AND HYPERBRANCHED POLYMERS

235

% Change in absorbance

100 80 G3 PEG Polyol G4 OH G4-NH2

60 40 20 0

0

1

2

3 4 TIme (hours)

5

6

7

6

7

% Change in absorbance

100 80 60 G4-OH Polyol G3 PEG G4-NH2

40 20 0 0

1

2

3 4 TIme (hours)

5

Figure 13.2. Effect of end groups on cellular entry profile: (top) supernatant analysis, (bottom) cell lysate analysis [13]. A549 cells were treated separately with each of the dendrimers and hyperbranched polymer was tagged with fluorescent dye. The supernatant was removed at times 0, 30, 60, 120, 240, and 360 min. The amount of tagged dendrimer in the supernatant was estimated by UV/Vis absorbance at 492 nm. The cells were then washed with PBS, trypsinized, and centrifuged to obtain a cell pellet. The cell pellet was then lysed with cell lysate buffer and the amount of tagged dendrimer in the lysate was quantified. An increase in the fluorescent activity in the cell lysate along with a corresponding decrease in activity in the supernatant indicates the intracellular entry of the tagged dendrimer.

entry rate of amine-terminated G4 PAMAM compared to G3 PAMAM was explained by the larger amount of surface charges on the G4 dendrimer. The other polymers studied, namely, hydroxyl-terminated G4 PAMAM, polyol, and G3 PAMAM-PEG, do not carry cationic surface charges and their cellular uptake is associated with nonspecific adsorption to cell membrane. The hydroxyl-terminated G4 PAMAM entered the cells more rapidly compared to other uncharged polymers, suggesting that the globular dendrimer structure is contributing to the enhanced cellular uptake rates. PEG modification of amine-terminated G3 PAMAM reduced its cellular uptake rates due to reduced ionic interaction with the cell membrane and therefore reduced

c13.indd 235

7/30/2010 2:19:13 PM

236

CELLULAR TRAFFICKING OF DENDRIMERS

surface adsorption that would lead to endocytosis. When the noncationic polymers were compared for their cellular uptake rates, the dendritic structure enhances the cellular uptake compared to hyperbranched polyol. These studies must be viewed as somewhat qualitative and relative, since quantification would require more detailed work.

13.3

SURFACE CHARGE

G4 PAMAM dendrimers with 64 (calculated) amine (cationic), carboxyl (anionic), or hydroxyl (neutral) surface groups were studied for their cellular uptake mechanisms on A549 lung epithelial cells, which are known to have a negative surface charge [14]. The three types of PAMAM dendrimers were tagged with fluoroisothiocyanate (FITC) and their cellular uptake was investigated by flow cytometry and fluorescence microscopy analysis. The cellular uptake rates of the three dendrimers were analyzed in comparison to each other (Figure 13.3). Rate of cell uptake was highest for the cationic dendrimer, followed by anionic and neutral dendrimers. In order to identify specific uptake mechanisms, inhibitors of specific endocytotic pathways were used in each group. Regardless of their surface charge, all PAMAM dendrimers studied were endocytosed by energy-dependent, fluid-phase endocytosis while their specific endocytosis uptake mechanisms varied. Anionic dendrimers were partly taken up by caveolae, while the cationic and anionic dendrimers were 100

%Cell entry

80 60 40 COOH OH NH

20 0 0

1

2 Time (h)

3

4

Figure 13.3. A549 cell entry profile of G4-NH2, G4-OH, and G3.5-COOH PAMAM dendrimers [14]. Rate of cell entry is determined as percent of (cell entry at time t/cell entry at 3 h). The cellular uptake was quantified by flow cytometry using fluorescence. A comparison of the time-dependent fluorescence intensity levels indicates that rate of cell uptake was highest for the cationic dendrimer, followed by anionic and neutral dendrimers. The cell uptake for cationic dendrimer plateaus off after 1 hour, unlike with the other two dendrimers where the cell entry increased more or less linearly with the treatment time, as shown in the graph. (See color insert.)

c13.indd 236

7/30/2010 2:19:13 PM

SURFACE CHARGE

237

(A)

(B)

(C)

Figure 13.4. Confocal microscopic images (63×) of A549 lung epithelial cells after treatment with different dendrimers and LysoTracker for 5 min followed by chase for 30 min in serum-free medium devoid of any dendrimers. Images indicate cells treated with (A) FITC-labeled PAMAM-G4-NH2 dendrimer and LysoTracker. (B) FITClabeled PAMAM-G4-OH dendrimer and LysoTracker. (C) FITC-labeled PAMAMG3.5-COOH dendrimer and LysoTracker. The left-hand panel shows the green fluorescence from the dendrimer, the middle panel shows the red fluorescence from the LysoTracker, and the right panel shows the yellow fluorescence due to the colocalization of green dendrimer–FITC and red LysoTracker in the lysosomes. LysoTracker dye preferentially partitions to the lysosome, giving an intense red color in the acidic environment of the lysosomes. From the colocalization studies, it is evident that the dendrimers are in the lysosomes within 30 min of their transport from the cell membrane [14]. (See color insert.)

taken up by a non-clathrin- and non-caveolae-mediated endocytosis mechanism in A549 cells. Furthermore, the surface charge of PAMAM dendrimers seems to play a role in their intracellular trafficking. After cellular uptake, extent of cationic dendrimer that resides in the lysosomes was less than that of anionic and neutral dendrimers, whereas the cationic dendrimer was found in greater extent in peripheral vesicles (Figure 13.4). This finding is

c13.indd 237

7/30/2010 2:19:13 PM

238

CELLULAR TRAFFICKING OF DENDRIMERS

significantly important for dendrimer–drug conjugate therapies that rely on lysosome enzyme activity for detachment of drug from its carrier. The greater lysosomal residence time for the anionic and neutral dendrimer can provide longer release times, whereas cationic dendrimer conjugates may have less time in the lysosome compartment, thus requiring faster releasing covalent linkages. Therefore the surface charge of the dendrimers can be utilized to modulate the cell entry kinetics, mechanisms, and residence time in intracellular compartments.

13.4

SURFACE MODIFICATION

Human lung epithelial carcinoma cells were also used to investigate the efficacy of PAMAM dendrimer conjugates of ibuprofen (Ibu) [15] and methylprednisolone (MP) [16]. The cellular uptake along with their in vitro efficacy were reported. MP was conjugated to carboxyl terminated G2.5 PAMAM and hydroxyl-terminated G4-OH PAMAM dendrimers while the conjugates were also tagged with FITC. The conjugates were rapidly internalized within minutes, while the uptake continued for up to 4 hours. The localization of the conjugates was studied using fluorescence and confocal microscopy. The in vitro efficacy of MP-PAMAM conjugates was studied by inhibition of prostaglandin secretion. The efficacy of the conjugated MP was comparable to free MP treatment, suggesting that the dendritic delivery approach was successful. Even though the dendrimer may enhance the uptake of MP, the drug has to be released from the dendrimer for prostaglandin suppression. The fact that the conjugate shows comparable efficacy at short times (4 hours), when the drug release is expected to be very small, suggests that the delivery is significantly better with the dendrimer. Ibuprofen complexes of G4 PAMAM dendrimers were also studied for their cell entry. Compared to free Ibu the PAMAM complexes had higher cell entry rates, and the complexes were taken up to significant levels within 1 hour. When ibuprofen was attached to hydroxyl-terminated G4 PAMAM dendrimer and the cellular uptake rates were studied, it was determined that the conjugates entered the cells rapidly with ∼30% uptake in 15 minutes. The punctuated distribution of fluorescence in the cytoplasm determined by confocal and fluorescence microscopy images suggested that the cellular uptake was through endocytotic pathways. Intracellular trafficking of G3 PAMAM dendrimers with amine functionality in the human colon adenocarcinoma HT-29 cell line was also studied. PAMAM dendrimer surface was modified with lauroyl chains, propranolol molecules, or both among with FITC tagging [17]. Cellular uptake of unmodified and propranolol-modified G3 PAMAM dendrimers was determined to be by both caveolae-dependent endocytosis and macropinocytosis pathways, whereas when both surface modifications were present internalization of

c13.indd 238

7/30/2010 2:19:13 PM

PERMEABILITY THROUGH BIOLOGICAL BARRIERS

239

dendrimer was caveolae dependent, and possibly clathrin-dependent. Cell entry pathways of lauroyl chain-modified G3 PAMAM were caveolae dependent, clathrin dependent, and macropinocytosis. All the modified G3 PAMAM dendrimers were trafficked to endosomes and lysosomes after internalization. Modification of G3 PAMAM dendrimer with lauroyl moieties increased the cellular uptake rates. Furthermore, lauroyl modification of dendrimer surface reduced the extent of lysosomal accumulation. The impact of PEG modification on cellular uptake kinetics of carboxylterminated PAMAM dendrimers was also studied with Caco-2 cells with focus on transepithelial transport [18–20]. Cellular uptake and transport of G3.5 and G4.5 PAMAM dendrimers with three levels of PEG surface modification were studied. PEG modification of the surface significantly decreased the transepithelial transport for both generations, whereas the cellular uptake showed generation dependency. G3.5 dendrimer cellular uptake was largely reduced by attachment of PEG to the surface in contrast to a strong reduction in uptake for G4.5 PAMAM with PEG modification. The degree of PEG modification did not create a significant difference in cellular uptake of G3.5 dendrimers and regardless of surface groups modified the cellular uptake was decreased. On the other hand, for G4.5 PAMAM dendrimers the lowest extent of PEG surface modification showed the most improvement over the unmodified dendrimer while further PEG modification actually reduced the cellular uptake compared to less surface modification. The conformation of flexible PEG chains on the surface are believed to be determining parameters for the different characteristics for the two generations studied. The reduction in transepithelial permeability was about 65% for both G3.5 and G4.5 PAMAM dendrimers upon PEG modification as compared to permeability of unmodified G3.5 and G4.5 PAMAM dendrimers.

13.5

PERMEABILITY THROUGH BIOLOGICAL BARRIERS

For oral delivery of dendrimer drug conjugates, it is important to evaluate the permeability though epithelial barriers. For determination of absorption and cell entry characteristics human epithelial colorectal adenocarcinoma cells (Caco-2) are often employed [21, 22]. To demonstrate the influence of size and charge on transport of PAMAM dendrimers across Caco-2 cells, extensive studies were carried out using positive, neutral, and negative charged PAMAM dendrimers [23]. Permeability increased with an increase in the number of anionic surface groups, thus generation number in the carboxyl-terminated PAMAM dendrimers also increased. Cationic G2 PAMAM had greater permeability than hydroxyl-terminated G2 and carboxyl-terminated G1.5 and G2.5 dendrimers. Anionic dendrimer G3.5 PAMAM and cationic G4 PAMAM modified with FITC molecules showed the highest transport rates. Increase in the attachment of hydrophobic FITC label increased the permeability and

c13.indd 239

7/30/2010 2:19:13 PM

240

CELLULAR TRAFFICKING OF DENDRIMERS

reduced the toxicity of cationic dendrimers. The overall permeabilities of various PAMAM dendrimers through epithelial cells were determined as G3.5COOH > G2NH2 > G2.5COOH > G1.5COOH > G2OH. The enhanced PAMAM permeability was attributed to opening of tight junctions in cells via paracellular transport across the Caco-2 cells. Furthermore, depending on their surface properties, PAMAM dendrimers are transported across epithelial barriers to a higher extent than some of the conventional linear watersoluble polymers. Epithelial permeability of cationic dendrimers decreases with size and in contrast the permeability of anionic dendrimers increases with their size. The charged dendrimers had greater permeability than neutral dendrimers, which has no net surface charge at physiological pH to enable its interaction with cell monolayers and between the cationic and anionic, the cationic dendrimers exhibit higher permeability. Furthermore, the cationic dendrimers are transported by a combination of paracellular and endocytotic mechanisms. The trafficking of PAMAM dendrimers to endosomal and lysosomal compartments in Caco-2 cells is rapid and mediated by a clathrin-dependent endocytosis mechanism. This was confirmed from the uptake of amineterminated G4 PAMAM using endocytosis inhibitors such as brefeldin A, colchicine, filipin, and sucrose. In the presence of these inhibitors the uptake and permeability of the G4 PAMAM were significantly lowered [24, 25]. The anionic G4 PAMAM dendrimers are taken up by caveolae-mediated endocytosis in A549 lung epithelial cells and the cationic and neutral G4 PAMAM dendrimers are internalized by a non-clathrin-, non-caveolae-mediated mechanism involving electrostatic interactions or other nonspecific fluid-phase endocytosis. These studies revealed that PAMAM dendrimer internalization by endocytosis is largely dependent on the types of cells targeted in addition to the surface charge, molecular weight, and generation considerations. Therefore the differences in cell uptake mechanism of dendrimers may be cell type dependent. This dependency requires that dendritic devices may need to be tailored for each application and for ultimate cell type being targeted for optimal cellular uptake kinetics. In another study, 125I-labeled anionic G1.5 and G2.5 PAMAM dendrimers were transported across everted rat intestinal tissue much faster than other linear polymers such as polyvinylpyrrolidone (PVP), poly(N-vinylpyrollidoneco-maleic anhydride) (NVPMA), and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers [26]. While 80% of PAMAM radioactivity was transferred directly across to the serosal fluid, 20% of the anionic PAMAM dendrimer remained associated with the tissue. On the other hand, when the studies were carried out with G3 and G4 amine-terminated cationic PAMAM dendrimers, 60% of the dendrimer radioactivity was found in the tissue whereas 40% was transferred into the serosal fluid. This observation clearly demonstrates the increased interactions between the cationic dendrimers and the cell membranes.

c13.indd 240

7/30/2010 2:19:13 PM

INTRACELLULAR TRAFFICKING AND EFFICACY

13.6

SIRNA

241

OLIGONUCLEOTIDE DELIVERY

siRNA therapeutics have generated a lot of interest in drug delivery research. However, their low resistance against enzymatic degradation, poor cellular uptake, and rapid renal and liver clearance have limited the in vivo applications. In order to address these issues, PAMAM dendrimers were investigated as carriers for antisense and siRNA oligonucleotides. Amine-terminated G5 PAMAM dendrimer was complexed with antisense and siRNA oligonucleotides using the cationic charge of the dendrimer surface and the negative charge of the oligonucleotides [27]. Dendrimer–oligonucleotide complexes were further modified by covalent attachment of a cell-penetrating peptide that is cationic in nature. BODIPY fluorescent molecules were attached to dendrimers for visualization, whereas Cy-5 fluorescent molecules were used for oligonucleotide tracking. The efficacies and the cellular uptake of the complexes were studied on NIH 3T3 fibroblast cells transfected with a plasmid containing the human MDR1 gene. G5 PAMAM–oligonucleotide complexes were effectively internalized and accumulated in intracellular vesicles as determined by confocal microscopy and flow cytometry. Conjugation of cellpenetrating peptide to the G5 PAMAM increased the cellular uptake by 25%, whereas this enhanced uptake did not improve the effectiveness of therapy, which was studied by inhibition of P-glycoprotein expression. G5 PAMAM delivery systems, both with and without peptide modification, were moderately effective in intracellular delivery of antisense oligonucleotide. As delivery vehicles, the effectiveness of dendrimers was comparable to Lipofectamine 2000 as a positive control. The confocal microscopy images suggested that both G5 PAMAM with and without peptide modification, complexed with either siRNA or antisense oligonucleotide, had similar intracellular distribution with most of the material associated with intracellular vesicles while a small portion of siRNA and antisense oligonucleotides were found in the cytoplasm and nucleus. Therefore amine-terminated G5 PAMAM was equally effective in carrying the oligonucleotides into the cells, but overall effectiveness varied due to the differences in siRNA and antisense nucleotide characteristics.

13.7 INTRACELLULAR TRAFFICKING AND EFFICACY Efficacy of the dendrimer–drug conjugates is usually dependent on the release of the active molecules from the dendritic carrier. The activity of small drugs are often suppressed or completely blocked in their conjugated form. It is important to design systems that will release their payload intracellularly at predefined rates. Intracellular trafficking characteristics of dendrimer-based delivery systems such as cellular uptake rates, lysosomal residence time, exocytosis rates, and ability to exit lysosomes to reach the cytoplasm determine the overall extent of drug release. The drug release rates of covalently linked

c13.indd 241

7/30/2010 2:19:13 PM

242

CELLULAR TRAFFICKING OF DENDRIMERS

dendrimer–drug conjugates vary with the intracellular conditions, such as availability and activity of the lysosomal enzymes and acidity of the intracellular compartment, along with time spent under these conditions. Therefore the intracellular activation of the drugs being delivered as well as the efficacy of the dendrimer conjugate system will be in direct relation with its intracellular trafficking properties. The effects of dendrimer surface functional groups on the efficacy of the dendrimer–drug conjugates were investigated with PAMAM dendrimer– methotrexate (MTX) conjugates [28]. The efficacy of amine- and carboxylterminated PAMAM–MTX conjugates were evaluated in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer–MTX conjugates were prepared with carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX and another between an amineterminated G3 dendrimer and the carboxylic acid group of the MTX. Carboxylic acid-terminated G2.5 dendrimer conjugate showed an increased drug activity compared to free MTX toward both sensitive and resistant cell lines, whereas amine-terminated dendrimer conjugate did not show significant activity on any of the cell lines. The successful enhancement in cellular entry of MTX by carboxyl-terminated dendrimer conjugation was evidenced by 8and 24-fold enhancement in IC50 values toward MTX-resistant CCRF-CEM and CHO cells, respectively, which otherwise had impaired MTX transport mechanisms. The difference in efficacy of these amide-bonded conjugates was associated with the intracellular drug release from the cationic dendrimer versus the anionic dendrimer, due to the differences in lysosomal residence times dictated by the surface functionality. Carboxyl-terminated dendrimer conjugates may have longer lysosomal residence time and longer period of drug release compared to cationic PAMAM conjugate. Dendrimer–drug conjugates that depend on cytoplasmic conditions rather than lysosomal drug release pathways were also designed. An amineterminated G4 PAMAM dendrimer-N-acetyl cysteine (NAC) conjugate that contains a disulfide linkage was synthesized and evaluated for its efficacy on activated BV-2 microglial cells [29, 30]. The drug release pathway of the conjugates were glutathione (GSH) dependent, which is the most abundant thiol species in the cytoplasm functioning as the major reducing agent in biochemical processes. G4 PAMAM–NAC conjugate rapidly internalized by the cells within 15 minutes and the internalization continued for up to 4 hours. The conjugates showed an order of magnitude increase in antioxidant activity compared to free NAC, which suggests that the conjugate was successfully exiting the lysosomes and releasing its payload in the reductive cytoplasmic environment. Since the antioxidant effect of NAC is associated with its thiol group, which is occupied when in conjugated form, the conjugate would have to release the NAC to have efficacy. After the conjugates are taken up by endocytosis, they will reside in the lysosomes for a period of time, where the release of NAC may be relatively slow, because of the lower thiol content

c13.indd 242

7/30/2010 2:19:13 PM

CONCLUSION

243

in lysosomes. As the conjugate escapes the lysosomal compartment, the NAC will be released into the cytoplasm. Therefore the lysosomal residence times of these types of dendrimer conjugates will ultimately affect their efficacy.

13.8

KEY ASPECTS IN DATA INTERPRETATION

Intracellular trafficking of dendrimers is often studied by attachment of some fluorescent molecules or other imaging agents followed by visualization of the uptake by confocal microscopy or quantification via flow cytometry techniques. Attachment of multiple copies of drugs to the functional surface groups of the dendrimers may alter their uptake characteristics. For a given dendrimer type, clearly the intracellular trafficking can be quite different if the dendrimer is conjugated to different drugs in two studies. The labeling of dendrimers with fluorescent molecules followed by in vitro visualization, identification of compartments, and colocalization studies via fluorescent or confocal microscopy techniques have some other limitations. Often, the fluorescent molecules have pH-dependent fluorescence or pH-dependent quenching properties. Concentration-dependent quenching is also observed in such studies. When considering the endocytotic uptake pathway of most dendrimers, such pH dependency can lead to artifacts, since the lysosomal pH is considerable different from that of the cytoplasm. Furthermore, the lysosomal accumulation could lead to a high enough concentration to introduce concentration-dependent quenching. Therefore it is important to evaluate such cellular uptake data with caution. The cellular uptake studies performed with such systems should be used as a guide for better delivery system designs, while efficacy of the ultimate therapeutic version of the dendritic delivery systems (without imaging agent) should be used for evaluating performance. Another consideration in studying cellular uptake and trafficking of dendrimers is the pathway inhibition strategies used for blocking certain pathways. These inhibition mechanisms are usually very complex and it is reported that one inhibitor can upregulate another cellular uptake mechanism under certain conditions. The experimental conditions can alter the uptake pathway of macromolecules completely even with the same cell type. The knowledge from a cell line that is used for the dendrimer uptake studies may not be readily transferable to other cell types due to variability in their membrane and intracellular trafficking characteristics.

13.9

CONCLUSION

Dendritic architecture creates opportunities for designing well-defined nanosized gene and drug delivery devices for tissue, cell, and even intracellular targeting. Due to their unique structure and advantages over linear or

c13.indd 243

7/30/2010 2:19:13 PM

244

CELLULAR TRAFFICKING OF DENDRIMERS

hyperbranched polymers in drug delivery applications, dendrimer research is receiving growing interest. In addition to cellular and intracellular targeting of dendritic devices, new applications are under investigation to overcome biological barriers such as gastrointestinal layers, amniotic membranes, and the blood–brain barrier. In order to create successful dendrimer-based designs, a solid understanding of intracellular trafficking and cellular uptake properties is required. The most important parameters to be optimized will be regarding surface properties of the dendrimers, such as size, overall charge, surface charge and surface charge density, the extent of drug or targeting moiety attachment, hydrophobicity, and hydrogen bonding capacity. Most of these parameters are interrelated with each other in every system; therefore researchers will have to gain strong fundamentals in engineering the surface of these globular macromolecules.

REFERENCES 1. Tomalia, D. A., Naylor, A. M., and Goddard, W. A. Starburst dendrimers: molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem. Int. Ed. Engl. 29: 138–175 (1990). 2. Hawker, C. J. and Fréchet, J. M. J. Preparation of polymers with controlled molecular architecture. A new convergent approach to dendritic macromolecules. J. Am. Chem. Soc. 112: 7638–7647 (1990). 3. de Brabander-van den Berg, E. M. M. and Meijer, E. W. Poly(propylene imine) dendrimers: large-scale synthesis by hetereogeneously catalyzed hydrogenations. Angew. Chem. Int. Ed. Engl. 32: 1308–1311 (1993). 4. Ihre, H., Hult, A., and Söderlind, E. Synthesis, characterization, and 1H NMR self-diffusion studies of dendritic aliphatic polyesters based on 2,2bis(hydroxymethyl)propionic acid and 1,1,1-tris(hydroxyphenyl)ethane. J. Am. Chem. Soc. 118: 6388–6395 (1996). 5. Grinstaff, M. W. Biodendrimers: new polymeric biomaterials for tissue engineering. Chemistry 8: 2838–2846 (2002). 6. Nilsen, T. W., Grayzel, J., and Prensky, W. Dendritic nucleic acid structures. J. Theor. Biol. 187: 273–284 (1997). 7. Li, Y., et al. Controlled assembly of dendrimer-like DNA. Nat. Mater. 3: 38–42 (2004). 8. Sadler, K. and Tam, J. P. Peptide dendrimers: applications and synthesis. J. Biotechnol. 90: 195–229 (2002). 9. Turnbull, W. B. and Stoddart, J. F. Design and synthesis of glycodendrimers. J. Biotechnol. 90: 231–255 (2002). 10. Svenson, S. and Tomalia, D. Dendrimers in biomedical applications—reflections on the field. Adv. Drug Deliv. Rev. 57: 2106–2129 (2005). 11. Duncan, R. and Izzo, L. Dendrimer biocompatibility and toxicity. Adv. Drug Deliv. Rev. 57: 2215–2237 (2005).

c13.indd 244

7/30/2010 2:19:13 PM

REFERENCES

245

12. Seib, F. P., Jones, A. T., and Duncan, R. Comparison of the endocytic properties of linear and branched PEIs, and cationic PAMAM dendrimers in B16f10 melanoma cells. J. Control. Release 117: 291–300 (2007). 13. Kolhe, P., et al. Drug complexation, in vitro release and cellular entry of dendrimers and hyperbranched polymers. Int. J. Pharm. 259: 143–160 (2003). 14. Perumal, O. P., et al. The effect of surface functionality on cellular trafficking of dendrimers. Biomaterials 29: 3469–3476 (2008). 15. Kolhe, P., et al. Preparation, cellular transport, and activity of polyamidoaminebased dendritic nanodevices with a high drug payload. Biomaterials 27: 660–669 (2006). 16. Khandare, J., et al. Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates. Bioconjug. Chem. 16: 330–337 (2005). 17. Saovapakhiran, A., et al. Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization. Bioconjug. Chem. 20: 693–701 (2009). 18. Sweet, D. M., Kolhatkar, R. B., Ray, A., Swaan, P., and Ghandehari, H. Transepithelial transport of PEGylated anionic poly(amidoamine) dendrimers: implications for oral drug delivery. J. Control. Release 138: 78–85 (2009). 19. Kim, Y., et al. Systematic investigation of polyamidoamine dendrimers surfacemodified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity. Bioconjug. Chem. 19: 1660–1672 (2008). 20. Kitchens, K. M., et al. Transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers: influence of size, charge and fluorescent labeling. Pharm. Res. 23: 2818–2826 (2006). 21. Kolhatkar, R. B., et al. Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability. Bioconjug. Chem. 18: 2054–2060 (2007). 22. Pisal, D. S., et al. Permeability of surface-modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers. Int. J. Pharm. 350: 113–121 (2008). 23. Yang, H., et al. Stealth dendrimers for drug delivery: correlation between PEGylation, cytocompatibility, and drug payload. J. Mater. Sci. Mater. Med. 19: 1991–1997 (2008). 24. Kitchens, K. M., et al. Endocytosis inhibitors prevent poly(amidoamine) dendrimer internalization and permeability across Caco-2 Cells. Mol. Pharm. 5: 364–369 (2008). 25. Kitchens, K. M., et al. Transepithelial and endothelial transport of poly(amidoamine) dendrimers. Adv. Drug Deliv. Rev. 57: 2163–2176 (2005). 26. Malik, N., Wiwattanapatapee, R., Klopsch, R., Lorenz, K., Frey, H., Weener, J. W., Meijer, E. W., Paulus, W., and Duncan, R. Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J. Control. Release 65: 133–148 (2000). 27. Kang, H., DeLong, R., Fisher, M. H., and Juliano, R. L. Tat-conjugated PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides. Pharm. Res. 2099–2106 (2005).

c13.indd 245

7/30/2010 2:19:13 PM

246

CELLULAR TRAFFICKING OF DENDRIMERS

28. Gurdag, S., et al. Activity of dendrimer–methotrexate conjugates on methotrexatesensitive and -resistant cell lines. Bioconjug. Chem. 17: 275–283 (2006). 29. Navath, R. S., et al. Dendrimer drug conjugates for tailored intracellular drug release based on glutathione level. Bioconjug. Chem. 19: 2446–2455 (2008). 30. Kurtoglu, Y. E., et al. Poly(amidoamine) dendrimer–drug conjugates with disulfide linkages for intracellular drug delivery. Biomaterials 30: 2112–2121 (2009).

c13.indd 246

7/30/2010 2:19:14 PM

CHAPTER 14

Endolysosomolytically Active pHSensitive Polymeric Nanotechnology HAN CHANG KANG and YOU HAN BAE Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah

14.1

SITE-SPECIFIC NANOTHERAPEUTICS

Interest in developing pharmaceutical nanotechnology has blossomed with the desire to develop effective systems for delivering various therapeutics (e.g., from low molecular weight chemical drugs and imaging agents to high molecular weight peptides, proteins, and genetic materials) to specific sites of interest (e.g., organs, tissues, cells, cytoplasm, mitochondria, perinuclear regions, and nucleus). Unlike traditional dosage forms, site-specific nanotherapeutics are designed to maximize the bioavailability of the delivered therapeutics at the target sites and have shown beneficial therapeutic efficacy in treating diseases with reduced side effects [1]. Delivering nanosized therapeutic carriers to their target sites can be achieved by exploiting differences in anatomy, pathology, or cellular events between target and nontarget sites. First, anatomical specificity of the organs or tissues of interest can passively drive major accumulation of therapeutics. Hydrodynamic injection for liver targeting [2] and the enhanced permeability and retention (EPR) effect for tumor accumulation [3] are known examples of passive organ/tissue targeting. Second, nanosystems can selectively target solid tumors using pathological differences. For example, the local pH of solid tumors is lower than the extracellular pH of normal tissues, creating the opportunity for pH targeted delivery [4]. (Please refer to Lee et al. [4] for more detailed information.) Third, targeting specific cells has been attained mostly by utilizing particular interactions between ligands and cell-specific or

Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

247

c14.indd 247

7/30/2010 4:12:17 PM

248

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

overexpressed receptors located on the plasma membrane. These ligand– receptor interactions accelerate the internalization rates of therapeutics and their carriers into the target cells and enhance the intracellular accumulation [5]. However, if the final destination of therapeutics carried by nanocarriers is subcellular compartments (e.g., cytoplasm, mitochondria, perinuclear areas, and nucleus), the therapeutics or their carriers should be able to escape from the endolysosomal pathways in order to achieve maximum therapeutic effects [6].

14.2

DESTABILIZATION OF ENDOLYSOSOMAL COMPARTMENTS

Therapeutics or nanocarriers larger than 1 kDa rarely cross the plasma membrane without the aid of cell-penetrating peptides [7]. These systems use other cellular entry mechanisms such as endocytosis: that is, the cellular membrane invaginates to engulf therapeutics or pharmaceutical nanosystems and forms intracellular membrane-bound vesicles (or endosomes). The therapeuticsloaded vesicles mature from early endosomes (neutral pH to approximately pH 6) to late endosomes (approximately pH 5 to pH 6) as acidification occurs due to cytosolic protons (H+) pumped into the endosomes by vacuolar ATPase-H+ pumps. As endosomes acidify even further, late endosomes merge with lysosomes (approximately pH 4 to pH 5), which contain various lytic enzymes [1, 7]. In lysosomes, the sequestrated therapeutics may be degraded by lytic enzymes, resulting in reduced bioavailability at the target site. To avoid such a scenario, therapeutics-carrying nanosystems should recognize endolysosomal characteristics, which are easily distinguishable from the extracellular and intracellular environment. One unique trait of endolysosomal compartments is pH—endolysosomal pH is generally lower than extracellular pH and cytosolic pH. To target specific pH ranges, materials containing specific functional groups (i.e., amines/imines [8–11], carboxylic acids [12–15], and sulfonamides [16] as shown in Figure 14.1) have often been used because the functional groups enable the transition between protonation/deprotonation states. Disrupting the endolysosomal membrane may be induced by the proton buffering capacity and/or fusogenicity of the “endolysosomolytic materials.” The “proton sponge” effect suggested by Boussif et al. [8] is related to inhibiting or delaying the acidification process of endolysosomes. When materials having protonable groups are entrapped in the endosomes, protons transferred by vacuolar ATPase-H+ pumps are mostly consumed by the materials themselves and do not contribute much toward endosomal maturation/ acidification. With further proton influx into the endosomes, chloride ions (Cl−) also accumulate in the endosomes, leading to osmotic imbalances between the cytoplasm and the endosomal compartments. This imbalance causes continuous water influx, endosomal swelling, and finally leads to endosomal rupture [17–20].

c14.indd 248

7/30/2010 4:12:17 PM

DESTABILIZATION OF ENDOLYSOSOMAL COMPARTMENTS

249

Amine/Imine-containing materials H N

H N

N H

N

N H H N

NH2 O H N CH C O

NH N

N

CH2

NH2 N

NH

NH

HN Poly (L-histidine) Branched PEI Carboxylic acid-containing materials C3H7 H2 C C

C2H5 H2 C C COOH

COOH

Poly (2-ethylacrylic acid)

Poly (2-propylacrylic acid)

Sulfonamide-containing materials CH3

S

H2 CH3 C C C=O NH

N

N

N N OCH3 Poly/oligo Poly/oligo sulfamethizole sulfadimethoxine R=

O=S=O NH R

OCH3

N

N

N

N

Poly/oligo sulfadiazine

CH3

Poly/oligo sulfamerazine

Figure 14.1. Examples of protonable oligomers/polymers that destabilize the endosomal membrane. (Copyright © 2008 Springer Science + Business Media, LLC [1].)

Fusogenic materials cause physical interactions between pH-sensitive membrane destabilizers and endosomal membranes. For example, positively charged polymers can interact with negatively charged phospholipid bilayers in the endosomal membrane, destabilizing the vesicle structure [17]. Polymers having pH-induced hydrophilic-to-hydrophobic transitions (i.e., anionic polyelectrolytes) show similar destabilization mechanisms, which were investigated using lipid bilayers (i.e., liposomes). That is, endosomal acidification increased the hydrophobicity of polymers, which could lead to interactions

c14.indd 249

7/30/2010 4:12:17 PM

250

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

with the hydrophobic tail region of the endosomal phospholipid bilayer membrane. This interaction stimulates lateral compression and forms pores in the bilayer, allowing the release of therapeutics into the cytoplasm [21–23].

14.3

ENDOLYSOSOMOLYTIC POLYMERIC NANOCARRIERS

Various materials (e.g., polymers, liposomes, peptides, viral components, and their hybrids) can induce the destabilization of endolysosomal compartments for effectively delivering therapeutics. This destabilization process is mostly triggered by a “pH” stimulus via various physical and chemical transitions of swelling/deswelling, hydrophilicity/hydrophobicity, micellization/demicellization, complexation/decomplexation, ionic/neutral charge, and α-helix/random coils conformational changes [1]. Target pH for endolysosomal rupture can range from extracellular pH to lysosomal pH while traversing the endolysosomal pathway. However, extracellular pH can vary depending on pathology (e.g., pH 7.4 for normal blood; for solid tumors, average pH 6.8 at normoglycemia and average pH 6.4 at hyperglycemia [24]; for brain, pH 7.2 (normal) and pH 6.4 (ischemic) [25]; for heart, pH 7.5 (normal) and approximately pH 6.8 (ischemic) [26]). Thus if certain materials can disrupt the endolysosomal membranes within specific pH ranges, nanocarriers with membrane destabilizers can be designed to treat specific diseases or target certain cells. Although there are various types of endolysosomolytic materials, this chapter will focus primarily on synthetic polymer/oligomer-triggered endolysosomal disruption. A brief summary of recent endolysosomolytic strategies developed by the Bae research group is presented with representative examples of chemical drug delivery nanocarriers (for low molecular weight therapeutics) and gene delivery nanocomplexes (for high molecular weight therapeutics). (For more information regarding other systems, please refer to their extensive reviews [6, 7, 14, 17, 27].) 14.3.1 Endolysosomolytic Polymeric Anticancer Drug Nanocarriers 14.3.1.1 Poly(L-histidine)-Based Micelles Histidine is an attractive amino acid for endosomolysis because its side chain, an imidazole ring, has pKa 6.0 and sharp proton buffering at pH 6.0 [28]. Also, as shown in Figure 14.2A, its homopolymer poly(l-histidine) (polyHis; see Figure 14.1 for its chemical structure) and various copolymers (i.e., polyHis-b-poly(ethylene glycol) (PEG)) show proton buffering over a broad pH range (pH 4 to pH 9) [29]. The apparent pKb values for polyHis5kDa and polyHis3kDa were about 6.5, whereas their PEGylated copolymers (i.e., polyHis5kDa-b-PEG2kDa and polyHis3kDa-b-PEG2kDa) showed a slightly higher apparent pKb of 7.0. These differences in apparent pKb values may result from differences in water content because the hydrophilic PEG block can induce more hydration [29].

c14.indd 250

7/30/2010 4:12:17 PM

ENDOLYSOSOMOLYTIC POLYMERIC NANOCARRIERS

251

Additional evaluation of polyHis for endolysosomolysis ability was performed using hemolysis because the membranes of red blood cells (RBCs) are similar to endolysosomal membranes. As shown in Figure 14.2B, polyHis5kDab-PEG2kDa disrupted 5–20% of total RBC number with decreasing pH from 7.4 to 6.75, whereas its hemolytic activity at pH 6.5 sharply increased to nearly 90%. However, poly(l-lactic acid) (PLLA)3kDa-b-PEG2kDa, which is used for preparing polyHis5kDa-b-PEG2kDa-containing mixed micelles, caused pHindependent membrane rupture of less than 5% in erythrocytes because the polymer does not possess pH-sensitive or endosomolytic groups [30]. To create a carrier for therapeutics (here, mostly hydrophobic chemical drugs), a polyHis block was conjugated to a hydrophilic PEG block. PolyHisb-PEG can maintain its micellar structure at basic pH due to its amphiphilicity, whereas at acidic pH its hydrophilicity can destabilize the micelles. At pH 8, micelles made from polyHis5kDa-b-PEG2kDa were more stable than those prepared from polyHis3kDa-b-PEG2kDa because the critical micelle concentration (CMC) of polyHis5kDa-b-PEG2kDa was much lower than polyHis3kDa-b-PEG2kDa (2.3 μg/mL for polyHis5kDa-b-PEG2kDa vs. 62 μg/mL for polyHis3kDa-b-PEG2kDa). However, the destabilization of polyHis5kDa-b-PEG2kDa-based micelles started at pH 7.4 and the extent of destabilization increased with decreasing pH [29]. This fact indicates that micelles fabricated from polyHis5kDa-b-PEG2kDa only are not suitable for destabilizing endolysosomes. To improve micelle stability at pH 7.4, the hydrophobic copolymer PLLA3kDa-b-PEG2kDa was introduced when preparing polyHis5kDa-b-PEG2kDa mixed micelles [31]. In mixed micelles made from polyHis5kDa-b-PEG2kDa and PLLA3kDa-b-PEG2kDa, the increased hydrophobicity (i.e., increased weight fraction of PLLA3kDa-b-PEG2kDa) lowered the pH that triggered micelle destabilization. PLLA3kDa-b-PEG2kDa (10 wt%) in the mixed micelles slightly improved micelle stability compared to micelles prepared from polyHis5kDa-bPEG2kDa. More PLLA3kDa-b-PEG2kDa (25 wt%) considerably enhanced micelle stability at pH 7.4 and initiated micelle destabilization below pH 7.0. Mixed micelles fabricated from 60 wt% of polyHis5kDa-b-PEG2kDa and 40 wt% of PLLA3kDa-b-PEG2kDa had a slightly lower destabilization pH than micelles containing 25 wt% PLLA3kDa-b-PEG2kDa. These results strongly correlated with the trigger pH for the release of doxorubicin (DOX, an anticancer drug). For the mixed micelles, DOX release could be triggered to release from pH 6.6 to pH 7.2 by modulating the weight fraction of PLLA3kDa-b-PEG2kDa [31]. These findings suggest that mixed micelles containing higher fractions of PLLA3kDa-b-PEG2kDa had weaker pH sensitivity although the micelle destabilization pH remained the same. Following the discovery that the pH at which micelle destabilization is triggered is closely linked to enhanced drug release, Yin et al. [32] investigated whether it was possible to predict pH-triggered micelle destabilization by monitoring pH-dependent changes in micelle size distribution using dynamic light scattering. The mixed micelles (75 wt% of polyHis5kDa-b-PEG2kDa and 25 wt% of PLLA3kDa-b-PEG2kDa) formed a spherical shaped secondary

c14.indd 251

7/30/2010 4:12:17 PM

(a) 14

(b) 120

NaCl polyHis5kDa-b-PEG2kDa polyHis5kDa polyHis3kDa-b-PEG2kDa polyHis3kDa

10

100 Hemolysis (%)

12

pH

8 6 4

PLLA3kDa-b-PEG2kDa

80 60 40 20

2 0

polyHis5kDa-b-PEG2kDa

0

200

400

600

800

0 7.4

7

1 N HCl (mL) (c)

6.75

6.5

pH

polyHis-b-PEG PLLA-b-PEG Proton

pH

pH

pH 7.4–7.0

pH 6.8–6.5

(d)

pH 6.0 (e)

7.6 DHPE

7.4

Lysotracker dye

Merged

7.2 pHt

PHIM-f

7.0 6.8 6.6 6.4

PHSM-f 0

10 20 30 40 50 PLLA3kDa-b-PEG2kDa fraction (wt.%) (g) Tumor volume (mm3)

(f) 120

Cell viability (%)

100 80 60 40 20 0 0.0001 0.001 0.01 0.1 1 DOX concentration (μg/ml)

c14.indd 252

3500 3000 2500 2000 1500 1000 500 0 0 3 6 9 12 15 18 21 24 27 30 Days after 1st i. v. injection

10

7/30/2010 4:12:17 PM

ENDOLYSOSOMOLYTIC POLYMERIC NANOCARRIERS

253

Figure 14.2. (A) Acid–base titration curves of polyHis and polyHis-b-PEG. (Reproduced from Ref. 29. Copyright © 2003, with permission from Elsevier B.V.) (B) Hemolytic activity of polyHis5kDa-b-PEG2kDa and PLLA3kDa-b-PEG2kDa. (Copyright © 2008 Springer Science + Business Media, LLC [30].) (C) Schematic illustrations of micelle destabilization. (Reproduced from Ref. 32. Copyright © 2008 with permission from Elsevier B.V.) (D) The effects of PLLA3kDa-b-PEG2kDa on micelle destabilizationtriggering pH (pHt) in mixed micelles made from polyHis5kDa-b-PEG2kDa and PLLA3kDab-PEG2kDa. (Reproduced from Ref. 32. Copyright © 2008 with permission from Elsevier B.V.) (E) In vitro endosomal disruption using fluorescein DHPE-encapsulated PHSM/f and PHIM/f and the LysoTracker® dye against A2780/DOXR MDR cells. (Copyright © 2008 Springer Science + Business Media, LLC [30].) (F) In vitro cytotoxicity of free DOX (䉬), DOX-loaded PHSM/f (䊉), and PHIM/f (䊏) against A2780/DOXR MDR cells after 48-h incubation. (Copyright © 2008 Springer Science + Business Media, LLC [30].) (G) In vivo tumor growth inhibition test of s.c. A2780/DOXR MDR cells-bearing BALB/c nude mice using control (䉬), free DOX (ⵧ), DOX-loaded PHSM/f (䊉), and DOX-loaded PHIM/f (䉱). Three intravenous doses of 10 mg/kg DOX equivalent dose were administered at Days 0, 3, and 6. (Copyright © 2008 Springer Science + Business Media, LLC [30].) (See color insert.)

structure in the range of pH 7.0 to pH 7.4 because individual core–shell micelles with relatively hydrophobic cores associated together. In the pH range 7.0–7.4, the mixed micelles had a narrow and unimodal size distribution. When pH was decreased from 6.8 to 6.5, there was an obvious increase in the size and aggregation number of the micelles caused by the destabilization of the micelle core. Their size distribution was broad and unimodal, but hydrophilic polyHis5kDa-b-PEG2kDa unimers were released from the micelles. Further decreases in pH (reaching pH 6.0) resulted in a bimodal distribution of micelle size because most polyHis5kDa-b-PEG2kDa unimers had dissociated from the micelle core (see Figure 14.2C for an illustration of pH-induced micelle destabilization) [32]. As shown in Figure 14.2D, when different amounts of PLLA3kDab-PEG2kDa were introduced into the mixed micelles, pH-triggered micelle destabilization decreased with increasing weight fraction of PLLA3kDa-bPEG2kDa (e.g., pHt 6.8 for 25 wt% of PLLA3kDa-b-PEG2kDa and pHt 6.5 for 40 wt% of PLLA3kDa-b-PEG2kDa) [32]. With endolysosomal acidification, the protonated polyHis5kDa-b-PEG2kDa can cause the endolysosomes to swell due to proton buffering of the polymer. In addition, according to the destabilization mechanism of the micelles suggested by Yin et al. [32], the polyHis5kDa-b-PEG2kDa unimers released from the micelles can interact with the endolysosomal membrane. The proton buffering and fusogenic characteristics of polyHis5kDa-b-PEG2kDa may disrupt the endolysosomal compartments as shown in Figure 14.2E. When mixed pH-sensitive micelles (PHSM) containing targeting folate (PHSM/f) (i.e., micelle systems prepared from 80% of polyHis5kDa-b-PEG2kDa and 20% of PLLA3kDa-bPEG2kDa-folate) and mixed pH-insensitive micelles (PHIM) with folate

c14.indd 253

7/30/2010 4:12:18 PM

254

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

(PHIM/f) (i.e., micelle systems prepared from 80% of PLLA3kDa-b-PEG2kDa and 20% of PLLA3kDa-b-PEG2kDa-folate) were administered to DOX-induced multidrug-resistant (MDR) ovarian carcinoma cell lines (i.e., A2780/ DOXR MDR cells), PHSM/f caused an even distribution of green fluorescent DHPE (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanoamine triethylammonium salt) and red fluorescent LysoTracker® dye in the cytoplasm. However, LysoTracker® and DHPE delivered using PHIM/f were scattered locally in the cytoplasm. This difference in the intracellular localization of DHPE and LysoTracker dye indicated that polyHis5kDa-b-PEG2kDa in the PHSM/f is an important component for the endolysosomal escape of therapeutics. Endolysosomolytic polyHis5kDa-b-PEG2kDa-containing PHSM/f effectively delivered DOX and killed both drug-sensitive tumor cell lines and MDR tumor cell lines [10, 30]. As shown in Figure 14.2F, DOX-loaded PHSM/f effectively killed in the range of tested DOX concentrations without displaying the cytotoxic effects of free DOX and DOX-loaded PHIM/f. It is noteworthy that DOX-loaded PHSM/f killed approximately 60% of A2780/DOXR MDR cells at 1 μg/mL of DOX, whereas free DOX and DOX-loaded PHIM/f killed approximately 10% and 20% of the cells, respectively [30]. For in vivo models of A2780/DOXR MDR cells-bearing BALB/c nude mice, PHSH/f treatment effectively inhibited tumor growth for 1 month, whereas free DOX and PHIM/f treatments did not suppress tumor growth (Figure 14.2G). 14.3.1.2 Poly(L-histidine-co-L-phenylalanine)-Based Micelles When PHSM/f were exposed to acidic extracellular pH (i.e., pH 6.4–6.8 for solid tumors and ischemia), micelles with up to 40 wt% of PLLA3kDa-b-PEG2kDafolate became destabilized in extracellular environments. Thus to destabilize micelles containing endosomolytic materials at pH lower than the destabilization pH of PHSM/f, polyHis was modified with the hydrophobic amino acid phenylalanine (Phe). As summarized in Table 14.1, pH-sensitive copolymers of His and Phe (i.e., poly(His-co-Phe) (PHP)) with various apparent pKb were synthesized. Their molecular weights were approximately 5 kDa and their apparent pKb values ranged from 6.7 to 4.8 as the hydrophobic Phe mole% in the copolymer was increased [33].

TABLE 14.1

Characterization of Poly(His-co-Phe)

Polymer

Phe (mol%) in Polymer

Apparent pKb

Buffering pH Range

10 16 22 27

6.7 6.3 5.7 4.8

8.2–4.8 7.6–4.7 6.5–4.0 5.8–3.2

PHP10 PHP16 PHP22 PHP27

Source: Reproduced with permission from Ref. 33. Copyright © 2008 Wiley-VCH Verlag GmbH & Co.KGaA.

c14.indd 254

7/30/2010 4:12:18 PM

ENDOLYSOSOMOLYTIC POLYMERIC NANOCARRIERS

1200

100

1000

80

800

Size (nm)

120

60 40

600

Intensity (%)

(B)

(A)

Transmittance (%)

255

30 25 20 15 10 5 0 1

100 10000 Size (nm)

400

20

200

0 3

4

5

6

7 8 pH

9 10 11 12

0

8

7.4

6.5 pH

6

5.5

Figure 14.3. (A) pH-dependent transmittance transition of PHSMPhe10 (䉫), PHSMPhe16 (䉱), PHSMPhe22 (ⵧ), and PHSMPhe27 (䊉). (B) pH-dependent particle size of mPHSMPhe16(5%) (black), mPHSMPhe16(10%) (gray), and mPHSMPhe16(20%) (white). The inset shows a bimodal particle size distribution at pH 6.5 using mPHSMPhe16(5%). (Reproduced with permission from Ref. 33. Copyright © 2008 Wiley-VCH Verlag GmbH & Co.KGaA.)

Like polyHis, poly(His-co-Phe) (PHP) was conjugated with PEG for encapsulating hydrophobic drugs. The pH-sensitive micelles (PHSMPhe) prepared from PEGylated PHP (i.e., PHP-b-PEG2kDa) showed transmittance transition at pH 7.0 for PHSMPhe10, at pH 6.5 for PHSMPhe16, at pH 5.8 for PHSMPhe22, and at pH 5.2 for PHSMPhe27 (Figure 14.3A). To obtain finer tuning of the destabilization pH, PLLA3kDa-b-PEG2kDa was mixed with PHP-b-PEG to prepare mixed micelles (mPHSMPhe). In the initial trial to target early endosomal pH values, PHP16-b-PEG2kDa was selected because its micelles were stable at pH 7.0 but became destabilized at pH 6.5. The micelles “mPHSMPhe16” prepared from various weight ratios of PHP16-b-PEG2kDa and PLLA3kDa-bPEG2kDa showed pH-dependent changes in micelle size, and more dramatic size changes were seen with increasing PLLA3kDa-b-PEG2kDa content. As shown in Figure 14.3B, the size of mPHSMPhe16(5%) (i.e., mPHSMPhe16 prepared from 95 wt% PHP16-b-PEG2kDa and 5 wt% of PLLA3kDa-b-PEG2kDa) prepared at pH 8 increased as pH dropped below pH 6.5, and the sizes of mPHSMPhe16(10%) and mPHSMPhe16(20%) increased below pH 6 and sharply below pH 5.5, respectively. At pH 6.5, a bimodal size distribution of mPHSMPhe16(5%) indicated that ionized PHP blocks were not miscible with PLLA3kDa-b-PEG2kDa and dissociated from PLLA3kDa-b-PEG2kDa micelles [33]. In vitro endolysosomal escape of mPHSMPhe16(20%)/f (i.e., mPHSMPhe16 prepared from 80 wt% PHP16-b-PEG2kDa and 20 wt% of PLLA3kDa-b-PEG2kDafolate) was similar to that of the PHSM/f system as shown in Figure 14.2E. That is, mPHSMPhe16(20%)/f caused even intracellular distribution of DHPE and LysoTraker dyes. This result may be induced by the PHP16-b-PEG2kDa

c14.indd 255

7/30/2010 4:12:18 PM

256

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

released from the mixed micelles. The micelle systems effectively killed both A2780 wild-type cells and A2780/DOXR MDR cells [33]. Based on the destabilization pH of mPHSMPhe16 with different amounts of PLLA3kDa-b-PEG2kDa, mPHSMPhe22 and mPHSMPhe27 prepared from PHP22 and PHP27 with PLLA3kDa-b-PEG2kDa could be designed to target late endosomes and lysosomes, respectively, because PHSMPhe22 and PHSMPhe27 fabricated from PHP22 and PHP27 showed micelle destabilization at pH 5.8 and pH 5.2, respectively. 14.3.1.3 Virus-Mimetic Nanogels Virus-mimetic (VM) nanogels, which can migrate from one cell to other cells, have been developed to effectively kill solid tumors. As shown in Figure 14.4A, the nanogels consist of a hydrophobic PHP core (i.e., poly(His32-co-Phe6) with nearly pKb 6.4) and two hydrophilic layers of PEG2kDa and bovine serum albumin (BSA). Like the capsid shell of a virus, the gel’s mimetic outer shell is comprised of the core and BSA bridged by multiple PEG chains. Interestingly, the nanogels have a reversible swelling/deswelling characteristic that is pH dependent. As shown in Figure 14.4B, the size of VM nanogels at pH 7.4 was approximately 55 nm with a narrow distribution. At pH 6.8, VM nanogels still showed similar size and size distribution to those at pH 7.4. However, upon exposure to pH 6.4 (close to early endosomal pH), the nanogels became bigger up to approximately 355 nm with a broader distribution due to the ionization and swelling of the PHP core. This pH-induced swelling/deswelling transition of VM nanogels modulated DOX release rates. As shown in Figure 14.4C, DOX release rates from the nanogels at pH 7.4 and pH 6.8 were similar because the solidified core at pH 7.4 and pH 6.8 entrapped hydrophobic drugs. However, pH 6.4 induced the ionization and swelling of the PHP core and allowed more DOX to release. The release rate at pH 6.4 was approximately threefold faster than at pH 7.4 and pH 6.8. In addition, acidic pH-induced volumetric expansion of VM nanogels and known proton buffering capacities of histidine-based polymers (i.e., poly(His16-co-Phe6)) induced endolysosomal disruption as shown in Figure 14.4D. Control nanopaticles (NPs) without endolysosomolytic characters were localized with LysoTracker dye, whereas VM nanogels showed even intracellular distribution. Acidic pH-induced DOX release profiles and the endolysosomolytic function of VM nanogels effectively killed A2780 wild-type cells and A2780/ DOXR MDR cells compared to free DOX and control NP treatments. As shown in Figure 14.4E, the antitumor activity of the nanogels in pH 6.8 medium demonstrated that the nanogels were stable at pH 6.8 and released DOX to effectively kill tumor cells. Interestingly, after killing tumor cells, the nanogels that still contained DOX migrated to kill other tumor cells (Figure 14.4F). This concept of a “nanogel” that is stable in an acidic extracellular environment and can be activated to release drugs and to disrupt endolysosomes

c14.indd 256

7/30/2010 4:12:18 PM

257

ENDOLYSOSOMOLYTIC POLYMERIC NANOCARRIERS

pH 7.4 pH 6.4 DOX released F: folate F F F PEG Inner shell F F F F F F F pH F F F F F F F F F FF FF F F F BSA outer shell F FF 355 nm 55 nm

(A)

F F F F

F

F

(B) pH 7.4

pH 6.8

0 100

pH 7.4

0 100

pH 7.4

pH 6.4

pH 6.8

0 100

0 100

pH 7.4

pH 6.4

0 100 0 200 400 600 800 0 100 0 200 400 600 800

Particle size (nm)

(D) 20

FITC

Lyso Tracker

Merged

Control NPs VM-nanogels

pH pulses relative rate of DOX (μg/hour)

(C) 15 10 5 0

pH 7.4 pH 6.8 pH 6.4

: appled pH for 1 hour each

Cell viability / %

80

**

**

60 40 20 0

**

**

A2780 A2780/ A2780 A2780/ DOXR DOXR MDR cells MDR cells

Blank DOX-loaded DOX-loaded VM-nanogels Free DOX control NPs VM-nanogels

(F) pH 6.8

pH 7.4

(E) 100

Infected

not Infected

Subsequent Infections

A

B-0

B-1

B-2

B-3

A

B-0

B-1

B-2

B-3

A

B-0

B-1

B-2

B-3

A

B-0

B-1

B-2

B-3

Figure 14.4. VM nanogel: (A) Schematic presentation. (B) pH-modulating size change: A (pH 7.4), B (pH 6.8), and C (pH 6.4). (C) pH-dependent DOX release rate from DOX-loaded VM nanogels. (D) In vitro endosomal escaping activity. (E) pHdependent cytotoxicity of DOX-loaded VM nanogel (white), DOX-loaded control nanoparticles (gray), and free DOX (black) against A2780 wild-type cells and A2780/ DOXR MDR cells after 48-h incubation (DOX dose = 1 μg/mL). (F) Migration of DOXloaded VM nanogels in A2780/DOXR MDR cells. (Reproduced with permission from Ref. 38. Copyright © 2008 Wiley-VCH Verlag GmbH & Co.KGaA.) (See color insert.)

while contained in the endolysosomal pathway could be expanded to treat ischemia in the brain and heart. In this case, although migration of the nanogel toward other cells might be difficult, the pH-dependent drug release profile of the nanogel could effectively prevent the progress of ischemia over a long time period because ischemic states are initiated by drops in pH.

c14.indd 257

7/30/2010 4:12:18 PM

258

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

14.3.2 Endolysosomolytic Polymeric Gene Nanocomplexes Kang and Bae have been interested in developing cell-customized endosomolytic agents for more effectively delivering therapeutics because endolysosomal characteristics such as endolysosomal pH [34, 35], endolysosomal membrane composition [36, 37], and acidification rate are cell specific. To investigate cell-specific endolysosomolytic agents, sulfonamides were selected. Sulfonamides have broad-range pKa values (3–11) and hydrophobicity conferred by various substituted groups, R (see Figure 14.1) [16]. For feasibility studies, sulfamethizole (SMT; pKa 5.45), sulfadimethoxine (SDM; pKa 6.1), sulfadiazine (SDZ; pKa 6.4), and sulfamerazine (SMZ; pKa 7.0) were selected because their pKa values fall within the endolysosomal pH range. Using chain transfer radical polymerization, the synthesized sulfonamide oligomers (designated as OSMT, OSDM, OSDZ, and OSMZ) had Mn = 1.8–2.5 kDa. Oligomeric sulfonamides (OSAs) showed different proton buffering and aqueous solubility transition within the endosomal/lysosomal pH range. In aqueous solubility transition studies of the OSAs (Figure 14.5A), OSMZ showed solubility transitions over a broad pH range, whereas OSMT, OSDM, and OSDZ showed relatively sharp transmittance changes within a narrow pH range. As shown in Figure 14.5B, OSMT and OSDZ demonstrated broad proton buffering over the pH ranges of 5.0–6.4 and 5.7–7.3, respectively, whereas OSDM and OSMZ buffered protons at a specific pH, 6.5 and 7.3, respectively. Their apparent pKa values were 5.7 (OSMT), 6.5 (OSDM and OSDZ), and 7.3 (OSMZ) and were slightly higher than their monomeric sulfonamides counterparts [16]. To understand the endolysosomolytic functions of anionic OSAs in polymeric nanocarriers, a well-known polymeric gene delivery system without endosomolytic function (here, poly(l-lysine) (PLL)-based polyplexes) was selected. Anionic OSAs cannot directly carry anionic genes, but OSAs can be incorporated within any polycation-based gene carriers. In intracellular distribution studies of polyplexes using pH-sensitive fluorescein pDNA (F-DNA), OSDZ-polyplexes (i.e., PLL/OSDZ/F-DNA) showed broad distribution in the cytoplasm, whereas PLL/F-DNA complexes were mostly localized (Figure 14.5C). These differences in fluorescence distribution demonstrate that OSA polyplexes caused more pDNA to escape from endosomal/lysosomal compartments than PLL/pDNA complexes. This faster endosomal release of OSDZ polyplexes resulted in faster gene expression than for PLL/pDNA complexes. In in vitro transfection studies using three different cell lines (i.e., human hepatoma HepG2 cells, human embryonic kidney HEK293 cells, and rat insulinoma RINm5F cells), OSA polyplexes showed 4–55-fold better transfection efficiency than control polyplexes (PLL/pDNA) as shown in Figure 14.5D. Interestingly, OSDM and OSDZ were more effective in transfecting HEK293 cells whereas OSMZ was the best for transfecting RINm5F cells. This study supports the need for cell-customized endosomolytic agents to achieve

c14.indd 258

7/30/2010 4:12:18 PM

CONCLUSION

8

PLL/OSDZ/F-DNA

(D)

150 mM NaCl OSMT OSDM OSDZ OSMZ OPAA

4

(C)

6 pH

PLL/F-DNA

7

20μm

150 mM NaCl OSMT OSDM OSDZ OSMZ OPAA

100 150 200 250 300 350 Added volume of 0.1 N HCI (μL)

80 70 60 50 40 30 20 10 0

HEK293 HepG2 RINm5F

PL

L PE O I SM O T SD M O SD O Z SM O Z PA A

20μm

5

Normalized Transfection Efficiency (Fold)

Transmittance (%)

100 90 80 70 60 50 40 30 20 10 0

(B) 11 10 9 8 7 6 5 4 3 2 50

pH

(A)

259

Polyplex type

Figure 14.5. (A) pH-dependent aqueous solubility transition of OSA and OPAA (oligomeric propylacrylic acid) solutions. (B) Acid–base titration curves of OSA and OPAA solutions. (C) In vitro intracellular localization studies of PLL/pDNA and PLL/ OSDZ/pDNA complexes using pH-sensitive F-DNA. (D) In vitro transfection studies for OSA-containing PLL/pDNA complexes (OSA polyplexes) containing a luciferase gene in HepG2, HEK293, and RINm5F cells. Dose of OSA and OPPA was 5 nmol (based on their monomeric units) per 1 μg pDNA. Normalized transfection efficiency was defined as (Absolute transfection efficiency of polyplexes)/(Absolute transfection efficiency of PLL/pDNA complexes) for a specific cell. Charge ratio (+/−) of polyplexes was 3 except for PEI/pDNA (+/− = 5). (Reproduced with permission from Ref. 16. Copyright © 2007 Wiley-VCH Verlag GmbH & Co.KGaA.)

clinically effective gene delivery [16]. In addition, these anionic materials could provide an opportunity to revisit the use of other biocompatible but less transfection-efficient gene carriers.

14.4

CONCLUSION

Endolysosomolytic materials are an indispensable component for effectively targeting subcellular compartments and maximizing therapeutic benefits. Because endolysosomal destabilization is mostly mediated by “pH,” understanding extracellular pH and endolysosomal pH of various pathological states

c14.indd 259

7/30/2010 4:12:18 PM

260

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

is important. Knowledge about the pH in the body, organs, tissues, cells, and extracellular and intracellular environments for healthy and disease states would aid the design of effective endolysosomolytic polymeric nanocarriers.

ACKNOWLEDGMENTS This work is partially supported by NIH grants (CA101850 and GM82866). The authors appreciate the help of Deepa Mishra, Dr. Haiqing Yin, and Dr. Dongin Kim for their critical reading.

REFERENCES 1. Kang, H. C., Lee, E. S., Na, K., et al. Stimuli-sensitive nanosystems: for drug and gene delivery. In: V. P. Torchilin, ed. Multifunctional Pharmaceutical Nanocarriers. Springer, New York, 2008, pp. 161–199. 2. Herweijer, H. and Wolff, J. A. Gene therapy progress and prospects: hydrodynamic gene delivery. Gene Ther. 14: 99–107 (2007). 3. Maeda, H., Bharate, G. Y., and Daruwalla, J. Polymeric drugs for efficient tumortargeted drug delivery based on EPR-effect. Eur. J. Pharm. Biopharm. 71: 409–419 (2009). 4. Lee, E. S., Gao, Z., and Bae, Y. H. Recent progress in tumor pH targeting nanotechnology. J. Control. Release 132: 164–170 (2008). 5. Nishikawa, M. Development of cell-specific targeting systems for drugs and genes. Biol. Pharm. Bull. 28: 195–200 (2005). 6. Breunig, M., Bauer, S., and Goepferich, A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm. 68: 112–128 (2008). 7. Bareford, L. M., and Swaan, P. W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Deliv. Rev. 59: 748–758 (2007). 8. Boussif, O., Lezoualc’h, F., Zanta, M. A., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA. 92: 7297–7301 (1995). 9. Pichon, C., Goncalves, C., and Midoux, P. Histidine-rich peptides and polymers for nucleic acids delivery. Adv. Drug Deliv. Rev. 53: 75–94 (2001). 10. Lee, E. S., Na, K., and Bae, Y. H. Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor. J. Control. Release 103: 405–418 (2005). 11. Yang, S. R., Lee, H. J., and Kim, J. D. Histidine-conjugated poly(amino acid) derivatives for the novel endosomolytic delivery carrier of doxorubicin. J. Control. Release 114: 60–68 (2006). 12. Jones, R. A., Cheung, C. Y., Black, F. E., et al. Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles. Biochem. J. 372: 65–75 (2003).

c14.indd 260

7/30/2010 4:12:18 PM

REFERENCES

261

13. Yessine, M. A., Lafleur, M., Meier, C., et al. Characterization of the membranedestabilizing properties of different pH-sensitive methacrylic acid copolymers. Biochim. Biophys. Acta. 1613: 28–38 (2003). 14. Yessine, M. A. and Leroux, J. C. Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug Deliv. Rev. 56: 999–1021 (2004). 15. Kiang, T., Bright, C., Cheung, C. Y., et al. Formulation of chitosan-DNA nanoparticles with poly(propyl acrylic acid) enhances gene expression. J. Biomater. Sci. Polym. Ed. 15: 1405–1421 (2004). 16. Kang, H. C. and Bae, Y. H. pH-tunable endosomolytic oligomers for enhanced nucleic acid delivery. Adv. Funct. Mater. 17: 1263–1272 (2007). 17. Cho, Y. W., Kim, J. D., and Park, K. Polycation gene delivery systems: escape from endosomes to cytosol. J. Pharm. Pharmacol. 55: 721–734 (2003). 18. Kang, H. C., Lee, M., and Bae, Y. H. Polymeric gene carriers. Crit. Rev. Eukaryot. Gene Expression 15: 317–342 (2005). 19. Pack, D. W., Hoffman, A. S., Pun, S., et al. Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 4: 581–593 (2005). 20. Sonawane, N. D., Szoka, F. C. Jr., and Verkman, A. S. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine–DNA polyplexes. J. Biol. Chem. 278: 44826–44831 (2003). 21. Xie, A. F. and Granick, S. Phospholipid membranes as substrates for polymer adsorption. Nat. Mater. 1: 129–133 (2002). 22. Tirrell, D. A., Takigawa, D. Y., and Seki, K. pH sensitization of phospholipid vesicles via complexation with synthetic poly(carboxylic acid)s. Ann. N. Y. Acad. Sci. 446: 237–248 (1985). 23. Thomas, J. L. and Tirrell, D. A. Polymer-induced leakage of cations from dioeoylphosphatidylcholine and phosphatidylglycerol liposomes. J. Control. Release 67: 203–209 (2000). 24. Volk, T., Jahde, E., Fortmeyer, H. P., et al. pH in human tumour xenografts: effect of intravenous administration of glucose. Br. J. Cancer 68: 492–500 (1993). 25. Sarantopoulos, C., McCallum, B., Sapunar, D., et al. ATP-sensitive potassium channels in rat primary afferent neurons: the effect of neuropathic injury and gabapentin. Neurosci. Lett. 343: 185–189 (2003). 26. Hunjan, S., Mason, R. P., Mehta, V. D., et al. Simultaneous intracellular and extracellular pH measurement in the heart by 19F NMR of 6-fluoropyridoxol. Magn. Reson. Med. 39: 551–556 (1998). 27. Wattiaux, R., Laurent, N., Wattiaux-De Coninck, S., et al. Endosomes, lysosomes: their impication in gene transfer. Adv. Drug Deliv. Rev. 41: 201–208 (2000). 28. Gilbert, H. F. Basic Concepts in Biochemistry: A Student’s Survival Guide, 2nd ed. McGraw-Hill Health Professional Division, New York, 1999. 29. Lee, E. S., Shin, H. J., Na, K., et al. Poly(l-histidine)-PEG block copolymer micelles and pH-induced destabilization. J. Control. Release 90: 363–374 (2003). 30. Kim, D., Lee, E. S., Park, K., et al. Doxorubicin loaded pH-sensitive micelle: antitumoral efficacy against ovarian A2780/DOXR tumor. Pharm. Res. 25: 2074–2082 (2008).

c14.indd 261

7/30/2010 4:12:18 PM

262

ENDOLYSOSOMOLYTICALLY ACTIVE PH-SENSITIVE POLYMERIC NANOTECHNOLOGY

31. Lee, E. S., Na, K., and Bae, Y. H. Polymeric micelle for tumor pH and folatemediated targeting. J. Control. Release 91: 103–113 (2003). 32. Yin, H., Lee, E. S., Kim, D., et al. Physicochemical characteristics of pH-sensitive poly(l-histidine)-b-poly(ethylene glycol)/poly(l-lactide)-b-poly(ethylene glycol) mixed micelles. J. Control. Release 126: 130–138 (2008). 33. Kim, D., Lee, E. S., Oh, K. T., et al. Doxorubicin-loaded polymeric micelle overcomes multidrug resistance of cancer by double-targeting folate receptor and early endosomal pH. Small 4: 2043–2050 (2008). 34. Rybak, S. L., Lanni, F., and Murphy, R. F. Theoretical considerations on the role of membrane potential in the regulation of endosomal pH. Biophys. J. 73: 674–687 (1997). 35. Rybak, S. L. and Murphy, R. F. Primary cell cultures from murine kidney and heart differ in endosomal pH. J. Cell Physiol. 176: 216–222 (1998). 36. Alberts, B., Johnson, A., Lewis, J., et al. Molecular Biology of the Cell, 4th ed. Garland Science, New York, 2002. 37. Evans, W. H. and Hardison, W. G. Phospholipid, cholesterol, polypeptide and glycoprotein composition of hepatic endosome subfractions. Biochem. J. 232: 33– 36 (1985). 38. Lee, E. S., Kim, D., Youn, Y. S., et al. A virus-mimetic nanogel vehicle. Angew. Chem. Int. Ed. Engl. 47: 2418–2421 (2008).

c14.indd 262

7/30/2010 4:12:18 PM

CHAPTER 15

Uptake and Intracellular Dynamics of Proteins Internalized by Cell-Penetrating Peptides ARWYN T. JONES Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, United Kingdom

15.1

INTRODUCTION

Many pathogens and therapeutic macromolecules need to enter cells to fulfill a requirement for, respectively, survival and replication or the treatment of disease. An ability to target specific cells and then overcome the barrier posed by the plasma membrane to reach the cytosol or a defined subcellular organelle, and to then mediate an effective biological response is, however, a feat limited to pathogens or pathogen proteins. An example is the HIV transactivator of transcription protein, HIV-Tat, that enters cells by endocytosis, escapes from the endolysosomal system to the cytosol, and then moves to the nucleus to mediate its effects on transcription. This protein highlights the extraordinary capacity of evolution to manufacture simple but highly efficient targeting systems. Essential for HIV-Tat to perform these feats is a basic protein transduction domain that can enter cells either alone or associated with cargo of various forms ranging from other proteins to nanoparticles. Hundereds of natural and synthetic protein transduction domains or cellpenetrating peptide (CPP) sequences have now been described and this chapter highlights the attempts that have been made to understand the cellular dynamics of proteins attached by various means to CPPs. An appreciation of the mechanisms by which these systems operate may hold answers to address a major pharmaceutical need for the design of more efficient systems for cellular delivery of therapeutic proteins. This chapter will describe endocytosis and endocytic pathways (Section 15.2), the need to deliver proteins into cells Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

263

c15.indd 263

7/30/2010 2:19:19 PM

264

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

(Section 15.3), our current understanding of the mechanism by which the HIV-Tat enters cells and reaches the nucleus (Section 15.4), delivery of proteins linked to other CPP sequences (Section 15.5), alternative mechanisms for covalent and noncovalent attachment of CPPs to proteins (Section 15.6), delivery of CPP proteins to lysosomes (Section 15.7), and a comparison of the cellular dynamics of CPP proteins with CPP fluorophores (Section 15.8).

15.2

ENDOCYTIC PATHWAYS

The plasma membrane is a highly effective and selective boundary between the inside and outside of cells. All cells, however, have a physiological need to internalize membrane-impermeable molecules and to do this they invaginate portions of their plasma membrane to internalize membrane and accompanying extracellular fluid. This process is called endocytosis and the internalized membrane or vesicular structures are then delivered together with the fluid cargo to a sorting station that directs further membrane traffic to one of a number of cellular destinations. Only recently are we beginning to appreciate the enormous complexities of endocytosis and endocytic pathways [1]. This process, regulated by a network of proteins and lipids, is essential for several often unrelated cellular functions, and this may be why a single cell has the capacity to internalize material through several different endocytic pathways (Figure 15.1). These include those originating from clathrin-, and non-clathrin-coated structures such as caveolae through to larger structures called macropinosomes that are formed following plasma membrane ruffling. It is beyond the scope of this chapter to discuss endocytosis at great length and those wishing to gain a good foundation on the current knowledge of endocytic pathways should consult the reviews in Refs 1–9 and also this informative web site at http://endocytosis.org/. 15.2.1 Using Endocytic Pathways to Deliver Therapeutics The effectiveness of using endocytosis to deliver therapeutic macromolecules is in part constrained by the fact that the fate of the therapeutic within one of these pathways is predetermined by the cell’s trafficking mechanisms and biological barriers posed initially by the plasma membrane and then by the endolysosomal network [10–12]. Thus fragile molecules such as proteins and genes may rapidly be delivered to hostile environments such as lysosomes and inactivated before they have any chance to reach and interact with their intended targets. Pathogens and pathogen-proteins especially toxins have been shown to exploit and manipulate these pathways to reach defined subcellular destinations, thus suggesting that therapeutics can also be delivered by similar mechanisms [13, 14]. Endocytic features such as low pH and high degradative activities can, however, act as activators of bioresponsive molecules to enhance translocation of the active entity before the cell’s endocytic pathway

c15.indd 264

7/30/2010 2:19:19 PM

ENDOCYTIC PATHWAYS

265

Figure 15.1. Endocytic pathways and cell entry mechanisms available for proteins attached to cell-penetrating sequences or natural cell-penetrating proteins such as HIV Tat. Numbers 1–6 represent defined pinocytic pathways and not described here or in the text is phagocytosis.1, Clathrin-coated vesicles; 2, caveolae; 3, fluid-phase uptake; 4, flotillin-1-dependent pathway; 5, other clathrin- and caveolin-independent pathway(s) [5]; 6, macropinocytosis, and 7, direct penetration and entry through the plasma membrane. The named proteins represent those required for the pathways to operate and thus offer the opportunity for their mutation or depletion via siRNA. This together with pharmacological inhibitors will allow for more accurate requirement of particular pathways for entry of macromolecules designed for intracellular delivery and targeting. It is likely that an early or sorting endosome exists to accept cargo delivered through all these endocytic pathways and some have been defined. It still remains to be determined as to whether entry via any particular pathway is advantageous for allowing subsequent escape from the endolysosomal system to locate the protein in the cytosol.

traffics it to its final destination. Enhancing the delivery of therapeutics through endocytosis is therefore dependent on acquisition of a high level of understanding of specific endocytic pathways inherent in the target cell, the traffic and fate of the molecule within endocytic organelles, the effect of the macromolecule on the dynamics of endocytic pathways, the downstream effects of these on the integrity of the cell, and if required the mechanism by which escape from the endolysosomal system occurs.

c15.indd 265

7/30/2010 2:19:19 PM

266

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

15.2.2 Identifying Endocytic Pathways Cell biologists studying endocytic pathways are consistently identifying new protein and lipid mediators and a goal for this remit that overlaps with that of drug delivery is to identify a protein that is only involved in regulating one distinct pathway. This then opens the possibility of either mutating this protein or inhibiting its manufacture to gain a better understanding of the pathway that it regulates and also the fate of the cargo that it helps to deliver. Much of the current information regarding the endocytic pathways utilized by drug delivery systems, including those using CPPs, comes from studies using microscopy and endocytic probes [15, 16] in conjunction with a library of pharmacological inhibitors of endocytic pathways [17]. Examples include methyl ß-cyclodextrin (MßCD), amiloride, chlorpromazine, and cytochalasins. At one time these agents were deemed to be specific for certain pathways but it is now clear that all suffer predominantly from a lack of specificity, and toxicity is also a common problem [17]. Increasingly, researchers are using molecular biology tools such as cell lines expressing dominant negative forms of endocytic proteins or siRNA-mediated depletion of proteins that regulate endocytic pathways. This siRNA approach has recently identified new pathways and roles in endocytosis for proteins such as flotillin-1 [18] and significantly added to the current knowledge on established endocytic proteins such as clathrin [19–21]. The use of siRNA depletion has its own drawbacks for those wishing to use the technology for analyzing endocytosis of their drug delivery systems and these include (1) the fact that a delivery system is first required to introduce the siRNA into the cytosol, (2) the possibility that the protein of interest regulates other membrane trafficking pathways, not necessarily endosomal (clathrin heavy chain and dynamin are good examples), and (3) the fact that depletion of the protein affects cell viability or dysregulates other endocytic pathways. Another important aspect of this approach is that a specific probe is required whose uptake is confined to one pathway; this allows researchers to identify whether expression of mutant proteins or siRNA depletion is having a biological response. Transferrin is a very well-established marker for uptake via clathrin-coated vesicles [22], both anti CD35 antibodies and poly(ethylenimine) complexes have been shown to be partially internalized in a flotillin-1-dependent manner [18, 23] and cholera toxin is often used as a marker for uptake via caveolae. A number of other poorly defined pathways exist and identifying specific endocytic probes for those not organized by clathrin has been difficult [5, 9]. For example, a number of studies show that cholera toxin can access through different pathways [9] and very careful analysis is required to ensure that uptake is confined to one route [24]. 15.3

DELIVERING PROTEINS INTO CELLS

Delivering a full-length protein that may have multiple domains, interactions, and effects is sometimes required to mediate a necessary biological response, whether for research or therapy. The realistic potential of delivering full-

c15.indd 266

7/30/2010 2:19:19 PM

HIV-TAT PROTEIN

267

length proteins was probably seeded when it was realized that many diseases were caused by malfunctioning or missing proteins and the technology became available for their purification, especially from transformed microorganisms such as Escherichia coli. Most proteins are membrane impermeable and a large fraction of them function inside cells; therefore systems are required for their delivery into their site of action, often the cytosol and in some cases subcellular organelles such as the nucleus and mitochondria. This therefore requires an intracellular delivery system and potential candidates for performing this function are short peptide sequences that have the capacity to enter cells either as single entities or attached to much larger cargo such as fulllength proteins. These are called protein transduction domains (PTDs) or cell-penetrating peptides. 15.3.1 Defining Cell-Penetrating Peptides and Protein Transduction Domains The term cell-penetrating peptide gained prominence around 20 years ago as did the use of the term protein transduction domain and there is still healthy discussion in meetings over the most appropriate terminology for what could generally be described as membrane active peptides. Peptides that interact with eukaryotic and prokaryotic membranes have, however, been described in the literature for decades before either of these now familiar terms gained prominence [25]. An excellent review written by Plank et al. [26] in 1998 described a number of peptides with potential for delivering macromolecules and examples include melittin, mastoparan, GALA, and KALA; these are more familiarly known as pore-forming, membrane active, or destabilizing peptides (Table 15.1). However, as previously noted, the distinction between these and what we now refer to as CPPs or PTDs can sometimes be difficult to define [27]. Hundereds of peptides have now been described that share a common ability to deliver cargo to cells and their heterogeneity with respect to sequence and physical and biological properties suggests that neither of these commonly used terms is appropriate for all. One of the best characterized is the cell-penetrating peptide from the protein transduction domain of the HIV-Tat protein.

15.4

HIV-TAT PROTEIN

The genome of HIV encodes proteins that are important for structure, organization of its envelope, enzyme regulators, and also transactivators such as HIV-Tat. This 101 amino acid protein is manufactured inside infected cells and gains entry into the nucleus, where it binds RNA to activate viral transcription [28]. Residues 1–86 are transactivation active and a number of laboratories have used this truncated form for their studies, rather than the full-length protein. The protein also appears to be released from intact virus-infected cells and this fraction can enter other cells via endocytosis

c15.indd 267

7/30/2010 2:19:19 PM

268

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

TABLE 15.1

Sequences of Membrane Active Peptidesa Described in This Chapter

Peptide HIV-Tat Bov-Tat JDV Tat Transportan TP10 pVEC Penetratin (pAntennapedia) MPG PEP-1 PTD-5 PDX-1 PTD VP22 GALA KALA Melittin Mastoparan HA2 HA2′

Sequence

Reference

GRKKRRQRRRPPQ RRRGTRGKGRRIRR RHDGRRKKRGTRGKGR GWTLNSAGYLLGKINLKALAALAKKIL AGYLLGKINLKALAALAKKIL LLIILRRRIRKQAHAHSK RQIKIWFQNRRMKWKK

127 54 53 83 106 84 78

GALFLGFLGAAGSTMGAWSQPKKKRKV KETWWETWWTEWSQPKKKRKV YARAARRAARR RHIKIWFQNRRMKWKK DAATATRGRSAASRPTERPRAPARSASRPRRPVE WEAALAEALAEALAEHLAEALAEALEALAA WEAKLAKALAKALAKHLAKALAKALKACEA IKITTMLAKLGKVLAHV INLKALAALAKKIL GDIMGEWGNEIFGAIAGFLG GLFEAIEGFIENGWEGMIDGWYG

89 88 81 62 128 92 92 25 25 58 64

a

Variations on these sequences are commonly used, especially the addition of terminal residues such as cysteine for conjugation to fluorophores. Bov, bovine immunodeficiency virus; JDV, Jembrane disease virus; PDX-1, pancreatic and duodenal homeobox factor-1 protein transduction domain.

[29–32]. But most importantly for those interested in drug delivery, the protein then escapes from the endolysosomal network to the cytosol and then on to the nucleus [33]. HIV-Tat has also been shown to interact with a number of other cell surface molecules to mediate biological effects, and its location on the plasma membrane and interaction with viral gp-120 may also aid in promoting viral entry [34, 35]. A crystal structure for HIV-Tat has not been published, but interestingly NMR studies reveal that the protein may be one of the many proteins that exist as an unfolded entity that may allow for multiple interactions with affectors and effectors [28, 36–38]. The basic domain is seen fully exposed on the surface (Figure 15.2) but it is likely that other parts of the molecule, perhaps the hydrophobic core, also aid in endosomal escape. 15.4.1 Cellular Dynamics HIV-Tat Protein The biology of HIV-Tat protein has been the subject of intense scrutiny and like other HIV proteins it is a target for anti-HIV drugs [28]. Its biology, either manufactured by the protein synthesis machinery inside a virus-infected cell or following uptake from the extracellular fluid, is extremely complex and over 250 HIV-Tat interacting partners have now been identified [39].

c15.indd 268

7/30/2010 2:19:19 PM

HIV-TAT PROTEIN

(A)

269

(B)

Figure 15.2. NMR structures of HIV-Tat 1–86. Both (A) [37] and (B) [36] show quite different structures but common is the exposure of the basic domain, residues 48–60, shown in blue. The deposited structures were obtained from the Protein Data Bank and were processed using Pymol software.

15.4.1.1 Binding to the Plasma Membrane A requirement for HIV-Tat endocytosis, endosomal escape, nuclear enrichment, and RNA binding is a short highly basic sequence spanning residues 47–57 (Table 15.1). This region interacts with negatively charged cell surface heparan sulfate proteoglycans and is thought to gain access to the cell as an ionic complex with these sugars [40–42]. The initial cellular uptake and endocytosis of HIV-Tat protein and of functional residues 1–72 were performed using E. coli expressed protein that was then purified and labeled with 125I [30, 31]. Uptake was partially characterized in a number of cell lines and found to be dependent on temperature and could be inhibited by competing anionic charge in the form of heparin. Of note is the fact that the protein at 1 nM (∼10 ng/mL) was able to promote transactivation—an amazing feat and one that bears consideration when looking at subsequent studies aiming to delineate in much more detail its uptake and intracellular dynamics and also of HIV-Tat fusion proteins and HIV-Tat peptide linked proteins. Immediate to the finding that HIV-Tat could enter cells were studies showing that major fractions of the protein or the basic region could be linked to the N or C terminus of proteins and promote their uptake as active entities both in vitro in a number of different cell lines, and also in vivo [43–45]. There was thus an obvious interest in elucidating the mechanism by which these proteins were able to overcome membrane bilayers. The creation of a HIVTat-Enhanced Green Fluorescent protein (EGFP) fusion protein confirmed that this domain interacted strongly with heparan sulfate proteoglycans and that attenuated uptake of the fusion protein and its capacity for transactivation was observed in Chinese hamster ovary (CHO) cells deficient in proteoglycan synthesis [42, 46]. Similar reduced uptake and transactivation was noted when

c15.indd 269

7/30/2010 2:19:19 PM

270

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

soluble glycosaminoglycans, most notably heparin, were added to cell culture media during incubations with HIV-Tat fusion proteins and with HIV-Tat alone [31]. Recent data in primary brain cells also demonstrate a degree of positive correlation between glycosaminoglycan expression and the ability of GFP-HIV-Tat peptide fusion to gain cell entry [47]. 15.4.1.2 Endocytosis The most extensive characterization of endocytosis and translocation of native HIV-Tat (residues 1–86) was performed in Jurkat T lymphocytes [35]. Uptake of the protein at 50 nM was inhibited by approximately 50% in cells treated with chlorpromazine, a well-known inhibitor of clathrin-coated uptake and it was localized, using immunocytochemistry, in clathrin-coated pits. In support of this, the ability of Tat to escape from the endolysosomal system was inhibited in cells expressing mutant proteins that affected clathrin-coated uptake. A cell-free translocation assay was developed to monitor Tat release from endosomes and this revealed that escape was dependent on endosomal acidification. 15.4.1.3 Fixing Artifacts It is fair to say that the field of cell-penetrating peptides shuddered somewhat when Lundberg and co-workers published two reports regarding the uptake and subcellular localization of GFP extended with cell-penetrating sequences from HIV-Tat and herpes virus protein VP22, octalysine and octaarginine [48, 49]. The studies highlighted the different capacities of these four sequences to label CHO cells but also suggested that fixing cells for microscopy and flow cytometry had major effects leading to an overestimation of the capacity of CPPs to deliver cargo to the inside of cells and the nucleus rather than to label, respectively, the plasma membrane and endocytic vesicles. These studies and others investigating fluorescently labeled CPPs [50] had a huge effect on this field and awoke researchers to the realization that studies with CPPs, (especially the cationic forms) needed to be performed in unfixed cells, and that stringent washing procedures with enzymes and anionic molecules were required to remove surface label. 15.4.1.4 Endocytic Pathways Utilized by HIV-Tat Protein The aforementioned study of HIV-Tat endocytosis in Jurkat cells was performed in fixed cells using anti HIV-Tat antibodies [35], but to perform live cell imaging there was a requirement to tag the protein with a fluorophore or to extend its sequence with a fluorescent protein such as GFP. In these and other studies, additional tags are added to the protein to aid in purification and these include His6 and glutathione S-transferase tags [49, 51] that may or not be linked to the protein of interest via protease sensitive linkages [52]; this minimizes the possibility of tag-mediated effects. This technology has recently been used to investigate the uptake and subcellular distribution of Tat proteins from HIV 1 and more recently other viruses [53, 54]. Comparative analysis has been performed on the uptake and intracellular localization of GST-Tat (full length)-EGFP and GST-Tat(48–60)EGFP in a

c15.indd 270

7/30/2010 2:19:19 PM

HIV-TAT PROTEIN

271

HeLa cell derivative HL3T1 [51, 55]. Uptake of both was dependent on endocytosis and the actin cytoskeleton and fluorescence was shown to colocalize in vesicles labeled with caveolin-1 and cholera toxin that is often used as a marker of uptake via caveolae. The problems of using cholera toxin as an endocytic pathway marker have been described above. The cholesterol-sequestering agent MßCD inhibited the uptake of both fusion proteins, suggesting that lipid rafts were necessary for uptake, and very little colocalization was observed with fluorescent transferrin, a marker of uptake via clathrin-coated vesicles. This data was rather different from studies in T cells showing that uptake of HIV-Tat alone (i.e., without cargo) was through clathrin-coated pits [35]. One important aspect of the study on the uptake of HIV-Tat in the absence of cargo was the fact that it was performed in Jurkat T cells that do not express caveolin-1 [56], and thus uptake in this cell line could not have been mediated by caveolae. HeLa cells, on the other hand, express low levels of caveolin-1 but it is difficult to visualize caveolae in this cell line [57]. Overall, the studies demonstrate that caveolae are not absolutely required for cell entry and escape from endocytic pathways. A role of clathrin and caveolae was disputed when a Tat-Cre recombinase assay was used to investigate the capacity of the basic domain of Tat to deliver the 38-kDa Cre recombinase protein to mediate expression of the EGFP reporter gene [58]. The purified Cre protein was labeled with Alexa488 and its uptake into cells was shown to be inhibited after treatment with the cholesterol-interacting agent nystatin, that along with MßCD also inhibited recombination. The study showed that Tat-Cre entry and function were not dependent on caveolin expression and thus uptake via caveolae. Recombination, however, was inhibited following cytochalasin D and amiloride treatment, suggesting that macropinocytosis may be involved in peptide uptake. This endocytic process usually occurs in growth factor stimulated cells and involves extensive actin reorganization and engulfment of large volumes of extracellular liquid that are then enclosed to form internal macropinosomes [4, 6, 59]. Vesicles formed from other endocytic pathways have diameters in the range of 60–120 nm but macropinosomes can be micrometers in diameter. These enlarged structures were not observed but in support of a role for this pathway in Tat-Cre uptake was an increased fluid phase uptake of 70 kDa dextran. This was an important observation suggesting that the basic domain of Tat was interacting with the plasma membrane to cause cytoskeletal activation, ruffling, and enhanced uptake of fluid-phase markers, and possibly itself. It is important to note here that 70-kDa dextran (hydrodynamic radius 5.5 nm) is not a specific marker for macropinocytosis, as is sometimes mentioned in the literature, but rather a good probe to monitor enhanced fluid-phase uptake that is characteristic of this endocytic event. The studies did suggest, however, that entry of this protein and indeed cationic CPPs was not a passive mechanism but one that involved activation and reorganization at the plasma membrane.

c15.indd 271

7/30/2010 2:19:19 PM

272

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

A later study using a similar Tat-Cre system also showed that energy and an intact actin cytoskeleton were required for cell entry [60]. The data also demonstrated the limitations of confocal microscopy for these kinds of analysis in so much that despite the fact that there was no visible (microscopical) evidence for cytosolic and nuclear Cre, the biological readout assay suggested otherwise. Conversely, confocal microscopy and laser-induced membrane damage and leakage can also potentially overestimate the fraction of an internalized entity that is released in the cytosol [61]. Macropinocytosis was also implicated in the uptake of the transcription factor pancreatic and duodenal homeobox factor 1 (PDX-1) [62] that has within its sequence a cell-penetrating domain (Table 15.1). Labeling recombinant PDX-1 with fluorescein isothiocyanate (FITC) and adding it to cells allowed for microscopical analysis of its distribution, and at 5-μM extracellular concentration the protein was found to label vesicles and the cytosol. Further analysis of the recombinant protein was not performed but amiloride was shown to inhibit uptake of the protein transduction domain of this protein. 15.4.2 Endocytosis of Tat from Other Viruses GFP fusion proteins of Jembrane disease virus and bovine immunodeficiency virus Tat have been generated and these also have basic rich sequences, and the Jembrane disease virus basic domain is longer than that of HIV-Tat (Table 15.1). Both of these proteins efficiently delivered GFP into cells but the uptake mechanism was not studied in detail. Interestingly, there was evidence that uptake of denatured Tat-EGFP constructs occurred via an energyindependent manner whereas uptake of the native proteins was inhibited in the absence of energy and therefore dependent on endocytosis. But this needs further analysis to reveal whether the proteins were actually located inside the cells rather than on the cell surface. 15.4.3 Escaping from the Endolysosomal Network If endosomal escape of HIV-Tat protein is dependent on acidification, then the possibility exists that the ultimate fate of the fraction that cannot escape will be degradation in lysosomes. One study utilized a mutant GFP (F64L, T203Y) that has a pH-dependent emission profile and its pH environment can then be monitored during endocytosis [63]. The mutant was extended with the HIV-Tat basic sequence and the protein was then added to cells to reveal that the fusion protein reached a compartment of pH 5.8 before the GFP fluorescence was quenched and the protein degraded. Extensive degradation had occurred within 8 hours. Degradation is a major issue if there is a requirement for the therapeutic to be delivered from the endolysosomal system, and beyond, and attempts have been made to enhance escape from endosomes. One study showed that coincubation of a membrane fusion peptide HA2 derived from the N terminus of influenza A hemagluttinin (Table 15.1) to

c15.indd 272

7/30/2010 2:19:19 PM

HIV-TAT PROTEIN

273

incubations with Tat-Cre enhanced its capacity for recombination [58]. A longer sequence that was also called HA2 (HA2 in Table 15.1) was shown to enhance the transcriptional activity of extracellularly delivered p53RRRRRRRRR fusion protein [64]. However, the approach here was quite different as in this case the CPP, fusion peptide, and delivered cargo were linked as one protein HA-p53-R9. Subsequent labeling of this protein with FITC and confocal microscopy analysis in live glioma cells showed it to be diffusely localized and enriched in the nucleus; this was 4 hours postincubation. The same fusion protein lacking the HA2 moiety was predominantly localized to the plasma membrane. The exact mechanism by which HIV-Tat protein in the absence of a defined fusion sequence escapes from the endolysosomal system is unknown but to aid viral replication a fraction must gain access to first the cytosol and then the nucleus. The previously mentioned GFP mutant study [63] demonstrated that the protein reached a compartment of pH 5.8 but whether the “active” fraction has already escaped before this pH is reached is unknown. It is technically difficult to analyze endosomal escape of a protein and there are many unanswered questions regarding the dynamics of this protein once it has gained access into cells. One interesting method employed fluorescent resonance energy transfer (FRET) to monitor the uptake and endosomal escape of the Tat peptide sequence conjugated to fluorescent protein mCherry that formed the acceptor fluorophore for FITC [65]. FRET between these two molecules was quenched by 4-(4′-dimethylaminophenylazo)benzoic acid (Dabcyl) and the fluorescence profile of the construct was dependent on whether it was intact or degraded in endosomes or the cytosol. Although this was a complex system, it offered an opportunity to monitor in real time the fate of delivered proteins fused with penetrating sequences. 15.4.4 Entry into the Nucleus Passive diffusion should allow cytosol–nucleus exchange for molecules <45 kDa [66] and full-length HIV-Tat protein should therefore be able to access by diffusion. EGFP is a 27-kDa protein and it is expected that EGFP-HIV-Tat protein fusion proteins (masses of 36 kDa or 37 kDa, depending on whether full length or the commonly used residues 1–86 are used) should also be able to access the nucleus by diffusion. There is, however, some dispute over whether nuclear entry of EGFP-HIV-Tat is in fact an active process. Early studies in cells overexpressing HIV-Tat protein-EGFP showed the protein to be highly enriched in the nucleus and when expression was very high it was also very noticeable in the nucleolus [67]. A number of studies have subsequently aimed to determine the mechanism by which the protein reaches the nucleus and whether the basic stretch of amino acids, in addition to promoting cell entry and endosomal escape, also harbors a bona fide nuclear localization sequence (NLS) [68, 69]. Typically, NLSs are monopartite or bipartite with respect to basic sequences [66] as exemplified,

c15.indd 273

7/30/2010 2:19:19 PM

274

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

respectively, by the NLS from SV40 T antigen PKKKRKV [70] and nucleoplasmin (KRPAATKKAGQAKKKKL) [71]. Studies using GFP and GFP dimers or tetramers containing, at the N terminus, the HIV-Tat basic domain sequence (YGRKKRRQRRR-GFP) suggest nuclear localization of GFP or GFP dimers occurs in the absence of energy and thus via passive diffusion [72]. However, by substituting the three arginines terminal to the basic sequence with glycines to give YGRKKRRQGGG-GFP, the nuclear localization of a 110-kDa GFP tetramer cargo, that was normally absent from the nucleus, could be achieved [73]. This suggests that the three consecutive arginines in the HIV-Tat sequence GRKKRRQRRRPPQ may in fact hinder nuclear entry. These studies have important implications for researchers attempting to deliver therapeutics to the nucleus.

15.5

PROTEIN DELIVERY BY OCTAARGININE

GFP variants have also been shown to be delivered to cells by extending the sequence with octaarginie (R8) that, like HIV-Tat, represents one of the most studied CPPs [49, 74]. R8-EGFP when introduced extracellularly to CHO cells was localized predominantly to the plasma membrane but the images only show data after 5 minutes uptake [49]. It is likely that longer incubations are needed to clearly visualize the protein in endosomes and possibly the cytosol. Flow cytometry analysis clearly showed that extending EGFP with R8 enhanced its cell association, but these studies did not use trypsinization to lift the cells from the plates and thus the fluorescence may have been overestimated. The addition of pyrenebutyrate, a hydrophobic negatively charged counterion, to the cell media before addition of Alexa488-R8 peptide significantly enhanced the fluorescence signal in the cytosol and there was also evidence that the same was true for R8-EGFP [74]. It was suggested that pyrenebutyrate interaction with the arginine guanidinium groups of R8, and with membranes, promoted R8-mediated delivery of EGFP to the cytosol. This effect, however, may be cell type dependent [75].

15.6 SYSTEMS FOR ATTACHING CELL-PENETRATING PEPTIDES TO PROTEINS Typically, cloning strategies are used to extend the N or C terminus of proteins to cell-penetrating peptide sequences. However, a number of other approaches have been used to link them to protein cargo (Figure 15.3). 15.6.1 Covalent Attachment 15.6.1.1 Disulfide Bonds The Tat sequence and R7 have also been attached to proteins such as tissue-specific plasminogen activator and

c15.indd 274

7/30/2010 2:19:19 PM

SYSTEMS FOR ATTACHING CELL-PENETRATING PEPTIDES TO PROTEINS

7

275

1

SS

6

2

AvBi

ion

3

lat

e ch

5 4

Figure 15.3. Protein delivery strategies using CPP sequences or mimetics. 1, CPP sequence attached to cargo via a peptide bond; 2, full-length or truncated transducing proteins fused to cargo proteins; 3, CPP-mimetic–protein complex; 4, ionic or hydrophobic CPP–protein complexes; 5, CPP–protein chelates; 6, CPP–protein avidin–biotin complexes; 7, CCP sequence is linked to a protein through an S–S bond. Center shows early endosomes (red) and nucleus (blue) of a liver Hep3B cell.

asparaginase using disulfide linkages formed with the crosslinking agent N-succinimidyl 3-(2-pyridyldithio)propionate [76, 77]. FITC-labeled Tat–S-Sasparaginase was shown to be diffusely distributed in HeLa cells but the cells were fixed for microscopy and the extracellular concentration of Tat was not provided [77]. 15.6.1.2 Cell-Penetrating Peptide Mimetics Efforts have also been made to mimic the translocation capacities of cell-penetrating peptides to design new “synthetic” entities based on the well-characterized tendency for CPPs to be amphipathic and to contain guanidinium groups. An example was described using the antennapedia sequence [78] (Table 15.1) and its helical structure as a template for the design of a biphenyl mimic small molecule carrier (SMoC). This was shown to effectively deliver FITC and geminin, a 23-kDa DNA replication inhibitor protein [79]. Delivery of this protein caused growth inhibition of cancer cells, suggesting successful delivery of the protein to the nucleus. The uptake of the SMoC was inhibited at low temperatures, showing a requirement for energy, but internalization was not inhibited by a range of endocytic inhibitors including cytochalasin D, chlorpromazine, and the amiloride analog EIPA. Similar data was obtained with SMoC protein

c15.indd 275

7/30/2010 2:19:19 PM

276

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

cargo with the exception that chlorpromazine inhibited uptake by 70%. This suggests that a large fraction entered cells via clathrin-coated vesicles. An intermediate of this SMoC could be derivatized to accommodate a wide range of attachment chemistries for conjugation to multiple types of cargo. 15.6.2 Noncovalent Linkages between Cell-Penetrating Peptides and Proteins 15.6.2.1 Avidin–Biotin Affinity GFP has been a very useful tool for analyzing the uptake and cellular dynamics of proteins containing CPP sequences. A number of studies aiming to better understand these processes have used the high affinity and specificity of avidin–biotin chemistry. One such study compared the capacity of biotinylated Tat, R4-12R, 4K-12K to deliver Alexa488-streptavidin and streptavidin-ß-galactosidase to five different cell lines [80]; also included here was a modified HIV-Tat basic sequence PTD-5 (Table 15.1) [81]. Despite the fact that these studies were preformed before the “fixing” papers came to press, some of the experiments employed trypsinization to remove surface-associated label. Tat, PTD-5, R6-12, and K6-12 were shown to deliver these cargoes but the extent by which they were able to do this was highly dependent on the cell line. Generally, the most efficient delivery sequences were either K8 or K10. This is interesting, as the literature shows that many more papers since this period have used oligoarginine and Tatbased sequences compared with oligolysines. Avidin–biotin chemistry was also used to compare the delivery capacity of HIV-Tat peptide, penetratin, transportan, and pVEC [82]. Penetratin is the protein transduction domain from the Drosophila Antennapedia protein [78], transportan is a chimera of sequences from peptides galanin and mastoparan [83], and pVEC is derived from murine cadherin [84]. Streptavidin was conjugated to the fluorophore FITC and this was preincubated with the biotinylated peptides to form avidin–biotin complexes that were then added to cells. Transportan and Tat had significantly higher delivery capacity than the other peptides but these experiments were only performed in HeLa cells. Interestingly, preincubating the cells with MßCD had only a minimal effect on uptake by HIV-Tat and transportan but despite having much lower delivering capacity, Antennapedia and pVEC mediated uptake seemed to be much more sensitive to cholesterol depletion. Extensive confocal and electron microscopy studies on biotinylated transportan, and its deletion analog TP10 [85] complexed with fluorescent or goldlabeled avidin, suggested that there was little overlap of the complexes with internalized transferrin and that labeled structures akin to macropinosomes were observed using electron microscopy. The ultimate fate of the complexes was deemed to be lysosomes but this study also raised the possibility that these CPPs could be delivering protein cargo directly across the plasma membrane and also highlighted the problems of complex aggregation in the extracellular

c15.indd 276

7/30/2010 2:19:19 PM

SYSTEMS FOR ATTACHING CELL-PENETRATING PEPTIDES TO PROTEINS

277

media, thus making data interpretation very difficult. The main problems with using electron microscopy is that the cells need to be fixed, but parallel live cell imaging and electron microscopy can give revealing information as described here. Later studies from the same group showed reduced uptake (∼30–50%) following siRNA depletion of caveolin-1 in HeLa cells and also reduced uptake in caveolin-1 knockout mouse embryonic fibroblasts compared to control cells [86]. Again confocal microscopy showed very little evidence of colocalization of these complexes with endocytosis markers such as rab5, transferrin, and flotillin-1 but some colocalization was observed with caveolin-1 using confocal and electron microscopy. 15.6.2.2 Cell-Penetrating Peptide–Protein Complexes Cell-penetrating peptides have also been shown to deliver biologically active proteins via complexation rather than through direct peptide bond linkage or through avidin– biotin chemistry. The most well described are amphipathic peptides MPG, Pep-1 (Table 15.1), and their derivatives [87–89]. Their sequence is deemed to allow for electrostatic and/or hydrophobic interactions with a number of different proteins, but once the complex is formed the peptides still have the necessary functionalities to interact with cells and allow delivery to the cytosol. Despite observations showing that these peptides, as single entities or associated with cargo, induce actin rearrangements [90], the current thinking is that delivery may not require any form of endocytosis and that the access route is directly across the plasma membrane [87, 89]. This is thought to be mediated via pore formation; however, studies investigating siRNA delivery with MPG suggest that endocytosis is very much involved [91]. The implications of a mechanism for delivering proteins directly across the plasma membrane are clear as it could reduce or negate degradation issues that are a feature of endocytosis if the cargo is fragile to the hostile environment of late endosomes and lysosomes. Other notable pore-forming peptides with delivery capacity are the KALA and GALA peptides (Table 15.1) and related sequences [26, 92]. Comparative studies on the uptake of avidin or streptavidin either as complexes with cell-penetrating sequences or linked via biotin was revealing in that it showed among other things that the capacity of cell-penetrating sequences to deliver cargo was very much dependent on the nature of the cargo [93, 94]. An uncharacterized synthetic CPP YTA2 (Table 15.1) was analyzed against a number of well-characterized CPPs and overall the studies show that the effectiveness of any particular sequence for delivering cargo, including full-length proteins, is very much dependent on the physical properties of the cargo and most probably the choice of studied cell line or tissue. The issue of CPP-mediated toxicity and membrane leakage has been discussed in some detail [95–97] and overall the studies suggest that great care needs to be taken when interpreting data measuring uptake of cargo without parallel viability assays.

c15.indd 277

7/30/2010 2:19:19 PM

278

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

15.6.2.3 Cell-Penetrating Peptide–Protein Chelates Linking expressed fusion proteins, with histidine residues is routine for purification of bacterially expressed proteins, but the Futaki group exploited this further when they manufactured a nitriliotraiacetic acid derivatized octaarginine peptide NTA. This forms noncovalent complexes with His residues and this affinity was shown to mediate the uptake of His6-EGFP into mammalian cells [52]. The derivatized peptide was also able to deliver a His6 tagged proapoptotic peptide PAD (KLAKLAK)2 to cells to inhibit viability. Numerous studies have shown effective in vitro and in vivo cell killing with CPP-PAD conjugates [80, 98–102] but the possibility exists in some cases that cationic CPPs and PAD, when linked together, synergistically disrupt the plasma membrane and cause necrosis rather than apoptosis [103].

15.7

TARGETING LYSOSOMES

In some situations it is beneficial to keep the protein in the endolysosomal system to allow it to reach its target or substrate in the lysosomes. This is especially true for diseases caused by a deficiency of a protein that naturally resides and functions in these organelles. An example is ßglucuronidase deficiency that causes mucopolysaccharide type VII syndrome, a lysosomal storage disease. This protein can gain access to cells via the mannose-6-phosphate receptor, which can then traffic it to the lysosome. A study investigated the uptake of this protein compared to the ß-glucuronidase sequence extended with the HIV-Tat peptide [104]. This C-terminal extension changed the endocytic uptake mechanism and generally uptake was reduced, but in tissues such as the kidney, higher levels of the chimera were observed and these correlated with enhanced clearance of glycosaminoglycans. This may be due to the reduced renal clearance that was observed for the chimera but it also showed effective intracellular delivery of this protein to the lysosomes and that the presence of the Tat domain negated the requirement for the receptor to deliver ß-glucuronidase to these organelles. Very similar results were obtained when galactocerebrosidase was extended with Tat peptide in an effort to treat Krabbe disease, another lysosomal storage condition [105].

15.8 COMPARING CPP FUSION PROTEINS WITH CPP FLUOROPHORES Despite the fact that a wealth of information exists on the capacity of CPPs to deliver active full-length or truncated proteins, only a few studies have carefully scrutinized uptake and biological activity in the same study. To analyze the uptake and intracellular traffic of CPP–protein conjugates is technically challenging as there is a requirement for cloning a cDNA for a

c15.indd 278

7/30/2010 2:19:19 PM

CONCLUSION

279

fusion protein, expressing this, for example, in E.coli, purifying the protein, and then monitoring its uptake as a fluorescent entity. Here there is a further requirement that its conjugation to a fluorescently labeled large (GFP) or relatively small (FITC/Alexa dyes, <1 KDa) fluorophores does not interfere with its cellular dynamics and activity. Labeling peptides with fluorophores and analyzing their uptake is, however, relatively simple and hence the process by which CPP fluorophores interact with cells and gain access to the cell interior and beyond has been the subject of intense scrutiny. Readers are referred to numerous reviews that have recently been published on experiments in vivo, in vitro, and in artificial systems such as membrane bilayers [4, 11, 27, 106–112]. A number of the endocytic pathways described in Figure 15.1 have been implicated as being involved in entry of CPPs attached to fluorophores such as FITC and Alexa dyes. Some common themes are now emerging that should help researchers in drug delivery and cell biology maximize their translocation capacities, especially in cell-based assays. For cationic peptides there is clear evidence that they can traverse the plasma membrane of some cell lines in the presence and absence of energy [113–115]. It is also clear that at 37 °C and at relatively high peptide concentrations (=5 μM) the plasma membranes of some cell lines are permeable to these peptides but not to dyes such as propidium iodide when these are coicubated with the CPPs [114, 116–118]. However, these concentrations are very close to those that then show a general increase in membrane porosity and thus propidium iodide entry [97]. The presence of serum in the media has significant effects owing to the general affinity of the peptides to proteins such as albumin that then reduces the effective peptide concentration at the plasma membrane [117]. An interesting question is how we can relate these studies to those described above, where the cargo is much larger than a fluorophore and where a requirement for endocytosis is more likely. With different cargoes, as previously suggested, the mechanism by which membrane bilayers are breached and especially the location of this translocation may be quite different.

15.9

CONCLUSION

Following the initial revelation that a CPP could deliver a 120 kDa protein to cells and different tissues [43, 45], we have witnessed a rapidly expanding literature on the potential of this technology for the delivery of proteins as therapeutics [108, 119–122]. Important questions regarding the usefulness of this approach for delivering proteins and a library of other cargoes have been asked [123–126] but those working in the field will strive to enhance their vector capacity. Future research in this area promises the usual mixture of interesting science, controversy, and hopefully news of CPP conjugates progressing from clinical trials to the clinic.

c15.indd 279

7/30/2010 2:19:19 PM

280

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

ACKNOWLEDGMENTS The author apologizes to those whose work was not mentioned here and who have contributed to this field, and would like to thank Nicola Zonta of Cardiff University for help in generating Figure 15.2.

REFERENCES 1. Doherty, G. J. and McMahon, H. T. Mechanisms of endocytosis. Annu. Rev. Biochem. 78: 857–902 (2009). 2. Cheng, Z. J., Singh, R. D., Marks, D. L., and Pagano, R. E. Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids. Mol. Membr. Biol. 23: 101–110 (2006). 3. Donaldson, J. G., Porat-Shliom, N., and Cohen, L. A. Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling. Cell Signal. 21: 1–6 (2009). 4. Jones, A. T. Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides. J. Cell Mol. Med. 11: 670–684 (2007). 5. Mayor, S. and Pagano, R. E. Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol. 8: 603–612 (2007). 6. Mercer, J. and Helenius, A. Virus entry by macropinocytosis. Nat. Cell Biol. 11: 510–520 (2009). 7. Rappoport, J. Z. Focusing on clathrin-mediated endocytosis. Biochem. J. 412: 415– 423 (2008). 8. Robertson, A. S., Smythe, E., and Ayscough, K. R. Functions of actin in endocytosis. Cell Mol. Life Sci. 66: 2049–2065 (2009). 9. Sandvig, K., Torgersen, M. L., Raa, H. A., and van Deurs, B. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem. Cell Biol. 129: 267–276 (2008). 10. Jones, A. T. Intracellular drug delivery. Workshop report from the 28th International Symposium on Controlled Release of Bioactive Materials, San Diego, 23–24 June 2001. Traffic 2: 917–920 (2001). 11. Jones, A. T. Gateways and tools for drug delivery: endocytic pathways and the cellular dynamics of cell penetrating peptides. Int. J. Pharm. 354: 34–38 (2008). 12. Jones, A. T., Gumbleton, M., and Duncan, R. Understanding endocytic pathways and intracellular trafficking: a prerequisite for effective design of advanced drug delivery systems. Adv. Drug Deliv. Rev. 55: 1353–1357 (2003). 13. Medina-Kauwe, L. K. “Alternative” endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery? Adv. Drug Deliv. Rev. 59: 798–809 (2007). 14. Sandvig, K. and van Deurs, B. Membrane traffic exploited by protein toxins. Annu. Rev. Cell Dev. Biol. 18: 1–24 (2002). 15. Watson, P. Live cell imaging for target and drug discovery. Drug News Perspect. 22: 69–79 (2009).

c15.indd 280

7/30/2010 2:19:19 PM

REFERENCES

281

16. Watson, P., Jones, A. T., and Stephens, D. J. Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells. Adv. Drug Deliv. Rev. 57: 43–61 (2005). 17. Ivanov, A. I. Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol. Biol. 440: 15–33 (2008). 18. Glebov, O. O., Bright, N. A., and Nichols, B. J. Flotillin-1 defines a clathrinindependent endocytic pathway in mammalian cells. Nat. Cell Biol. 8: 46–54 (2006). 19. Hinrichsen, L., Harborth, J., Andrees, L., Weber, K., and Ungewickell, E. J. Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 278: 45160–45170 (2003). 20. Huang, F., Khvorova, A., Marshall, W., and Sorkin, A. Analysis of clathrinmediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 279: 16657–16661 (2004). 21. Motley, A., Bright, N. A., Seaman, M. N., and Robinson, M. S. Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162: 909–918 (2003). 22. Maxfield, F. R. and McGraw, T. E. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5: 121–132 (2004). 23. Payne, C. K., Jones, S. A., Chen, C., and Zhuang, X. Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 8: 389–401 (2007). 24. Pelkmans, L., Burli, T., Zerial, M., and Helenius, A. Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118: 767–780 (2004). 25. Bernheimer, A. W. and Rudy, B. Interactions between membranes and cytolytic peptides. Biochim. Biophys. Acta 864: 123–141 (1986). 26. Plank, C., Zauner, W., and Wagner, E. Application of membrane-active peptides for drug and gene delivery across cellular membranes. Adv. Drug Deliv. Rev. 34: 21–35 (1998). 27. Henriques, S. T., Melo, M. N., and Castanho, M. A. Cell-penetrating peptides and antimicrobial peptides: how different are they? Biochem. J. 399: 1–7 (2006). 28. Campbell, G. R. and Loret, E. P. What does the structure–function relationship of the HIV-1 Tat protein teach us about developing an AIDS vaccine? Retrovirology 6: 50 (2009). 29. Arya, S. K., Guo, C., Josephs, S. F., and Wong-Staal, F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229: 69–73 (1985). 30. Frankel, A. D. and Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55: 1189–1193 (1988). 31. Mann, D. A. and Frankel, A. D. Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J. 10: 1733–1739 (1991). 32. Sodroski, J., Patarca, R., Rosen, C., Wong-Staal, F., and Haseltine, W. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science 229: 74–77 (1985). 33. Peruzzi, F. The multiple functions of HIV-1 Tat: proliferation versus apoptosis. Front. Biosci. 11: 708–717 (2006).

c15.indd 281

7/30/2010 2:19:19 PM

282

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

34. Marchio, S., Alfano, M., Primo, L., Gramaglia, D., Butini, L., Gennero, L., De Vivo, E., Arap, W., Giacca, M., Pasqualini, R., and Bussolino, F. Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein. Blood 105: 2802–2811 (2005). 35. Vendeville, A., Rayne, F., Bonhoure, A., Bettache, N., Montcourrier, P., and Beaumelle, B. HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses. Mol. Biol. Cell 15: 2347– 2360 (2004). 36. Bayer, P., Kraft, M., Ejchart, A., Westendorp, M., Frank, R., and Rosch, P. Structural studies of HIV-1 Tat protein. J. Mol. Biol. 247: 529–535 (1995). 37. Gregoire, C., Peloponese, J. M., Jr., Esquieu, D., Opi, S., Campbell, G., Solomiac, M., Lebrun, E., Lebreton, J., and Loret, E. P. Homonuclear 1H-NMR assignment and structural characterization of human immunodeficiency virus type 1 Tat Mal protein. Biopolymers 62: 324–335 (2001). 38. Shojania, S. and O’Neil, J. D. HIV-1 Tat is a natively unfolded protein: the solution conformation and dynamics of reduced HIV-1 Tat-(1-72) by NMR spectroscopy. J. Biol. Chem. 281: 8347–8356 (2006). 39. Ptak, R. G., Fu, W., Sanders-Beer, B. E., Dickerson, J. E., Pinney, J. W., Robertson, D. L., Rozanov, M. N., Katz, K. S., Maglott, D. R., Pruitt, K. D., and Dieffenbach, C. W. Cataloguing the HIV type 1 human protein interaction network. AIDS Res. Hum. Retroviruses 24: 1497–1502 (2008). 40. Chang, H. C., Samaniego, F., Nair, B. C., Buonaguro, L., and Ensoli, B. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 11: 1421–1431 (1997). 41. Fittipaldi, A. and Giacca, M. Transcellular protein transduction using the Tat protein of HIV-1. Adv. Drug Deliv. Rev. 57: 597–608 (2005). 42. Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J. Biol. Chem. 276: 3254–3261 (2001). 43. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B., and Barsoum, J. Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. U. S. A. 91: 664–668 (1994). 44. Nagahara, H., Vocero-Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy, S. F. Transduction of fulllength TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4: 1449–1452 (1998). 45. Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285: 1569–1572 (1999). 46. Silhol, M., Tyagi, M., Giacca, M., Lebleu, B., and Vives, E. Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat. Eur. J. Biochem. 269: 494–501 (2002). 47. Simon, M. J., Gao, S., Kang, W. H., Banta, S., and Morrison, B., 3rd. TATmediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression. Biotechnol. Bioeng. 104: 10–19 (2009).

c15.indd 282

7/30/2010 2:19:20 PM

REFERENCES

283

48. Lundberg, M. and Johansson, M. Positively charged DNA-binding proteins cause apparent cell membrane translocation. Biochem. Biophys. Res. Commun. 291: 367–371 (2002). 49. Lundberg, M., Wikstrom, S., and Johansson, M. Cell surface adherence and endocytosis of protein transduction domains. Mol. Ther. 8: 143–150 (2003). 50. Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278: 585–590 (2003). 51. Ferrari, A., Pellegrini, V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol. Ther. 8: 284–294 (2003). 52. Futaki, S., Niwa, M., Nakase, I., Tadokoro, A., Zhang, Y., Nagaoka, M., Wakako, N., and Sugiura, Y. Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine-tagged proteins. Bioconjug. Chem. 15: 475–481 (2004). 53. Deng, G., Qiao, W., Su, Y., Sha, R., Geng, Y., and Chen, Q. Internalization of Jembrana disease virus Tat: possible pathway and implication. Virus Res. 121: 122–133 (2006). 54. Deng, G., Su, Y., Mu, J., Sha, R., Geng, Y., Qiao, W., and Chen, Q. Molecular basis of the internalization of bovine immunodeficiency virus Tat protein. Virus Genes 36: 85–94 (2008). 55. Fittipaldi, A., Ferrari, A., Zoppe, M., Arcangeli, C., Pellegrini, V., Beltram, F., and Giacca, M. Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J. Biol. Chem. 278: 34141–34149 (2003). 56. Scherer, P. E., Lewis, R. Y., Volonte, D., Engelman, J. A., Galbiati, F., Couet, J., Kohtz, D. S., van Donselaar, E., Peters, P., and Lisanti, M. P. Cell-type and tissuespecific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J. Biol. Chem. 272: 29337–29346 (1997). 57. Skretting, G., Torgersen, M. L., van Deurs, B., and Sandvig, K. Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor. J. Cell Sci. 112 (Pt 22): 3899–3909 (1999). 58. Wadia, J. S., Stan, R. V., and Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10: 310–315 (2004). 59. Kerr, M. C. and Teasdale, R. D. Defining macropinocytosis. Traffic 10: 364–371 (2009). 60. Tunnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., and Cardoso, M. C. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J. 20: 1775–1784 (2006). 61. Maiolo, J. R., 3rd, Ottinger, E. A., and Ferrer, M. Specific redistribution of cellpenetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination. J. Am. Chem. Soc. 126: 15376–15377 (2004). 62. Noguchi, H., Matsumoto, S., Okitsu, T., Iwanaga, Y., Yonekawa, Y., Nagata, H., Matsushita, M., Wei, F. Y., Matsui, H., Minami, K., Seino, S., Masui, Y., Futaki, S., and Tanaka, K. PDX-1 protein is internalized by lipid raft-dependent macropinocytosis. Cell Transplant. 14: 637–645 (2005).

c15.indd 283

7/30/2010 2:19:20 PM

284

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

63. Serresi, M., Bizzarri, R., Cardarelli, F., and Beltram, F. Real-time measurement of endosomal acidification by a novel genetically encoded biosensor. Anal. Bioanal. Chem. 393: 1123–1133 (2009). 64. Michiue, H., Tomizawa, K., Wei, F. Y., Matsushita, M., Lu, Y. F., Ichikawa, T., Tamiya, T., Date, I., and Matsui, H. The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction. J. Biol. Chem. 280: 8285–8289 (2005). 65. Lee, Y. J., Datta, S., and Pellois, J. P. Real-time fluorescence detection of protein transduction into live cells. J. Am. Chem. Soc. 130: 2398–2399 (2008). 66. Pouton, C. W., Wagstaff, K. M., Roth, D. M., Moseley, G. W., and Jans, D. A. Targeted delivery to the nucleus. Adv. Drug Deliv. Rev. 59: 698–717 (2007). 67. Stauber, R. H. and Pavlakis, G. N. Intracellular trafficking and interactions of the HIV-1 Tat protein. Virology 252: 126–136 (1998). 68. Efthymiadis, A., Briggs, L. J., and Jans, D. A. The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. J. Biol. Chem. 273: 1623–1628 (1998). 69. Truant, R. and Cullen, B. R. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals. Mol. Cell Biol. 19: 1210–1217 (1999). 70. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. A short amino acid sequence able to specify nuclear location. Cell 39: 499–509 (1984). 71. Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64: 615–623 (1991). 72. Cardarelli, F., Serresi, M., Bizzarri, R., Giacca, M., and Beltram, F. In vivo study of HIV-1 Tat arginine-rich motif unveils its transport properties. Mol. Ther. 15: 1313–1322 (2007). 73. Cardarelli, F., Serresi, M., Bizzarri, R., and Beltram, F. Tuning the transport properties of HIV-1 Tat arginine-rich motif in living cells. Traffic 9: 528–539 (2008). 74. Takeuchi, T., Kosuge, M., Tadokoro, A., Sugiura, Y., Nishi, M., Kawata, M., Sakai, N., Matile, S., and Futaki, S. Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate. ACS Chem. Biol. 1: 299–303 (2006). 75. Nelson, A. R., Borland, L., Allbritton, N. L., and Sims, C. E. Myristoyl-based transport of peptides into living cells. Biochemistry 46: 14771–14781 (2007). 76. Kwon, Y. M., Li, Y., Naik, S., Liang, J. F., Huang, Y., Park, Y. J., and Yang, V. C. The ATTEMPTS delivery systems for macromolecular drugs. Expert Opin. Drug Deliv. 5: 1255–1266 (2008). 77. Kwon, Y. M., Li, Y. T., Liang, J. F., Park, Y. J., Chang, L. C., and Yang, V. C. PTDmodified ATTEMPTS system for enhanced asparaginase therapy: a proof-ofconcept investigation. J. Control. Release 130: 252–258 (2008). 78. Derossi, D., Chassaing, G., and Prochiantz, A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8: 84–87 (1998). 79. Okuyama, M., Laman, H., Kingsbury, S. R., Visintin, C., Leo, E., Eward, K. L., Stoeber, K., Boshoff, C., Williams, G. H., and Selwood, D. L. Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells. Nat. Methods 4: 153–159 (2007).

c15.indd 284

7/30/2010 2:19:20 PM

REFERENCES

285

80. Mai, J. C., Shen, H., Watkins, S. C., Cheng, T., and Robbins, P. D. Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate. J. Biol. Chem. 277: 30208–30218 (2002). 81. Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res. 61: 474–477 (2001). 82. Saalik, P., Elmquist, A., Hansen, M., Padari, K., Saar, K., Viht, K., Langel, U., and Pooga, M. Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug. Chem. 15: 1246–1253 (2004). 83. Pooga, M., Hallbrink, M., Zorko, M., and Langel, U. Cell penetration by transportan. FASEB J. 12: 67–77 (1998). 84. Elmquist, A., Lindgren, M., Bartfai, T., and Langel, U. VE-cadherin-derived cellpenetrating peptide, pVEC, with carrier functions. Exp. Cell Res. 269: 237–244 (2001). 85. Soomets, U., Lindgren, M., Gallet, X., Hallbrink, M., Elmquist, A., Balaspiri, L., Zorko, M., Pooga, M., Brasseur, R., and Langel, U. Deletion analogues of transportan. Biochim. Biophys. Acta 1467: 165–176 (2000). 86. Saalik, P., Padari, K., Niinep, A., Lorents, A., Hansen, M., Jokitalo, E., Langel, U., and Pooga, M. Protein delivery with transportans is mediated by caveolae rather than flotillin-dependent pathways. Bioconjug. Chem. 20: 877–887 (2009). 87. Crombez, L., Morris, M. C., Deshayes, S., Heitz, F., and Divita, G. Peptide-based nanoparticle for ex vivo and in vivo drug delivery. Curr. Pharm. Des. 14: 3656–3665 (2008). 88. Morris, M. C., Depollier, J., Mery, J., Heitz, F., and Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19: 1173–1176 (2001). 89. Morris, M. C., Deshayes, S., Heitz, F., and Divita, G. Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol. Cell 100: 201–217 (2008). 90. Gerbal-Chaloin, S., Gondeau, C., Aldrian-Herrada, G., Heitz, F., GauthierRouviere, C., and Divita, G. First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling. Biol. Cell 99: 223– 238 (2007). 91. Veldhoen, S., Laufer, S. D., Trampe, A., and Restle, T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucleic Acids Res. 34: 6561–6573 (2006). 92. Li, W., Nicol, F., and Szoka, F. C., Jr. GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv. Drug Deliv. Rev. 56: 967–985 (2004). 93. El-Andaloussi, S., Jarver, P., Johansson, H. J., and Langel, U. Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study. Biochem. J. 407: 285–292 (2007). 94. Myrberg, H., Lindgren, M., and Langel, U. Protein delivery by the cell-penetrating peptide YTA2. Bioconjug. Chem. 18: 170–174 (2007).

c15.indd 285

7/30/2010 2:19:20 PM

286

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

95. Saar, K., Lindgren, M., Hansen, M., Eiriksdottir, E., Jiang, Y., Rosenthal-Aizman, K., Sassian, M., and Langel, U. Cell-penetrating peptides: a comparative membrane toxicity study. Anal. Biochem. 345: 55–65 (2005). 96. Sugita, T., Yoshikawa, T., Mukai, Y., Yamanada, N., Imai, S., Nagano, K., Yoshida, Y., Shibata, H., Yoshioka, Y., Nakagawa, S., Kamada, H., Tsunoda, S. I., and Tsutsumi, Y. Comparative study on transduction and toxicity of protein transduction domains. Br. J. Pharmacol. 153: 1143–1152 (2008). 97. Tunnemann, G., Ter-Avetisyan, G., Martin, R. M., Stockl, M., Herrmann, A., and Cardoso, M. C. Live-cell analysis of cell penetration ability and toxicity of oligoarginines. J. Pept. Sci. 14: 469–476 (2008). 98. Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., Bredesen, D. E., and Pasqualini, R. Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5: 1032–1038 (1999). 99. Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., and Arap, W. Reversal of obesity by targeted ablation of adipose tissue. Nat. Med. 10: 625–632 (2004). 100. Law, B., Quinti, L., Choi, Y., Weissleder, R., and Tung, C. H. A mitochondrial targeted fusion peptide exhibits remarkable cytotoxicity. Mol. Cancer Ther. 5: 1944– 1949 (2006). 101. Nakase, I., Niwa, M., Takeuchi, T., Sonomura, K., Kawabata, N., Koike, Y., Takehashi, M., Tanaka, S., Ueda, K., Simpson, J. C., Jones, A. T., Sugiura, Y., and Futaki, S. Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther. 10: 1011–1022 (2004). 102. Nishimura, S., Takahashi, S., Kamikatahira, H., Kuroki, Y., Jaalouk, D. E., O’Brien, S., Koivunen, E., Arap, W., Pasqualini, R., Nakayama, H., and Kuniyasu, A. Combinatorial targeting of the macropinocytotic pathway in leukemia and lymphoma cells. J. Biol. Chem. 283: 11752–11762 (2008). 103. Watkins, C. L., Brennan, P., Fegan, C., Takayama, K., Nakase, I., Futaki, S., and Jones, A. T. Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD. J. Control. Release 140: 237–244 (2009). 104. Orii, K. O., Grubb, J. H., Vogler, C., Levy, B., Tan, Y., Markova, K., Davidson, B. L., Mao, Q., Orii, T., Kondo, N., and Sly, W. S. Defining the pathway for Tatmediated delivery of beta-glucuronidase in cultured cells and MPS VII mice. Mol. Ther. 12: 345–352 (2005). 105. Zhang, X. Y., Dinh, A., Cronin, J., Li, S. C., and Reiser, J. Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain. J. Neurochem. 104: 1055–1064 (2008). 106. El-Andaloussi, S., Holm, T., and Langel, U. Cell-penetrating peptides: mechanisms and applications. Curr. Pharm. Des. 11: 3597–3611 (2005). 107. Fischer, R., Fotin-Mleczek, M., Hufnagel, H., and Brock, R. Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides. Chembiochem. 6: 2126–2142 (2005). 108. Foged, C. and Nielsen, H. M. Cell-penetrating peptides for drug delivery across membrane barriers. Expert Opin. Drug Deliv. 5: 105–117 (2008).

c15.indd 286

7/30/2010 2:19:20 PM

REFERENCES

287

109. Futaki, S., Nakase, I., Tadokoro, A., Takeuchi, T., and Jones, A. T. Arginine-rich peptides and their internalization mechanisms. Biochem. Soc. Trans. 35: 784–787 (2007). 110. Gump, J. M. and Dowdy, S. F. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol. Med. 13: 443–448 (2007). 111. Nakase, I., Takeuchi, T., Tanaka, G., and Futaki, S. Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cellpenetrating peptides. Adv. Drug Deliv. Rev. 60: 598–607 (2008). 112. Patel, L. N., Zaro, J. L., and Shen, W. C. Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm. Res. 24: 1977–1992 (2007). 113. Al-Taei, S., Penning, N. A., Simpson, J. C., Futaki, S., Takeuchi, T., Nakase, I., and Jones, A. T. Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors. Bioconjug. Chem. 17: 90–100 (2006). 114. Fretz, M. M., Penning, N. A., Al-Taei, S., Futaki, S., Takeuchi, T., Nakase, I., Storm, G., and Jones, A. T. Temperature-, concentration- and cholesterol-dependent translocation of l- and d-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem. J. 403: 335–342 (2007). 115. Watkins, C. L., Schmaljohann, D., Futaki, S., and Jones, A. T. Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell penetrating peptide. Biochem. J. 420: 179–189 (2009). 116. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8: 848–866 (2007). 117. Kosuge, M., Takeuchi, T., Nakase, I., Jones, A. T., and Futaki, S. Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug. Chem. 19: 656–664 (2008). 118. Watkins, C. L., Schmaljohann, D., Futaki, S., and Jones, A. T. Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide. Biochem. J. 420: 179–189 (2009). 119. Deshayes, S., Morris, M., Heitz, F., and Divita, G. Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. Adv. Drug Deliv. Rev. 60: 537– 547 (2008). 120. Edenhofer, F. Protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins. Curr. Pharm. Des. 14: 3628–3636 (2008). 121. Murriel, C. L. and Dowdy, S. F. Influence of protein transduction domains on intracellular delivery of macromolecules. Expert Opin. Drug Deliv. 3: 739–746 (2006). 122. Wadia, J. S. and Dowdy, S. F. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Deliv. Rev. 57: 579–596 (2005). 123. Foerg, C. and Merkle, H. P. On the biomedical promise of cell penetrating peptides: limits versus prospects. J. Pharm. Sci. 97: 144–162 (2008).

c15.indd 287

7/30/2010 2:19:20 PM

288

UPTAKE AND INTRACELLULAR DYNAMICS OF PROTEINS INTERNALIZED

124. Leifert, J. A. and Whitton, J. L. “Translocatory proteins” and “protein transduction domains”: a critical analysis of their biological effects and the underlying mechanisms. Mol. Ther. 8: 13–20 (2003). 125. Trehin, R. and Merkle, H. P. Chances and pitfalls of cell penetrating peptides for cellular drug delivery. Eur. J. Pharm. Biopharm. 58: 209–223 (2004). 126. Wagstaff, K. M. and Jans, D. A. Protein transduction: cell penetrating peptides and their therapeutic applications. Curr. Med. Chem. 13: 1371–1387 (2006). 127. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277–284 (1985). 128. Elliott, G. and O’Hare, P. Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88: 223–233 (1997).

c15.indd 288

7/30/2010 2:19:20 PM

CHAPTER 16

Cargo Transport by Teams of Molecular Motors: Basic Mechanisms for Intracellular Drug Delivery MELANIE J. I. MÜLLER, FLORIAN BERGER, STEFAN KLUMPP, and REINHARD LIPOWSKY Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

16.1

INTRODUCTION

16.1.1 Barriers to Gene Therapy and Drug Delivery The delivery of drug carriers or viral and nonviral vectors for gene therapy is hampered by many extracellular and intracellular barriers. The drug or DNA carrier must find its way from the injection site to the target cell, avoiding sequestration and destruction, for example, by cells of the immune system. Upon reaching the target cell, the carrier must pass the plasma membrane to enter the cell, traverse the cytoplasm to its intracellular destination, possibly enter a target organelle like the nucleus or a mitochondrion, and then perform its function. Traditionally, research has focused on overcoming membrane barriers: how to get the drug carriers or transfection vectors into the cell and, once inside the cell, how to get these particles into the target organelle. However, the cytoplasm itself is also a major obstacle to delivery, especially for nonviral gene therapy: After a transfection vector has been released into the cytoplasm, it may have difficulties reaching the nucleus [1, 2]. 16.1.2 Inefficient Transport Via Diffusion One fact that is often overlooked is that drug or gene carriers are too large to diffuse through the cytoplasm at a reasonable rate. The cytoplasm is a crowded, Organelle-Specific Pharmaceutical Nanotechnology, Edited by Volkmar Weissig and Gerard G. M. D’Souza Copyright © 2010 John Wiley & Sons, Inc.

289

c16.indd 289

7/30/2010 2:19:25 PM

290

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

viscous medium consisting of proteins, filament meshworks, and organelles, which limit the diffusion of large particles [3]. For example, using experimentally determined cytoplasmic diffusion coefficients of DNA [4], a 1000-bp DNA needs about 30 min to diffuse a distance of 10 μm, compared to about 18 s in water. DNA strands that are larger than 2000 bp are practically immobile in the cytoplasm. Gene vectors used in gene transfer have typically several thousand base pairs. Consequently, microinjected plasmids do not diffuse far from their site of injection [5], and transfection increases dramatically if DNA is microinjected into or very close to the nucleus instead of a cytoplasmic location far from the nucleus [5, 6]. Therefore efficient transport to the nucleus cannot be achieved by diffusion and requires an active mechanism. 16.1.3 Viruses as Hijackers Some viruses face barriers similar to gene therapy: they must enter the host cell and deliver their genetic material to the nucleus. Many viruses have solved the problem of intracellular transport by hijacking the host cell’s active transport system [2, 7, 8], as shown in Figure 16.1A. After entering the cell via endocytosis or membrane fusion, the virus particle recruits molecular motors, which act as cellular “nanotrucks” and pull the virus along microtubule filaments toward the nucleus. Similarly, when leaving the cell, the virus uses molecular motors to travel from the site of assembly to the cell membrane. This active transport allows the virus to travel at a velocity of about 1 μm/s [8,

(A)

(B) entry

egress cargo dynein

tubule micro actin

MTOC

kinesin

nucleus 25nm delivery

microtubule

Figure 16.1. Intracellular trafficking. (A) Microtubule filaments (black) form long “highways” from the microtubule-organizing center (MTOC) near the nucleus to the cell periphery, while a dense meshwork of actin filaments (cyan) provides “side roads” for short-range traffic. Plus-end directed (blue) and minus-end directed (yellow) molecular motors pull various cellular cargoes along these filaments. Viruses hijack the cellular transport machinery: after entry into the cell, a virus particle (red) recruits motors from the cytoplasm to travel along microtubules toward the nucleus, where it delivers its genetic material (white). In order to leave the cell, the newly assembled virus uses motors to travel back to the plasma membrane. (B) Cargo particle (red) that is transported along a microtubule by one kinesin-1 (blue) and one cytoplasmic dynein (yellow) motor. (See color insert.)

c16.indd 290

7/30/2010 2:19:25 PM

INTRACELLULAR TRAFFIC OF MOLECULAR MOTORS

291

9] so that it can overcome a distance of 10 μm in about 10 s. For comparison, diffusion of a 100-nm virus capsid in the cytoplasm over the same distance would take a few hours. 16.1.4 Understanding Intracellular Transport Hijacking the cellular transport machinery is an elegant solution to the problem of transport through the crowded cytoplasm, which is employed by many viruses, such as human immunodeficiency virus, herpes simplex virus, and adenoviruses [2, 7, 8]. These viruses are commonly used in viral gene therapy, in which the gene vector is packaged into a virus capsid. However, also nonviral vectors and drug carriers can bind to molecular motors via adaptor proteins recruited from the cytoplasm, and then travel actively along microtubule filaments [2]. In order to improve gene and drug delivery, it is therefore essential to design carrier systems that can recruit the cellular transport machinery in an appropriate way. As a prerequisite for such a design, a good understanding of intracellular transport by molecular motors is required. 16.1.5 Overview This chapter is organized as follows. The basic features of molecular motors and cytoskeletal filaments are briefly described in Section 16.2.1. The challenges encountered by motor transport are summarized in Section 16.2.2. The different mechanisms for cooperative transport by motor teams are discussed in Section 16.3. In this latter section, we will distinguish three such mechanisms that we have recently elucidated by the construction and analysis of explicit theoretical models: (1) cargo transport by one team of identical motors [10, 11]; (2) transport by two antagonistic teams of motors that both work on the same type of filament [12, 13]; and (3) cargo transport by two teams of motors that work on different filaments [14]. At the end, we give a brief outlook on open questions and possible applications of the teamwork of molecular motors.

16.2

INTRACELLULAR TRAFFIC OF MOLECULAR MOTORS

16.2.1 Molecular Motors and Their Tracks The complex internal structure of cells depends to a large extent on active transport: vesicles, as well as RNA, filaments, and protein complexes, move between different compartments, travel from the cell center to the cell periphery or vice versa [15]. This active transport is mainly accomplished by molecular motors. These molecular motors can be compared to road trucks: they travel along a “road network” consisting of cytoskeletal filaments and consume ATP as molecular “fuel.”

c16.indd 291

7/30/2010 2:19:25 PM

292

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

16.2.1.1 Cytoskeletal Filaments The road network for the molecular motors is formed by the cytoskeleton. The cytoskeleton is a meshwork built up from three types of filaments—microtubule, actin, and intermediate filaments—which form a complex meshwork throughout the cell. Only microtubules and actin filaments are used in motor transport. Kinesin and dynein motors walk on microtubules, while myosin motors travel along actin filaments. The microtubules form a very structured network spanning the whole cell from the center to the periphery, see Figure 16.1A, and serve as “highways” for the long-range traffic. The actin filaments, on the other hand, form a dense meshwork concentrated near the cell cortex and serve as “side-roads” for short-range traffic. Both types of filaments are polar: their building blocks are asymmetric so that each filament possesses an intrinsic directionality. Each filament has a “plus end,” at which it polymerizes faster than at its “minus end.” 16.2.1.2 Cytoskeletal Motors Each cytoskeletal motor works on a certain filament and recognizes the polarity of this filament. Thus each motor walks either to the filament’s “plus” or “minus” end. The cytoplasmic dynein motor, for example, walks to the microtubule minus end, while the kinesin-1 motor walks to the microtubule plus end; see Figure 16.1B. 16.2.1.3 Kinesin-1 The best-studied motor, kinesin-1, was first identified in the 1980s in a biochemical fractionation of squid axonal tissue [16, 17]. It is a heterotetramer composed of two identical heavy chains and two identical light chains [18]. The heavy chains dimerize to form an α-helical coiled-coil stalk. Each of the 120-kDa heavy chains includes a globular “head” at its N terminus, with both a microtubule-binding domain and an ATP-binding domain. The two heads perform the actual motor activity of the protein. The light chains associate with the heavy chains near the C terminus and form the motor tail, which is involved in cargo binding. 16.2.1.4 Kinesin Stepping When traveling along a microtubule, kinesin-1 rather “steps” than “drives” [19]. A microtubule filament consists of 13 protofilaments aligned in parallel, each of which provides a track for the motor with a binding site every 8 nm. For each step, kinesin moves the trailing head forward to the next binding site in front, while the other head remains bound to the microtubule. In this so-called hand-over-hand mechanism, each head covers a distance of 16 nm, while the center-of-mass of the motor makes a forward step of 8 nm. During each step, the motor hydrolyzes a single ATP molecule. 16.2.1.5 Cytoplasmic Dynein The structure of cytoplasmic dynein, which was discovered as a minus end microtubule motor in the 1980s [20], is more complex than that of kinesin [18, 21], as indicated in Figure 16.1B. Dynein also has two heads, which can bind to the microtubule, but its ATP-binding

c16.indd 292

7/30/2010 2:19:25 PM

INTRACELLULAR TRAFFIC OF MOLECULAR MOTORS

293

domains reside in rings of seven globular domains, which are connected to the heads by a short coiled-coil stalk. Several light and intermediate chains are attached near the cargo binding end of dynein and are involved in the binding of cargo and of accessory proteins, making dynein a huge multisubunit protein of 1.2 MDa. 16.2.1.6 Other Types of Motors Kinesin-1 and cytoplasmic dynein are the most prominent members of a whole “zoo” of molecular motors, consisting of three motor families—kinesins, dyneins, and myosins—as reviewed in Refs. 18, 22, and 23. While the dynein family consists of only two subclasses, the kinesin family has more than ten subclasses. All dyneins walk to the microtubule minus end, whereas most kinesins walk to the microtubule plus end. Several classes of myosins mediate transport on actin filaments. 16.2.2 Challenges for Motor Transport The various motors described in the last section provide a versatile toolbox for intracellular traffic [18]. These motors transport an enormous amount of vastly different cargoes through the cell, ranging from endosomes and other types of vesicles to RNAs and filaments as well as to whole organelles such as mitochondria [15]. Each cargo has to recruit the right type of motor and an appropriate motor number in order to be shuttled to the correct destination. In this section, we will discuss how the motors perform these challenging tasks. 16.2.2.1 Motor Processivity Native motor molecules have a linear extension of about 100 nm; see Figure 16.1B. Because of their relatively small size, their motion is strongly affected by thermal fluctuations and viscous forces: molecular motors continuously collide with other molecules and thus walk “in a strong storm.” Because of this thermal noise, molecular motors unbind from their track from time to time. Upon unbinding they lose their ability to perform directed motion and randomly diffuse in the surrounding solution until they finally rebind to a filament. For kinesin-1, unbinding happens on average after a run time of about 1 s, or equivalently a run length (or walking distance) of about 1 μm. A cellular cargo, however, must accomplish distances of tens of micrometers, and in some extremely large cells like neurons even up to a meter [24]. 16.2.2.2 Load Forces Molecular motors can generate forces in the range of a few piconewtons [22, 25, 26]. This has to be compared to the frictional force, Ffr = φfr v with φfr = 6πηR = kBT D

(16.1)

experienced by the cargo particle of size R as it moves through a medium with dynamical viscosity η. The parameter D is the diffusion coefficient of the

c16.indd 293

7/30/2010 2:19:25 PM

294

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

(A) Fibroblast cell

(B) Epithelial cell apical

(C) Neuronal cell dendrite

basolateral

(mixed orientation)

unipolar orientation cell body

axon

synapse

Figure 16.2. Organization of microtubules (black lines with indicated plus ends) in three different cell types for which the nuclei are represented as black ellipses and the microtubule-organizing centers or centrosomes as white rectangles. (A) In fibroblast cells, microtubule minus ends nucleate at the centrosome and the plus ends point radially outwards. (B) In epithelial cells, microtubules form a parallel array with minus ends pointing to the apical and plus ends to the basolateral surface. (C) In long axons of neuron cells, microtubules form an isopolar array with the plus ends oriented toward the synapses at the axon tips.

(unbound) cargo, and kBT is Boltzmann’s constant times absolute temperature. In water with dynamical viscosity η = 1 mPa · s, the force needed to pull a 1-μm sized cargo at a velocity of v = 1 μm/s is equal to 0.01 pN, which is negligible for a molecular motor that is able to generate several piconewtons of force. However, since diffusion in the cytoplasm is slowed down by a factor of 10–1000 depending on the cargo size [47], it follows from Equation 16.1 that frictional forces in the cytoplasm can be in the range of 0.1–10 pN, which represents a large load for a molecular motor. For these estimates, we assumed that the diffusion coefficient D as measured for cargo diffusion in the cytoplasm also applies to motor transport; see, however, Ref. 27. 16.2.2.3 Bidirectional Transport Cells typically have an “isopolar” microtubule cytoskeleton [28]; see Figure 16.2. In a “round” cell such as a fibroblast cell, for example, microtubules are arranged radially, with their minus ends close to the nucleus at the cell center and their plus ends directed outward to the cell periphery. In epithelial cells, microtubules form a parallel array with their minus ends pointing to the apical and their plus ends to the basolateral surface. The isopolar microtubule arrangement is most pronounced in long cellular subcompartments such as neuronal axons or fungal hyphae. In these long and relatively narrow membrane tubes, the microtubules are arranged parallel to the tube axis with their minus ends pointing to the cell body and the plus ends pointing to the tip. Single molecular motors walk along these tracks only into one direction: dyneins to the minus ends, and most kinesins to the plus ends. However, many cellular cargoes travel in both directions [29, 30], a transport mode that avoids accumulation of cargo at either the cell periphery or the cell center [31, 32]. Cargoes like endosomes, mitochondria, and viruses are observed to move bidirectionally, reversing direction every few seconds [29, 30]. These cargoes have both plus- and minus-end directed motors attached. Bidirectional

c16.indd 294

7/30/2010 2:19:25 PM

TEAMWORK OF MOLECULAR MOTORS

295

motion can lead to net transport if the runs in one direction are on average longer or faster than runs in the other direction. Therefore bidirectional transport requires an appropriate balance between the activity of plus and minus motors. 16.2.2.4 Transport Regulation Cellular regulation of cargo transport often leads to changes in the run lengths, that is, the distances covered during runs into the plus or the minus direction. This has been observed for the directional regulation of mitochondria in axons [33], of lipid droplets during embryo development [34], of virus targeting during entry and egress [35], and of melanosomes during dispersion and aggregation [36]. In the latter example, melanosome redistribution in specialized pigment cells of fish or frog allows the organism to adapt its skin color to the environment. During “aggregation,” minus-end motion dominates, and the melanosomes accumulate in the cell center. During “dispersion,” the melanosomes spread out over the whole cell because of a decrease in minus run length [36]. Aggregation and dispersion are triggered by hormonal stimulation, which is transmitted to the motors via a signal cascade involving cAMP and protein kinase A (PKA) [37]. 16.2.2.5 Transport on Two Types of Tracks Intracellular transport usually proceeds in two steps [38, 39]; see Figure 16.1A. Long-range transport from the cell center to the periphery and vice versa is mediated by kinesins and dyneins walking along the long microtubule highways, while short-range delivery is the task of myosin motors using the dense actin meshwork in the cell cortex. This “dual transport” requires that cargoes are able to switch from microtubule to actin tracks, as has indeed been observed for various cellular cargo such as mitochondria, pigment granules, and synaptic vesicles, reviewed in Refs. 38 and 39. These cargoes need to balance the activities of actin- and microtubule-based motors. A prominent example is again the traffic of melanosomes, which are carried by kinesin-2 and cytoplasmic dynein along microtubules and by myosin V along actin filaments [38]. During dispersion, myosin V is upregulated, which leads to enhanced switching of melanosomes to actin filaments [36, 40]. During aggregation, downregulation of myosin V “recollects” the melanosomes to the microtubules, on which they are then transported toward the cell center.

16.3

TEAMWORK OF MOLECULAR MOTORS

16.3.1 Different Levels of Motor Motility In general, the behavior of molecular motors involves several levels of motility [41] related to (i) the stepping of single motors, (ii) the cooperative transport of cargo by motor teams, and (iii) the traffic of many motor-propelled cargoes. At the level (i) of a single motor, most progress has been made in understand-

c16.indd 295

7/30/2010 2:19:25 PM

296

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

ing the structure and the stepping mechanism of kinesin-1, in particular, the coordination of its two heads [19, 21, 42]. The level (iii) of cell wide cargo trafficking has received mainly theoretical attention. On the cellular length scale, each cargo is viewed in a course-grained manner as a particle with the ability to bind and actively move along cytoskeletal filaments, while diffusing randomly in the surrounding solution. This description allows one to calculate quantities such as the densities and fluxes of cargoes throughout the cell [43–46], which are important to characterize the intracellular traffic. For drug delivery, gene therapy, or virus trafficking, another important quantity is the mean first passage time from the point of entry into the cell to the intracellular target [47, 48].

16.3.2 Motor Teams The level (ii) of cooperative transport by several molecular motors will be the focus of this chapter. Many cellular cargoes are attached to more than one motor, and to more than one type of motor [29, 30, 38, 39]. The number of motors in a team is small, typically between 1 and 10 [27]. Cargo transport by small teams of molecular motors is a topic of current research both experimentally [27, 49–54] and theoretically [11, 13, 14, 50, 55, 56]. In the following, we will review our recent theoretical studies of cargo transport by small teams of molecular motors [11–14]. After introducing our model for a single motor in the next section, we will first examine the transport by one cooperating team of molecular motors that all belong to the same species. We will then discuss transport by two antagonistic teams of molecular motors that walk into opposite directions, and finally transport by two teams of motors that can walk on different types of filaments.

16.3.3 Transport Properties of a Single Motor 16.3.3.1 Different Subprocesses of Motor Transport Since we are interested in cargo motion on time scales ranging from seconds to minutes and length scales from a few to hundreds of micrometers, we can describe the motors in a coarse-grained manner. We neglect the details of their protein structure and stepping cycle but take their finite binding time to the track into account. The motors exhibit three basic subprocesses: they walk along the filament with velocity v, unbind from this filament with rate ε, and rebind to it with rate π; see Figure 16.3A. If the motor experiences no load force, these parameters are of the order of π0 ∼ 1/s, vf ∼ 1 μm/s, and ε0 ∼ 1/s, respectively; see Table 16.1. If the motor has to work against a load force F, both the motor velocity v and its unbinding rate ε strongly depend on this force; see Figure 16.3B,C. This force dependence has been investigated experimentally for some motors using optical traps or tweezers. An optical trap uses a focused laser beam, the radiation pressure of which is able to exert forces in the piconewton-range on dielectric objects of nanometer to micrometer size. If a

c16.indd 296

7/30/2010 2:19:25 PM

v(F)

π0

ε(F)

Vf

vf(1–F/Fs)

0.5 Fs 0 0

(A)

vb(1–F/Fs)

(B)

2 4 6 8 10 12 load force F [pN]

unbind. rate ε [1/s]

F

velocity v [μm/s]

TEAMWORK OF MOLECULAR MOTORS

(C)

8

ε0exp[F/Fd]

6 4

ε0

2 0

297

0

2 4 6 load force F [pN]

Figure 16.3. Transport properties of a single motor. (A) A single motor walks along the filament with velocity v, unbinds from the filament with rate ε, and rebinds to it with rate π = π0. If the cargo experiences a load force F, both the velocity v and the unbinding rate ε depend on F. (B) Motor velocity v as a function of load force F as parameterized in Equations 16.2 and 16.3. The velocity decreases with increasing F until it vanishes at the stall force Fs. For superstall forces F > Fs, the motor steps backwards with a relatively small velocity. (C) Motor unbinding rate ε as a function of F as in Equation 16.4. The unbinding rate increases exponentially with increasing F. (See color insert.) TABLE 16.1 Single Motor Parameters for Conventional Kinesin or Kinesin-1 and Cytoplasmic Dyneina Parameter Stall force Detachment force Unbinding rate Binding rate Forward velocity Backward velocity

Symbol

Kinesin

Dynein

Fs Fd ε0 π0 vf vb

6 pN [25, 7, 58] 3 pN [59] 1/s [59] 5/s [10, 52] 1 μm/s [25, 7, 58] 6 nm/s [25, 7]

7 pN [26] 3 pN ? 0.25/s [60] 1.5/s [60] 1 μm/s [26, 60] 6 nm/s ?

a

These are abbreviated here as “kinesin” and “dynein,” respectively. The experimentally determined values for the stall force Fs of kinesin [25, 7, 58] vary between 5.5 pN and 7.6 pN. For dynein, the parameters with a question mark are currently not available from experimental studies; in the latter case, we used the same parameters for dynein as for kinesin.

single motor is bound to such an object, the movement of this motor can be studied under a constant load force F. 16.3.3.2 Single Motor Parameters The results of optical trapping experiments imply that the motor velocity v decreases approximately linearly with force F and is well described by the functional form v ( F ) = vf (1 − F Fs ) for 0 ≤ F ≤ Fs

(16.2)

which vanishes at the “stall force” F = Fs [25, 59]. For higher load forces F > Fs, the motor walks backwards very slowly [25, 57]; we use the parametrization [13] v ( F ) = vb(1 − F Fs ) for Fs ≤ F

(16.3)

with vb << vf.

c16.indd 297

7/30/2010 2:19:25 PM

298

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

The unbinding rate of the motor from the filament increases exponentially with applied force F according to [11] ε ( F ) = ε 0 exp ( F Fd )

(16.4)

with the detachment force Fd as obtained, for positive F, from the measurements of the walking distance of a single motor as a function of load [59] in agreement with Kramers’ rate theory [61]. Such an exponential form has also been used in the context of receptor-ligand binding at membranes [62]. The binding rate of the motor to the microtubule is taken to be independent of the load force and given by [11] π (F ) = π0

for all F .

(16.5)

In total, a single motor of a certain motor species is described by six parameters as explained in Figure 16.3. For the plus motor kinesin-1 and the cytoplasmic minus motor dynein, most of these single motor parameters have been determined experimentally; the corresponding parameter values are summarized in Table 16.1 [13]. This theoretical description for a single motor incorporates all results of single molecule experiments that are relevant for large-scale cargo transport. 16.3.4 One Team of Identical Motors In this section, we consider cargo transport by one team of motors, that is, N motors of the same type are attached to one cargo as shown in Figure 16.4. Since each motor binds to and unbinds from the filament in a stochastic manner, the number n of motors that crosslink the cargo to the filament and thus can actively pull on it varies with time between n = 0 and n = N as illustrated in Figure 16.4. The motors are taken to act independently. If n motors are bound to the filament in the absence of load, the unbinding of one of these

Figure 16.4. Transport by one team of identical motors. Each motor is characterized by a long stalk (grey), by which it is firmly attached to the cargo particle (red), and by two motor heads (blue), which can bind to the filament and then actively pull on the cargo. In this example, the total number of motors is N = 3 but the actual number n of crosslinking motors fluctuates between n = 0 and n = 3 because of thermally induced motor unbinding and rebinding. (See color insert.)

c16.indd 298

7/30/2010 2:19:26 PM

299

TEAMWORK OF MOLECULAR MOTORS

(A)

65 μm

100 10

1

311 μm

Velocity [μm/s]

Run length Δxb[μm]

1000

14 μm 3.5 μm 1 μm

1 0

1 2 3 4 Motor number N

5

6 (B)

0.8 N=10

0.6

N=

0.4 0.2 0 0

5

N=1 10 20 30 40 50 Stokes friction φfr [pNs/μm]

Figure 16.5. Transport by one team of identical motors. (A) The cargo’s run length Δxb increases exponentially with the total number N of motors attached to the cargo. (B) The average cargo velocity decreases with increasing friction coefficient φfr, but less so for larger motor number N. The single motor parameters correspond to kinesin-1 as given in Table 16.1.

bound motors is governed by the unbinding rate nε0, and the rebinding of one of the N − n unbound motors back to the filament takes place with rate (N − n)π0 [11]. 16.3.4.1 Processivity Enhancement A major problem of cargo transport by a single motor is the motor’s finite run length Δxb ∼ 1 μm, which is too small to cover typical cellular distances of tens of micrometers. Cargo transport by several molecular motors leads to a dramatic increase in the cargo’s run length; see Figure 16.5A. Intuitively, if one motor unbinds from the filament, the cargo remains attached to the filament as long as it is still crosslinked by another motor, giving the unbound motor a chance to rebind; compare Figure 16.4. An explicit calculation shows that the average run length Δxb is given by [11] Δxb =

vf ⎡(1 + π 0 ε 0 )N − 1⎤⎦ Nπ 0 ⎣

(16.6)

that is, it increases exponentially with the motor number N for large N. If the cargo is transported by kinesin-1 motors, only three such kinesins are required to cross a cell of 10-μm diameter; see Figure 16.5A. The increase of run length with increasing motor number has been observed in vitro [63, 64], but it has been difficult to determine the number of motors pulling the cargo. In recent experiments, the motor number was determined by force measurements [54] or dynamic light scattering [10]. In the latter case, the theory described here was used to describe the data quantitatively. 16.3.4.2 Frictional Forces Another feature of cargo transport by several motors is the ability to sustain large forces arising, for example, from hydrodynamic friction. For a cargo particle of radius R = 1 μm in water with

c16.indd 299

7/30/2010 2:19:26 PM

300

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

dynamical viscosity η = 1 mPa·s, the friction coefficient is φfr ∼ 10−2 pN · s/μm, which does not significantly change the velocity of a cargo particle that is transported by a single motor; see the curve for N = 1 in Figure 16.5B. However, as discussed in Section 16.2.2, particle diffusion in the crowded cytoplasm is reduced by a factor of 10–1000 [3], depending on the size of the particle. Since the friction coefficient in Equation 16.1 is inversely proportional to the diffusion constant, the cytoplasmic friction coefficient is 10–1000 times larger than the one in water and leads to a significant reduction of the cargo velocity if the cargo particle is pulled by only one motor; see Figure 16.5B. When the cargo is pulled by N > 1 motors, these N motors can share the force, so that the average cargo velocity remains relatively high even for large friction coefficient φfr; see Figure 16.5B. 16.3.5 Two Teams of Antagonistic Motors As discussed in Section 16.2.2, many cellular cargoes undergo fast transport both toward the plus end and toward the minus end of the microtubules. This bidirectional transport implies that these cargo particles must be attached to both plus-end and minus-end directed motors. 16.3.5.1 Tug-of-War Between Motors We now consider a cargo particle that is attached to N+ plus and N− minus motors. Because each motor unbinds from and rebinds to the filament in a stochastic manner, both the number of active plus motors and the number of active minus motors now fluctuate; see Figure 16.6. Three situations are possible: in the (+) states, only plus motors are active, which can pull the cargo into the plus direction without any opposition from the minus motors; in the (−) states, only minus motors are active and the cargo exhibits fast minus motion; and in the (0) states both types of motors are bound to the filament. In the latter case, the motors pull the cargo into opposite directions, leading to slow cargo movement. Since many bidirectional cargoes are observed to exhibit long periods of fast unidirectional motion, the motors must “cooperate” in some way to avoid the (0) states. Two mechanisms for bidirectional transport have been proposed [29, 30]: (1) coordination by a putative protein complex, which ensures that only one type of motors is active at any given time; and (2) tug-of-war between the motors that pull on each other until the stronger team wins and determines the direction of motion for a certain period of time. Previously, it was thought that a tug-of-war would lead to a prevalence of (0) states with slow cargo motion and thus would be inconsistent with the experimental observation of fast cargo motion [29, 30]. However, we have developed a realistic tug-of-war model, which is based on the single motor description as presented in Section 16.3.3 as well as on the assumptions that (1) each motor team exerts load forces on the other team and (2) motors of one team share the load force generated by the antagonistic motor team [12, 13]. Such a tug-of-war, which

c16.indd 300

7/30/2010 2:19:26 PM

TEAMWORK OF MOLECULAR MOTORS

(–)

(0)

(0)

(–)

(0)

(0)

(0)

(+)

(+)

301

Figure 16.6. Transport by two antagonistic teams of motors. The motors with blue heads are plus motors that pull to the right, the ones with yellow heads are minus motors that pull to the left. Both types of motors are firmly attached to the cargo particle (grey) via their long stalks. The total number of blue plus motors and yellow minus motors is N+ = 2 and N− = 2, respectively. The cargo particle exhibits (N+ + 1)(N− + 1) = 9 different states and undergoes transitions between these states arising from the unbinding and rebinding of single motors. (See color insert.)

does not require any coordination complex, is consistent with all experimental observations [13, 65]. 16.3.5.2 Patterns of Movements As shown in Refs. 12 and 13, a cargo transported by two teams of motors can exhibit different patterns of motility (or “motility states”) depending on the single motor parameters. For the single motor parameters given in Table 16.1, the cargo transport exhibits a dynamic instability, which effectively makes the (0) states very unlikely: if both types of motors are active simultaneously, they exert forces on each other and tend to pull each other off the filament. If, for example, a minus motor unbinds, each of the remaining minus motors has to sustain a larger force arising from the opposing team. Since the unbinding rate in Equation 16.4 increases exponentially with the force, the remaining minus motors now have a high unbinding probability, and undergo an unbinding cascade until all minus motors are detached. A similar unbinding cascade can happen for the plus motors. Because of these unbinding cascades, the cargo is unlikely to stay in a (0) state, in which both types of motors are attached to the filament, but is likely to attain a (+) or (−) state, in which only plus or only minus motors are bound to filament. Therefore the spatial displacement (or trajectory or kymograph) of a cargo pulled by several kinesin-1 and several dynein motors exhibits

c16.indd 301

7/30/2010 2:19:26 PM

302

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

alternating periods of fast plus and fast minus motion at speeds of micrometers/ second (μm/s); see Figure 16.7A. Switching between the two directions happens when a series of motor binding and unbinding events leads to the predominance of the opposing team, which is then stabilized by another unbinding cascade. 16.3.5.3 Advantages for Cargo Transport The tug-of-war performed by two motor species has several advantages for the cell. First, fast bidirectional motion is possible without any coordination complex. Second, cargo transport can easily be regulated, since a change in a single motor parameter affects the competition of the two teams and can lead to net plus or net minus motion by increasing or decreasing the corresponding run lengths, as found experimentally [33–36]. Third, the transport by teams of motors again leads to larger force generation and to enhanced processivity [66]. In particular, the binding time of a cargo increases exponentially with the numbers N+ and N− of attached plus and minus motors, respectively. The tug-of-war model described here leads to the binding time Δtb as given by Δtb( N +, N − ) ≅

(1 + π 0 + ε 0 + ) N + + (1 + π 0 − ε 0 − ) N − − 2

(16.7)

N+ π0+ + N− π0−

where the indices “+” and “−” label plus and minus motor parameters, respectively. Fourth, the stochastic switching of bidirectional transport leads to enhanced diffusion on long time and large length scales; see Figure 16.7B. On short time scales, the cargo particle moves ballistically, that is, the mean square discplacement increases quadratically with time, while it increases linearly on long time scales, with a diffusion coefficient of about 1 μm2/s. This diffusion constant is similar to the diffusion constant of a 1-μm sized cargo particle in

10

MSD [μm2]

Distance [μm]

15 5 0 −5 0 (A)

10

20 30 Time t [s]

40

104 102 10−2 10−4 0.01

(B)

~t

1 ~t2 1 103 Time t [s]

104

Figure 16.7. Transport by two antagonistic teams of motors. (A) Spatial displacement (or trajectory or kymograph) of cargo particle as a function of time. The cargo is pulled by 4 dyneins and 3 kinesins with single motor parameters as in Table 16.1 and exhibits fast transport in both the plus direction and minus direction. (B) Mean square displacement (MSD) of cargo as a function of time. The cargo is pulled by N+ = 4 plus and N− = 4 minus motors, which are both characterized by kinesin parameters as in Table 16.1. The cargo’s MSD grows quadratically and linearly with time for short and long times, respectively.

c16.indd 302

7/30/2010 2:19:26 PM

TEAMWORK OF MOLECULAR MOTORS

303

water, and about 100–1000 times larger than the diffusion constant of such a particle in the cytoplasm. This enhanced diffusion should be especially useful for cargoes in search of their destination, or for cargo particles such as mitochondria or pigment granules that must be distributed over the whole cell [29, 36, 67]. Finally, one would intuitively expect that bidirectional transport helps to reduce jams in the dense traffic of cargo particles as found in eukaryotic cells. Indeed, if a jam builds up in one direction, for example, because of an obstacle, the cargo particles at the very end of the jam may then move in the opposite direction and, in this way, start to dissolve the jam. 16.3.6 Two Teams of Motors Working on Different Tracks As discussed in Section 16.2.2, tracks for the long- and short-ranged transport of intracellular cargo particles are provided by microtubules and actin filaments, respectively. In order to switch between these two filament systems without interruptions, many cargo particles are attached to both microtubuleand actin-based motors [38, 39]. The probability of switching between the two filaments depends on the number and types of motors, on the cargo, as well as on cellular regulation [36, 40, 68]. 16.3.6.1 Processivity Enhancement During transport on one type of filament, both types of motors may be crosslinked to this filament. Indeed, myosin V, which walks along actin filaments, can also bind to microtubules and diffuse randomly on these latter filaments, whereas kinesin-1, which walks along microtubules, exhibits a weak affinity for actin filaments as well [49, 69]. This type of transport is illustrated in Figure 16.8A for a cargo particle that is

Cargo Myosin

Kinesin

-

Distance [μm]

6

+

4

2

0 0

(A)

Microtubule

(B)

3

6

9

12

Time t [s]

Figure 16.8. Cargo transport by one active and one “passive” motor. (A) A cargo particle (grey) is crosslinked to a microtubule by one kinesin-1 (blue) and one myosin V (red). The kinesin-1 motor is actively stepping whereas the myosin V motor is passively diffusing along the filament. (B) The trajectory of such a cargo exhibits fast plus motion interrupted by diffusive events. For this example, the total binding time was about 11 s, much longer than in the absence of the “passive” myosin V motor [14]. (See color insert.)

c16.indd 303

7/30/2010 2:19:26 PM

304

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

crosslinked to a microtubule by both one kinesin-1 motor, which actively walks along the microtubule, and one myosin V, which passively diffuses along this filament. Such a cargo particle exhibits fast plus motion interrupted by diffusive events; see Figure 16.8B. It is then necessary to distinguish the run length, which the cargo particle exhibits during its plus motion, from the binding length of the composite cargo motion. The binding length of a cargo particle that is transported by one kinesin-1 motor and one myosin V motor is more than two times larger than that of a cargo particle transported by only one kinesin motor [49]. This processivity enhancement can be understood from the single motor properties of kinesin and myosin (compare Section 16.3.3), provided the myosin motor is characterized by its bound diffusion constant rather than by its velocity [14]. The increase in run length arises in a similar way as for cargo transport by a team of identical motors; see Section 16.3.4. If the kinesins unbind, the myosins still act as crosslinkers between cargo and microtubules, thereby preventing the cargo from diffusing away from the filament and giving the kinesins a chance to rebind. Furthermore, in contrast to the competition between kinesin and dynein as described in Section 16.3.5, the simultaneous crosslinking by kinesin-1 and myosin V does not lead to a tug-of-war: because of the relatively weak affinity of myosin V to microtubules, myosin V motors can easily be dragged along by kinesin motors toward the plus end of the microtubule [14].

16.4

CONCLUSION AND DISCUSSION

Cellular cargoes, including endosomes, RNAs, protein complexes, and filaments as well as whole organelles, travel through the cell with the help of molecular motors that pull them along cytoskeletal filaments: kinesin and dynein motors mediate long-ranged transport on microtubule filaments, while local delivery is accomplished by myosin motors walking on a meshwork of actin filaments. 16.4.1 Team Work of Motors As described in this chapter, molecular motors work in teams in order to meet the challenges of intracellular transport. Three types of teamwork have been identified and elucidated. First, one team of identical motors as shown in Figure 16.4 is able to transport large cargoes through the viscous cytoplasm at considerable speeds of micrometers/second over distances of many micrometers; see Figure 16.5. Second, two teams of antagonistic motors that walk into opposite directions, as in Figure 16.6, accomplish bidirectional transport along an isopolar cytoskeletal network of motor tracks as illustrated in Figure 16.7A. Third, two teams of motors that can walk on different filaments (see

c16.indd 304

7/30/2010 2:19:26 PM

CONCLUSION AND DISCUSSION

305

Figure 16.8) allow a smooth transit from the long-range microtubule traffic to the short-range delivery on actin filaments and vice versa. The presence of several motor teams on one cargo allows versatile transport and fast reaction to cellular regulation. A regulation cascade that targets the motor proteins can change the transport properties of the cargo “on the fly,” making it travel on a microtubule to the minus instead of the plus direction by downregulating kinesin, or switching to actin filaments by upregulating myosin. 16.4.2 Virus, Gene, and Drug Delivery Many viruses hijack the intracellular transport systems and use them for their own purposes. These viruses recruit molecular motors from the host’s cytoplasm and, in addition, “know” how to activate these motors to ensure transport to the desired destination. Copying this viral strategy would dramatically increase the efficiency of gene therapy and drug delivery, because transport through the crowded and viscous cytoplasm would be strongly enhanced. Virus-based expression vectors should include all factors that allow the virus to use the host’s transport machinery during entry and transcription, but not those involved in virus replication and egress. Nonviral vectors and drug carriers should be able to recruit motor proteins from the cytoplasm, for example, by coating them with receptor proteins for molecular motors. Although many of the motor–cargo interactions still remain to be elucidated, an increasing catalog of receptor proteins is emerging [8]. Ideally, the appropriate receptors should be selected from such a catalog in order to bind the correct number and types of motors from the cytoplasm to the gene or drug carrier. The choice of motors should reflect the cargo destination, and possible responses to intracellular or externally applied regulatory stimuli. 16.4.3 Outlook: Open Questions and Possible Applications In order to construct such carrier and delivery systems, a good understanding of molecular motor traffic is necessary. Although much progress has been made during the last couple of years, many open questions remain. These include the details of the molecular stepping mechanisms of the different motors, the understanding of motor cooperativity, as well as the understanding of overall cellular traffic. A particularly challenging topic is the coupling of motor transport to cellular regulation. Such an understanding of molecular motor traffic would also be useful for other types of applications in medicine and nanotechnology. One example is provided by antiviral therapies: efficiency of virus infection would be greatly reduced if the viruses were prevented from hijacking the cellular transport machinery in an appropriate way. Likewise, understanding of molecular motor traffic could be useful in order to treat diseases in which improper intracellular transport plays an important role, such as Alzheimer’s disease or lissencephaly

c16.indd 305

7/30/2010 2:19:26 PM

306

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

[70, 71]. Finally, molecular motors are possible building blocks for nanotechnological applications. One example is provided by molecular motors that carry cargoes on lab-on-a-chip devices [72, 73].

REFERENCES 1. Cohen, R. N., Rashkin, M. J., Wen, X., and Szoka, F. C. Molecular motors as drug delivery vehicles. Drug Discov. Today 2: 111–118 (2005). 2. Vaughan, E. E., DeGiulio, J. V., and Dean, D. A. Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import. Curr. Gene Ther. 6: 671–681 (2006). 3. Luby-Phelps, K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. Surv. Cell Biol. 192: 189–221 (2000). 4. Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275: 1625–1629 (2000). 5. Dowty, M. E., Williams, P., Zhang, G. F., Hagstrom, J. E., and Wolf, J. A. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes. Proc. Natl. Acad. Sci. U.S.A. 92: 4572–4576 (1995). 6. Capecchi, M. R. High-efficiency transformation by direct micro-injection of DNA into cultured mammalian-cells. Cell 22: 479–488 (1980). 7. Campbell, E. M. and Hope, T. J. Gene therapy progress and prospects: viral trafficking during infection. Gene Ther. 12: 1353–1359 (2005). 8. Dohner, K., Nagel, C. H., and Sodeik, B. Viral stop-and-go along microtubules: taking a ride with dynein and kinesins. Trends Microbiol. 13: 320–327 (2005). 9. Greber, U. F. and Way, M. A superhighway to virus infection. Cell 124: 741–754 (2006). 10. Beeg, J., Klumpp, S., Dimova, R., Gracià, R. S., Unger, E., and Lipowsky, R. Transport of beads by several kinesin motors. Biophys. J. 94: 532–541 (2008). 11. Klumpp, S. and Lipowsky, R. Cooperative cargo transport by several molecular motors. Proc. Natl. Acad. Sci. U.S.A. 102: 17284–17289 (2005). 12. Müller, M. J. I., Klumpp, S., and Lipowsky, R. Motility states of molecular motors engaged in a stochastic tug-of-war. J. Stat. Phys. 133: 1059–1081 (2008). 13. Müller, M. J. I., Klumpp, S., and Lipowsky, R. Tug-of-war as a cooperative mechanism for bi-directional cargo transport by molecular motor. Proc. Natl. Acad. Sci. U.S.A. 105: 4609–4614 (2008). 14. Berger, F., Müller, M. J. I., and Lipowsky, R. Enhancement of the processivity of kinesin-transported cargo by myosin V. EPL 87: 28002 (2009). 15. Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. Essential Cell Biology. An Introduction to the Molecular Biology of the Cell. Garland, New York, 1998. 16. Brady, S. T. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature 317: 73–75 (1985).

c16.indd 306

7/30/2010 2:19:26 PM

REFERENCES

307

17. Vale, R. D., Reese, T. S., and Sheetz, M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42: 39–50 (1985). 18. Vale, R. D. The molecular motor toolbox for intracellular transport. Cell 112: 467–480 (2003). 19. Block, S. M. Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. Biophys. J. 92: 2986 (2007). 20. Paschal, B. M., Sheptner, H. S., and Vallee, R. B. MAP-1C is a microtubule- activated ATPase which translocates microtubules invitro and has dynein-like properties. J. Cell Biol. 1057: 1273–1282 (1987). 21. Gennerich, A. and Vale, R. D. Walking the walk: how kinesin and dynein coordinate their steps. Curr. Opin. Cell Biol. 21: 59–67 (2009). 22. Howard, J. Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates, Sunderland, MA, 2001. 23. Mallik, R. and Gross, S. P. Molecular motors: strategies to get along. Curr. Biol. 14: R971–R982 (2004). 24. Goldstein, L. S. B. and Yang, Z. Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23: 39–71 (2000). 25. Carter, N. J. and Cross, R. A. Mechanics of the kinesin step. Nature 435: 308–312 (2005). 26. Toba, S., Watanabe, T. M., Yamaguchi-Okimoto, L., Toyoshima, Y. Y., and Higuchi, H. Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. Proc. Natl. Acad. Sci. U.S.A. 103: 5741–5745 (2006). 27. Gross, S. P., Vershinin, M., and Shubeita, G. T. Cargo transport: two motors are sometimes better than one. Curr. Biol. 17: R478–R486 (2007). 28. Lane, J. and Allan, V. Microtubule-based membrane movement. Biochim. Biophys. Acta 1376: 27–55 (1998). 29. Gross, S. P. Hither and yon: a review of bi-directional microtubule-based transport. Phys. Biol. 1: R1–R11 (2004). 30. Welte, M. A. Bi-directional transport along microtubules. Curr. Biol. 14: R525– R537 (2004). 31. Klumpp, S., Nieuwenhuizen, Th. M., and Lipowsky, R. Self-organized density patterns of molecular motors in arrays of cytoskeletal filaments. Biophys. J. 88: 3118– 3132 (2005). 32. Müller, M. J. I., Klumpp, S., and Lipowsky, R. Molecular motor traffic in a halfopen tube. J. Phys. Condens. Matter 17: S3839–S3850 (2005). 33. Morris, N. R. and Hollenbeck, P. J. The regulation of bi-directional mitochondrial transport is coordinated with axonal outgrowth. J. Cell Sci. 104: 917–927 (1993). 34. Gross, S. P., Welte, M. A., Block, S. M., and Wieschaus, E. F. Dynein-mediated cargo transport in vivo: a switch controls travel distance. J. Cell Biol. 148: 945–955 (2000). 35. Smith, G. A., Murphy, B. J., Gross, S. P., and Enquist, L. W. Local modulation of plus-end transport targets herpesvirus enry and egress in sensory axons. Proc. Natl. Acad. Sci. U.S.A. 101: 16034–16039 (2004). 36. Gross, S. P., Tuma, M. C., Deacon, S. W., Serpinskaya, A. S., Reilein, A. R., and Gelfand, V. I. Interactions and regulation of molecular motors in Xenopus melanophores. J. Cell Biol. 156: 855–865 (2002).

c16.indd 307

7/30/2010 2:19:26 PM

308

CARGO TRANSPORT BY TEAMS OF MOLECULAR MOTORS

37. Tuma, M. C. and Gelfand, V. I. Molecular mechanisms of pigment transport in melanophores. Pigment Cell Res. 12: 283–294 (1999). 38. Brown, S. S. Cooperation between microtubule- and actin-based motor proteins. Annu. Rev. Cell Dev. Biol. 15: 63–80 (1999). 39. Goode, B. L., Drubin, D. G., and Barnes, G. Functional cooperation between