Erythrocyte Engineering for Drug Delivery and Targeting (Biotechnology Intelligence Unit, 6)

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Mauro Magnani, Ph.D.

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BIOTECHNOLOGY INTELLIGENCE UNIT 6

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Erythrocyte Engineering for Drug Delivery and Targeting

R.G. LANDES C O M P A N Y

BIOTECHNOLOGY INTELLIGENCE UNIT 6

Erythrocyte Engineering for Drug Delivery and Targeting Mauro Magnani, Ph.D. Istituto di Chimica Biologica "Giorgio Fornaini" Universitá degli Studi di Urbino Urbino, Italy

LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.

EUREKAH.COM AUSTIN, TEXAS U.S.A.

ERYTHROCYTE ENGINEERING FOR DRUG DELIVERY AND TARGETING Biotechnology Intelligence Unit Eurekah.com Landes Bioscience Designed by Jesse Kelly-Landes Copyright ©2002 Eurekah.com All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: Eurekah.com / Landes Bioscience, 810 South Church Street Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 www.Eurekah.com www.landesbioscience.com ISBN:

1-58706-061-2 (hardcover) 1-58706-117-1 (softcover)

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Erythrocyte engineering for drug delivery and targeting / [edited by] Mauro Magnani. p. ; cm. -- (Biotechnology intelligence unit ; 6) Includes bibliographical references and index. ISBN 1-58706-061-2 (hardcover) -- ISBN 1-58706-117-1 (softcover) 1. Erythrocytes--Biotechnology. 2. Drug carriers (Pharmacy) 3. Bioreactors. 4. Drug targeting. [DNLM: 1. Drug Delivery Systems. 2. Drug carriers. 3. Erythrocytes--metabolism. QV 785 E73 2002] I. Magnani, Mauro. II. Series. TP248.65.B56 E79 2002 615' .19--dc21

CONTENTS 1. Red Blood Cell Loading: A Selection of Procedures ................................. 1 Luigia Rossi, Sonja Serafini and Mauro Magnani Introduction .......................................................................................... 1 Main Procedures For the Entrapment of Drugs in Erythrocytes ............ 1 Electroporation ..................................................................................... 2 Development of Apparatus For Clinical Applications of Loaded Erythrocytes .................................................................... 10 Conclusions ......................................................................................... 13 2. Towards Activated Release of Payloads From Carrier Erythrocytes ........ 19 A.M. Rollan and A.P. McHale Introduction ........................................................................................ 19 Current Exploitation of Erythrocytes in Delivery and Targeting .................................................................................. 19 The Case For an Activatable Payload Release Mechanism From Carrier Erythrocytes ............................................................... 20 Photodynamic Activation and Activated Payload Release From a Light-Sensitive Erythrocyte Carrier System ......................... 21 Electric Field-Mediated Stimulation of Payload Release From HPD-Sensitised Human Erythrocytes .................................... 23 Challenges Associated With Activated Release From Erythrocyte Carriers ............................................................... 25 3. Targeting Drug Loaded Red Blood Cells................................................ 29 Mauro Magnani, Luigia Rossi and Giorgio Brandi Introduction ........................................................................................ 29 Red Blood Cells as Drug Delivery System ........................................... 30 Targeting Drug-Loaded Red Blood Cells ............................................ 30 Drug Targeting to Macrophages by Carrier Red Blood Cells ............... 31 Targeting New Anti-Herpetic Drugs ................................................... 31 Targeting Corticosteroid Analogues .................................................... 32 Targeting Peptides ............................................................................... 33 Conclusions ......................................................................................... 34 4. Streptavidin-Mediated Coupling of Therapeutic Proteins to Carrier Erythrocytes ......................................................................... 37 Vladimir R.Muzykantov and Juan-Carlos Murciano Introduction: RBC as Drug Carrier ..................................................... 37 Loading of Drugs Inside RBC and Coupling of Therapeutic Proteins to RBC Surface .......................................... 38 Destruction and Elimination of Modified RBC by Complement and Phagocytes ...................................................... 39 Biocompatibility of RBC Modified With Non-Specific Cross-Linkers .................................................... 41 Coupling of Active Therapeutic Proteins to RBC via Streptavidin-Biotin .................................................................... 43

Biocompatibility of RBC Modified With Biotin and (strept)Avidin: In Vitro Vtudies ................................................ 47 Biocompatibility of RBC Modified with Biotin and Streptavidin: In Vivo Studies .................................................... 49 Prolonged Circulation of Therapeutic Proteins Coupled to RBC ............................................................................................ 55 Conclusion and Perspectives ................................................................ 58 Acknowledgements .............................................................................. 62 5. Vaccination Strategy Using Red Blood Cells as Antigen Delivery System .................................................................. 68 Laura Chiarantini Introduction ........................................................................................ 68 Immobilization of Antigen on Red Blood Cells ................................... 69 Immunological Response to RBC Coupled With Proteins .................. 70 Immunological Response to RBC Coupled With Recombinant Proteins ............................................................ 70 Immunological Response to RBC Coupled With Surface Particles of Virus ........................................................ 71 Antigen Delivery System for Human Dendritic Cells .......................... 71 Conclusions ......................................................................................... 72 Acknowledgements .............................................................................. 73 6. Engineered Nanoerythrosomes as a Novel Drug Delivery System .......... 75 Sanjay Jain and N. K. Jain Introduction ........................................................................................ 75 Advantages .......................................................................................... 76 The Erythrocyte and Erythrocyte Membrane ...................................... 76 Requirements for Encapsulation .......................................................... 76 Isolation of Erythrocytes, Preparation of Erythrocyte Ghosts and Nanoerythrosomes ................................. 77 Inside-Out Red Cell Membraneous Vesicles ........................................ 79 In Vitro Characterization .................................................................... 81 Routes of Administration .................................................................... 83 Stability Studies ................................................................................... 83 In Vivo Studies and Toxicity ............................................................... 85 Immunological Considerations ............................................................ 87 Tardeting Potential and Applications of Nanoerythrosomes ................ 88 Advances, Conclusion and Future Prospects ........................................ 88 7. Red Blood Cells as Carriers of Antiviral Agents ...................................... 90 A. Fraternale, A. Casabianca and M. Magnani Introduction ........................................................................................ 90 Red Blood Cells for a Slow Release of Antiviral Drugs ......................... 90 Red Blood Cells for Targeting of Antiviral Drugs ................................ 91 Red Blood Cells as Carriers of Antiretroviral and Antiherpetic Drugs ................................................................... 93

Conclusions ......................................................................................... 94 Acknowledgements .............................................................................. 95 8. Erythrocytes as Carriers of Anthracycline Antibiotics In Vitro and In Vivo ............................................................................. 99 Victor M. Vitvitsky Introduction ........................................................................................ 99 Anthracycline Antibiotics .................................................................... 99 Carrier Macromolecules .................................................................... 100 Liposomes ......................................................................................... 100 Carrier Erythrocytes .......................................................................... 101 Conclusions ....................................................................................... 105 9. Drug-Loaded Red Blood Cells for the Control of the Inflammatory Response: Selective Targeting !B (NF-! !B) ........................................................... 109 of Nuclear Factor-! R. Crinelli, A. Antonelli, M. Bianchi, L. Gentilini and M. Magnani Introduction ...................................................................................... 109 Targeting Glucocorticoids to Macrophages: Inhibition of NF-!B Activation and Cytokine Release ................................... 110 Targeting Ubiquitin Analogues to Macrophages: A New Approach to Interfering with NF-!B Activation ................ 112 Conclusions ....................................................................................... 115 10. Design and Synthesis of New Pro-Drugs to be Used With Carrier Red Blood Cells ............................................................ 118 S. Scarfì, G. Damonte and U. Benatti Introduction ...................................................................................... 118 Dimeric Fluoropyrimidine Synthesis and Intraerythrocytic Biochemical Pathway .................................. 119 AZTp2AZT Homodinucleotide Synthesis and Characterization ..................................................................... 121 AZTp2ACV Heterodinucleotide Synthesis and Characterization ......................................................................... 123 AZTp2EMB Heterodinucleotide Synthesis and Characterization .................................................................... 125 Conclusions ....................................................................................... 126 11. Engineered Red Blood Cells as Circulating Bioreactors ........................ 130 P. Ninfali and E. Biagiotti Introduction ...................................................................................... 130 The Entrapped Enzymes ................................................................... 131 Coentrapment of Two Enzymes ........................................................ 134 Enzymes Bound to RBC Membrane ................................................. 136 Enzymes Encapsulated in RBC to Lower Alcohol Toxicity ................ 136 Conclusion ........................................................................................ 137 Acknowledgement ............................................................................. 140

EDITOR Mauro Magnani, Ph.D. Istituto di Chimica Biologica "Giorgio Fornaini" Universitá degli Studi di Urbino Urbino, Italy Chapters 1, 7, 9

CONTRIBUTORS A. Antonelli Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

A. Casabianca Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 9

Chapter 7

U. Benatti Department of Experimental Medicine University of Genoa Genoa, Italy

L. Chiarantini Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 10

E. Biagiotti Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 5

Chapter 11

R. Crinelli Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

M. Bianchi Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

G. Damonte Department of Experimental Medicine University of Genoa Genoa, Italy

Chapter 9

Chapter 9

Chapter 10

G. Brandi Institute of Hygiene Universitá di Urbino Urbino, Italy

A. Fraternale Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 3

Chapter 7

L. Gentilini Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

P. Ninfali Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 9

Chapter 11

N.K. Jain Department of Pharmaceutical Sciences Dr. H.S. Gour University Sagar, India

A.M. Rollan Science Business Incubator Unit Gendel Ltd. Coleraine, Northern Ireland

Chapter 6

Chapter 2

Sanjay Jain Department of Pharmaceutical Sciences Dr. H.S. Gour University Sagar, India

L. Rossi Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy

Chapter 6

Chapters 1, 3

A.P. McHale School of Biomedical Sciences University of Ulster Coleraine, Northern Ireland Chapter 2

S. Scarfi Department of Experimental Medicine University of Genoa Genoa, Italy Chapter 10

Juan-Carlos Murciano Department of Pharmacology University of Pennsylvania Philadelphia, Pennsylvania, U.S.A. Chapter 4

Vladimir R. Muzykantov Institute for Environmental Science University of Pennsylvania Philadelphia, Pennsylvania, U.S.A. Chapter 4

S. Serafini Istituto di Chimica Biologica "Giorgio Fornaini" Universitá di Urbino Urbino, Italy Chapter 1

Victor M. Vitvitsky National Research Center for Hematology Moscow, Russia Chapter 8

PREFACE

T

he therapeutic potential of blood has been recognized since antiq uity. However, it was only in the 20th century that relatively safe procedures for blood transfusion were developed. Since then an increasing understanding of cell physiology and of the biochemistry of red cells and platelets, alongside the development of large-scale plasma fractionation methods and freezing procedures, has greatly enhanced the use of blood. However, these procedures, and in particular those involving red blood cells, have had only one major biomedical use: that is, transfusion medicine. In other words, blood cells or blood components are collected, isolated and reinfused in the same donor or in compatible recipients only to restore their normal levels. Recently this approach and the biomedical use of blood have moved into a new era. Blood is now processed for the isolation of circulating stem cells to be reinfused but also to be expanded in vitro or modified by selective gene transfer. Increasing attention is being dedicated to the isolation of circulating immature dendritic cells from blood, as these cells are excellent antigen-presenting cells and thus very useful in vaccination studies, including in vitro priming. Furthermore, all lymphocyte populations are not only invaluable in diagnostic procedures, but are also increasingly seen as chemokine- and cytokine-producing cells. Finally, red blood cells have been considered by several researchers for use as circulating bioreactors for the degradation of toxic metabolites or the inactivation of xenobiotics, as drug delivery systems, as carriers of antigens for vaccination, and in several other biomedical applications. Many of these applications are only possible thanks to the introduction of procedures for the transient opening of pores across the red cell membrane. The use of resealed red blood cells was first reported in 1973 for enzyme-replacement therapy in inborn errors of metabolism (Ihler G.M., Glw R.H. and Schnure F.W. Proc Natl Acad Sci USA 70, 1973, 2663). Several improvements have been made to the procedure since then and new methods have been developed. These methods and a review of their biomedical applications are summarized in this book. Many of the procedures were described in details in a special volume of Methods in Enzymology (Vol. 149B, 1987, pp 217) and several books have been published over the years reporting the proceedings of the Society for the Use of Resealed Erythrocytes (ISURE). Herein, we will summarize the old and new biomedical applications of engineered red blood cells based on the specific functions of the processed cells. The reader will appreciate the incredible number of potential biomedical applications red cells can have in addition to their main physiological role of oxygen transporters. Unfortunately, only a few of these applications have reached the clinic. Nonetheless, the availability of a new

procedure for the processing of small volumes of autologous blood to be re-infused into the same donor (see Chapter 1) and the development of new biomedical applications of engineered red cells for the delivery of drugs, antigens, immunomodulators or diagnostic agents make me optimistic about the future of engineered red blood cells. Mauro Magnani

CHAPTER 1

Red Blood Cell Loading: A Selection of Procedures Luigia Rossi, Sonja Serafini and Mauro Magnani

I

n this Chapter a selection of procedures for the encapsulation of a wide range of molecules into red blood cells is reported. Electroporation, drug-induced endocytosis, osmotic pulsing and hypotonic hemolysis are described. Among the hypotonic hemolysis procedures, three different methods (dilutional, preswell dilutional and dialysis) are described. A critical comparison of several parameters suggests that the dialysis method, expecially that based on a high hematocrit dialysis procedure, is the preferred one since it permits one to obtain both a good percentage of drug incorporation and a good red cell recovery. However, when a procedure moves from the laboratory to the clinic, the availability of appropriate equipment becomes very important. Finally, a new procedure and a new apparatus for the encapsulation of drugs for human therapy, based on two sequential hypotonic dilutions followed by concentration with hemofilter, is described.

Introduction The introduction of procedures for the transient opening of pores across the red cell membrane provides the extraordinary opportunity to manipolate erythrocytes (RBC) for different biomedical applications. Until recently, the only biomedical uses of erythrocytes were in fact in transfusion medicine and in the preparation of blood products while with this new technology drugs, chemicals or macromolecules can be loaded into erythrocytes, offering a further step towards the optimal use of a natural resource as is blood. Erythrocytes submitted to the loading procedures have been proposed as circulating bioreactors for the degradation of toxic metabolites, as carriers of antigens for vaccination and particularly as drug delivery system. Erythrocytes are biodegradable, can circulate for long periods of time (months), have a large capacity and a high percentage of encapsulations can be obtained. Furthermore, the morphological, immunological and biochemical properties of carrier erythrocytes are similar to those of native cells. Moreover, besides the biomedical applications, the technology of opening and resealing of the erythrocytes also provides the opportunity to specifically investigate basic biochemical problems by the encapsulation of enzymes that generate new metabolic abilities,1 antibodies that inactivate single metabolic steps2 or chemicals that affect oxygen delivery.3 Several procedures for the entrapment of chemicals, drugs, proteins etc. in erythrocytes have been proposed. Since many of these procedures have already been described in detail,4 in this Chapter only the main characteristics of each method will be reported. Furthermore, a method that permits one to work with as little as 1 ml of blood for laboratory use will be described. Finally, a new loading procedure that allows the encapsulation of drugs into human erythrocytes for clinical use, starting from as little as 50 ml of autologous blood, will be discussed.

Main Procedures For the Entrapment of Drugs in Erythrocytes The majority of the methods for the entrapment of chemicals, drugs, proteins etc. in erythrocytes take advantage of the remarkable capacity of this cell for reversible shape changes and Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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Erythrocyte Engineering for Drug Delivery and Targeting

for reversible deformation under stress allowing transient opening of pores large enough to be crossed by externally placed macromolecules. In this Chapter, the following procedures: 1. Electroporation 2. Drug-Induced Endocytosis 3. Osmotic Pulse Method 4. Hypotonic Hemolysis (Dilutional, Preswell Dilutional, and Dialysis Methods) will be examined. Finally, a new Hypotonic Hemolysis method, developed in our laboratory and named Preswell Dilutional with Concentration, will be described.

Electroporation The electric modification of membrane permeability for drug loading into living cells was examined and described in detail by T.Y. Tsong.5 Here, only a limited description of the main features of the procedure is reported. Electrical methods for the entrapment of drugs consist of exposure of cells to a transient high intensity field (a few kV) of a short duration (a few ∝second) in appropriate isotonic solutions. When the externally applied field strength exceeds a certains threshold value, breakdown occurs in the membrane, resulting in a temporary increase in permeability. This permeability increase depends on the field strength, pulse duration and the composition of the external solution.6-10 If a drug to which the cell is otherwise impermeable is present in the external solution, during the high permeable state of the cell, it will diffuse inside the cell. The increased permeability of the pulsed cells can be maintained for 30-60 minutes at the melting ice temperature to allow the equilibration of the drug inside the cell. However, on incubation of these cells at 37°C for a few minutes up to an hour, the cell's original impermeability can be restored, thus entrapping the drug inside the cell. Un-entrapped drug present in the external solution can be removed by washing the resealed cells. The main steps of the electroporation procedure are as follows: • Fresh human blood is drawn in heparin. • Erythrocytes are washed three times by 50 volumes of 150 mM NaCl and 7 mM phosphate buffer at pH 7.0. • Packed cells are resuspended to 10-20% hematocrit in a “pulsation medium” and kept at 4°C. A pulsation medium is a mixture of isotonic saline (150 mM) and isosmotic sucrose (300 mM). The salt content of the pulsation medium is critical for obtaining different pores sizes and is adjusted by mixing the two isosmotic buffers.6-8 • 0.15 ml of an erythrocyte suspension at 10-20% hematocrit in pulsation medium is transferred into the pulsation chamber with the drug to be encapsulated and treated with a single electric pulse of up to 4 kV/cm and of duration up to 100 ∝sec at 25°C. The suspension is then kept at 4°C. • Spontaneous resealing of pores takes place at 37°C in an osmotically balanced me dium.7,8 The resealing properties of membranes, however, depend on the nature of the compound to be entrapped. An important observation of the electric method is that once complete resealing is achieved, the erythrocytes appear indistinguishable from the normal, untreated red cells in terms of shape, volume, transport activities and survivability.7,8 It is worth noting that while ions, saccharides and tetrasaccharides can easily enter erythrocytes, macromolecules, such as enzymes, require more severe conditions. Tsong and coll.10 have demonstrated that in human erythrocytes during membrane puncture by a kilovolt electric field, Na+, K+-ATPase is damaged. Kinosika and Tsong6,9 had demonstrated that once the red blood cells were rendered permeable to Na+ and K+ they swelled and eventually lysed because of the colloid osmotic pressure of its macromolecular contents. For example, the colloid osmotic pressure of hemoglobin is about 30 mOsm and this pressure drives water and ion influx. As a result, the cell swells while membrane is ruptured when the cell volume reaches 155% of its original volume. Thus, cell lysis is a secondary effect of electric modification of the membrane. Because all lysis is due to the colloid osmotic swelling, balancing

Red Blood Cell Loading

3

Figure 1. Colloid osmotic hemolysis of electrically perforated red cells and resealing pores. When RBC are treated with an electric pulse of greater than a threshold value (2 kV/cm for a 20 ∝sec pulse), their membrane become permeable to ions and small molecules (blue dots). A) The red cells swell, due to the colloid osmotic pressure of large cytoplasmic macromolecules (hemoglobin) and the membranes eventually rupture. B) When large molecules, BSA for example (•), are added to the suspension, the colloid osmotic pressure of cytoplasmic macromolecules is balanced and the cells will not swell even after their membranes are perforated with small pores. If drugs of small molecular weight (blue dots) are added at this point, they will permeate into the red cells. Reprinted with permission from: Tsong TY. In: Methods in Enzymology, Drug and Enzyme Targeting, 149 (part B):248-259. ∀1987 Academic Press, Inc.

the colloid osmotic pressure of cellular macromolecules, lysis can be prevent. This strategy is schematized in Figure 1. The tetrasaccharide stachyose or proteins such as bovine serum albumin (BSA) were used to counteract the colloid osmotic swelling of electrically perforated erythrocytes. Under these osmotically balanced conditions, pores will stay open at 4°C for few days.

Drug-Induced Endocytosis Endocytosis is a process by which most cell types internalize small amounts of external fluid. The plasma membrane invaginates and, subsequently, an intracellular vesicle is created when the two external surfaces of the plasma membrane approach each other and then fuse across the neck of the invaginated pouch. The external fluid, including proteins, ions and other dissolved substances, is entrapped in the intracellular vesicle along with any substances bound to the portion of the external membrane which subsequently forms the vesicle. As with most cell types, nucleated erythrocyte precursors are active in endocytosis. Erythroblasts, for example, actively incorporated ferritin bound to the cell surface by endocytosis, and ferritin-containing vacuoles can be found even at the reticulocyte stage of maturation. Apparently, young reticulocytes and even neonatal erythrocytes also retain some endocytic activity.11 Mature erythrocytes, however, are not generally active in endocytosis, so that the activity of the

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Erythrocyte Engineering for Drug Delivery and Targeting

Figure 2. Primaquine induced endocytosis in erythrocyte. Reprinted with permission from: Schrier SL. In: Methods in Enzymology, Drug and Enzyme Targeting, 149 (part B):260-270. ∀1987 Academic Press, Inc.

endocytic system must diminish greatly as the erythrocyte matures. However, an important element of endocytosis, that of fusion between opposed membranes, is observed in mature erythrocytes. In fact, membrane fusion can easily occur in erythrocytes if the cellular membrane is brought into contact with itself, that is if endocytosis is induced. Ginn et al12 discovered drug-induced endocytosis. A variety of amphipathic cations can produce first stomatocytosis and then, mostly at the advancing lip of the stome, inside-out endocytic vacuoles appear.13 Several classes of drugs can produce this phenomenon. The best studied are primaquine and related 8-aminoquinolines,13 vinblastine,13 chloropromazine14 and other cationic phenothiazines, hydrocortisone,13 propanol,15 tetracaine,15 and vitamin A.16 In Figure 2, primaquine induced endocytosis in erythrocytes is shown. After exposure of erythrocytes to membrane-active drugs, endocytic vacuoles form and substances, either bound to the membrane or dissolved in the extracellular fluid, may be entrapped

Red Blood Cell Loading

5

within endocytotic vescicles.13 This entrapment procedure may have considerable potential since cellular lysis and loss of cytoplasmic constituents do not occur. The erythrocyte membrane internalization is a metabolic process dependent on drug concentration, temperature and pH. In particular, low and prehemolytic concentrations of primaquine seem to stabilize membranes against hypotonic hemolysis, probably through expansion of the membrane, while high concentrations cause hemolysis.17 The optimal temperature for vacuole formation is around 37°C, while no vacuoles are seen at temperatures lower than 23°C and their formation is reduced even at temperature above 45°C. The optimum pH for vacuole formation was found to be 7.9-8.1 and at pH’s below 6.4-6.5 vacuole formation is abolished. The trapping of material in the vacuoles for the quantitative assessment of drug-induced endocytosis was studied in detail by S. L. Schrier.18 M.G. Ihler and coll. developed an entrapped procedure, employing induced endocytosis, which permits large substances, such as DNA, to be entrapped.19 Since erythrocytes can be readily fused in vitro with a variety of other cell types, drug-induced erythrocyte endocytosis might provide a route for the introduction of DNA, especially cDNA.

Osmotic Pulse Method In the osmotic pulse method the cells are submitted to a short but intense period of osmotic stress.20,21 Usually the osmotic pulse method utilizes dimethylsulfoxide (DMSO) to create a large, transient osmotic gradient across the RBC membrane, thus allowing entry of molecules. The incorporation procedure may be divided into several steps: • step 1: DMSO incubation • step 2: isotonic dilution with molecules to be encapsulated • step 3: post-dilution incubation • step 4: return to the original shape These steps are schematized in Figure 3. The first step (step 1) of the procedure is the addition of DMSO to RBC to establish a high intracellular and extracellular osmolality (~1500 mOsm for 8% DMSO).22 Next, the cell suspension is mixed rapidly and uniformly with an isotonic solution, containing the molecules to be encapsulated (step 2). The DMSO concentration of the extracellular fluid is decreased immediately upon mixing and creates a transient gradient of DMSO concentration and osmolality across the RBC membrane until DMSO diffusion from the cells establishes a new equilibrium. This gradient causes an influx of water and cellular swelling, which results in increased membrane permeability and the transport of the substance to be encapsulated into the cells and of hemoglobin out (step 3). The condition for this mechanism is that transport of DMSO out of the cell is slower than transport of H2O into the cell. This is reasonable considering the high permeability of water through the red cell membrane. Once the DMSO has left the cells, the osmotic balance is restored and the cells return to the original shape (step 4). The osmotic pulse method was largely investigated by Franco et al.20,23,24 in order to incorporate inositol hexaphosphate (IHP) into erythrocytes, thus preparing low O2 affinity cells for use in clinical research.23 IHP, binding more tightly than 2,3-bisphosphoglycerate (2,3-BPG) to deoxyhemoglobin, markedly decreases the affinity of hemoglobin for oxygen, leading to the release of the oxygen even at higher oxygen partial pressure (PO2). IHP encapsulation was also obtained by a continuous-flow method, allowing the treatment of clinically relevant volumes of RBC.20 This method will be described in the next section: ”Development of loading apparatus for clinical applications of engineered erythrocytes”. The continous-flow hypotonic dialysis is probably more suitable for processing quite large amounts of blood (one unit at a time) and requires more time (two days); while the DMSO method can be easily performed within one day and is more appropriate for smaller amounts of blood.25

Hypotonic Hemolysis

Three variations of the hypotonic hemolysis procedures are known: the dilutional,26 preswell dilutional27 and dialysis methods.28,29 However, all these procedures are based on the same

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Erythrocyte Engineering for Drug Delivery and Targeting

Figure 3. Osmotic pulse method steps.

physical-chemical features of red blood cells. When placed in the presence of a hypotonic solution, RBC increase in volume because of a faster water influx and a slow salt efflux and their normal morphology of biconcave discocytes is converted to spherocytes. As the erythrocytes are not able to synthesize additional plasma membrane and don’t possess redundant or internal membranes, their surface area remain necessarily fixed. Since RBC have little capacity to resist volume increases greater than 50-75% of the initial volume, when placed in solutions less than 150 mOsm/kg the membranes rupture with formation of large pores (200-500 Å in diameter), permitting escape of the cellular contents.30,31 By raising the salt concentration to its original level, the membranes can be resealed and the resealed erythrocytes can reassume their normal biconcave shape and their normal impermeability features. Remarkably, the resulting resealed erythrocytes have an almost normal life span in circulation. At the same time that the intracellular substances leave during hypotonic hemodialysis, equilibrating themselves with the external solution, externally added compounds may enter the erythrocytes. After resealing, these substances are trapped into erythrocytes. The main steps of the hypotonic hemolysis are summarized in Figure 4. Generally, erythrocytes submitted to hypotonic hemolysis are obtained from fresh blood collected in EDTA or heparin. Stored blood may also be loaded. The main sequential steps of the hypotonic processes to obtain lysed and resealed erythrocytes are: a. Separation of RBC and plasma b. Removal of leukocytes and platelets c. Several washings of the RBC suspension d. Hypotonic lysis e. Resealing and reannealing of the cells f. Additional washings of lysed and resealed RBC g. Resuspension of the cells in plasma or phosphate buffered saline (PBS) solution Step d is different accordingly to the specific method used and it is as follows: Dilutional: To one volume of washed erythrocytes, 2-20 volumes of hypotonic buffer or water containing the compounds to be loaded are added. The cell suspension is kept at 0°C for 5 min. Preswell Dilutional: Hemolysis is carried on in two steps. First, one volume of washed cells is suspended in five volumes of diluted PBS (six volumes of PBS plus five volumes of water) to a final salt concentration of 0.6% NaCl. Under these conditions, only a small percentage of the cells lysed, probably the most fragile population of erythrocytes. Most cells remain intact but have an increased cell volume averaging about 150% of normal. After 5 min at 0°C the cells are recovered by gentle centrifugation and the swelling procedure repeated once. Second, the swelled cells are pelletted, all of the overlying swelling solution is removed and a very hypotonic lysing

Red Blood Cell Loading

7

Figure 4. Hypotonic hemolysis steps.

solution containing the molecule to be loaded is added. The volume required for complete hemolysis is as little as the volume of the packed cell pellet, due to the preswell step, thereby conserving precious materials. Following vigorous vortex mixing to resuspend the pellet, the cells are allowed to lyse for 10 min at 0°C. The preswell dilutional hemolysis procedure is schematized in Figure 5. Dialysis: Erythrocytes at a hematocrit varing from 5 to 80% are placed in a dialysis tubing together with the substance to be loaded in isotonic media. The bag is then inflated with air and the bag sealed so that the erythrocyte suspension occupied no more than 75% of the internal volume. Proper mixing during dialysis is important for optimal encapsulation, expecially when high hematocrits are used. The dialysis bag is immersed in a hypotonic solution (50x erythrocyte volume) which is stirred continously. A schematic representation of apparatus for entrapping proteins in erythrocytes is shown in Figure 6. The substance to be encapsulated is added before dialysis only if the molecular weight is greater than the cut-off of the dialysis tube. Otherwise, if the substance is rapidly dialysable, it should be added to the external dialysing buffer, if large amounts of it are available, or after the dialysis step, incubating dialysed RBC with the substance directly in a tube. In the latter condition, the maximum concentration to be loaded may be limited by the need to avoid high osmolarities which interfere with the lysis procedure. The exact composition of the hypotonic dialysis medium seems to be relatively unimportant since a high concentration of hemoglobin provides substantial buffering capacity inside the bag. De Loach and Ihler28 utilized water for lysis; Furusawa et al32 used diluted PBS; and De Loach et al33 used 10 mM phosphate, pH 7.0, with 2 mM glucose and 0.5 mM CaCl2. Lysis time has been shown to be a function of the hematocrit present in the dialysis bag.29 For example, 45 min gives total lysis with a 50% hematocrit, whereas 75 min is required with an 80% hematocrit. Step e: The resealing of erythrocytes can be accomplished by adding sufficient 1.54 M KCl to achieve isotonicity. Reverse dialysis against isosmotic buffer can also be used to restore the osmotic pressure.28,29,34 However, in experiments, where preservation of energy metabolism within the cells is desirable, 4 mM MgCl2, 10 mM glucose, 2 mM adenosine are also included. Resealed erythrocytes are then allowed to sit at 37°C for 30-60 min (to allow cellular reannealing). Combining low temperatures (4°C) for lysis and high temperature (37°C) for resealing and sufficient energy supply represents the optimal method for loaded erythrocyte preparation including high entrapment, high cell recovery and the highest percentage of biconcave discocytes. In fact the shape of the erythrocyte is the result of divergent forces, and temperature during resealing was found to be an important factor. The theoretical maximum encapsulation percentage is limited to the packed volume of RBC, i.e., the hematocrit and by the loss of RBC in the dialysis process. The loss of RBC is due to intrinsic properties of the different RBC and

8

Erythrocyte Engineering for Drug Delivery and Targeting

Figure 5. Preswell dilutional method.

100% recovery is never achieved. Typically, cell recovery is 50 to 90%.33,35,36 Thus, the maximum encapsulation percentage for RBC at 70% Ht varies from 35 to 63%. Percent of encapsulation is calculated as follows: (amount encapsulated/amount added to RBC) x 100 The opened/resealed erythrocytes had almost normal hematological parameters37 with the exception of a reduced cellular volume and are slightly hypocromic. The choice of the dilutional method or the preswell dilutional method or the dialysis method depends on the characteristics of the agent to be loaded and the use of loaded erythrocytes. Usually, the dilutional method is suitable for encapsulation of low molecular weight (< 130,000 Da) substances and it is fast and simple. However, due to the large extracellular volume compared to the small intracellular volume, a large amount of starting material is needed and a low percentage of encapsulation is obtained. Moreover, this procedure causes a substantial loss of intracellular content, such as erythrocyte enzymes and hemoglobin. The preswell dilutional method is simple and quick too and produces erythrocytes with a good in vivo survival. However, a low percentage of encapsulation is reported. On the other hand, the dialysis method, particularly when a high hematocrit is used, results in highest percentage of encapsulation. Furthermore, by this method loaded erythrocytes have a very good in vivo survival perhaps because the gradual decrease in ionic strength (due to the fact that the RBC suspension is confined in a dialysis bag) better maintains the structural integrity of the membrane. Generally, loading by the dialysis method is very simple to perform, but some peculiarities must be kept in mind:

Red Blood Cell Loading

9

Figure 6. Schematic representation of apparatus for entrapping proteins into erythrocytes. The dialysis bag is tied tight to produce as rigid a bag as possible and contains an air bubble representing 20% of the volume. If the dialysis bag is small relative to the bottle, it may be anchored to a glass rod as shown to ensure rotation of the bag while the bottle turns. The rotating platform is submerged to its axle in an ice-water bath to ensure that the lysis buffer in the bottle remains at 0°C. Reprinted with permission from: Dale GL. In: Methods in Enzymology, Drug and Enzyme Targeting, 149 (part B):229-234. ∀1987 Academic Press, Inc.

1. 2.

It is important to work with a high hematocrit level (70%) during the dialysis step. To evaluate the osmolarity of dialysis buffer (it has to be about 60-70 mOsm); it must be kept in mind that a correct RBC osmolarity (~120 mOsm) after the dialysis step is crucial to obtain good entrapment and good recovery. 3. To evaluate the time of dialysis and modify it, or the mOsm of dialysis buffer, or both, to optimize the results; it must be remembered that a longer time of dialysis or a decrease in osmolarity of dialysis buffer leads to a decrease in RBC recovery. 4. The loaded RBC must be washed in physiological saline solution at 500 g instead of 1900 g because their fragility. 5. “Unloaded” erythrocytes should be used as control because addition of the resealing solution causes some metabolic perturbations. Usually, steady-state metabolite concentrations are better measured after the first hour of incubation. In addition, now we’ll describe a procedure based on the dialysis method developed in our laboratory to operate with few erythrocytes (1 ml) for research use. Here, we report the example of encapsulation of human hexokinase (HK) in erythrocytes1 but the same procedure can be used to encapsulate whatever substance with a molecular weight greater than the cut-off of the dialysis tube. This procedure involves three sequential steps, i.e., hypotonic hemolysis,

10

Erythrocyte Engineering for Drug Delivery and Targeting

isotonic resealing and reannealing of erythrocytes. Briefly, blood (2 ml) was collected in heparin immediately before use and centrifuged to separate the plasma, which was then maintaned at 0°C until use. Erythrocytes are washed twice in 5 mM sodium phosphate buffer (pH 7.4), containing 0.9% (w/v) NaCl and 5 mM glucose and finally resuspended in the same buffer containing hexokinase (10 IU/ml packed erythrocytes) at a hematocrit of 70% in a dialysis tube (Spectrapor, U.S.A. molecular size cut off, 12-14 kDa). Hypotonic lysis of erythrocytes is obtained by dialysis of 1 ml of cell suspension in a 50 ml tube containing 10 mM sodium phosphate/10 mM sodium bicarbonate/20 mM glucose (pH 7.4) and rotated at 15 rpm for 1 h at 4°C. The hemolysate is then collected and 1 vol of resealing solution (5 mM adenine/100 mM inosine/100 mM sodium pyruvate/100 mM sodium phosphate/100 mM glucose/12% (w/v) NaCl pH 7.4, named PIGPA.C) is added to every 10 vols of hemolysate. Reannealing of the cells is then performed at 37°C by incubation of cell suspension for 30 min. Three additional washes of lysed and resealed erythrocytes are performed at 4°C with physiological saline solution. Finally, erythrocytes are resuspended in their native plasma or in physiological saline solution and utilized for metabolic studies. In Table 1.1 the main features of HK-loaded erythrocytes are shown.

Development of Apparatus For Clinical Applications of Loaded Erythrocytes Up to now, in human therapy, only a limited series of experiments have been reported for clinical use of lysed and resealed RBC. Franco et al20 developed a method based on osmotic pulse in order to incorporate inositol hexaphosphate (IHP) into erythrocytes thus preparing very low affinity cells for use in respiratory disease. This method, shown in Figure 7, consists in: the washed and packed red blood cells are mixed with IHP/DMSO solution to give an hematocrit of approximately 50% and the desired DMSO concentration (5-8%). The cell suspension is drawn into a 20 cc syringe which is driven by a syringe pump. The diluent solution (IHP/polyethylene glycol, PEG, this last to improve hemoglobin recovery) is placed in two syringes which are driven simultaneously by a two-channel syringe pump. The cell suspension and the diluent are mixed in a disposable plastic three-way valve, flowed through a short piece of tubing, and collected in a receiving vessel. The parts of this apparatus which contact the solution may be easily assembled from sterile and disposable syringes. After a short time at room temperature (5-15 min), the cells are washed in succession with 37°C and roomtemperature washing buffers and then with autologous plasma. Finally, the cells are suspended in autologous plasma for the measurement of the oxygen dissociation curve. The DMSO incorporation procedure significantly alters the red cell indices, resulting in macrocytic cells with moderately reduced hemoglobin content. (When a 6% DMSO concentration is used, there is a 15% decrease in the mean cellular hemoglobin and a 14% increase in the mean cellular volume, which cause a sharp decrease in the mean cellular hemoglobin concentration). Battle et al38 have successfully treated lead poisoning through the administration of #-amino levulinate dehydratase loaded erythrocyte ghosts. Green et al39 improved the delivery of desferroxamine to the RES (reticuloendothelial system) of patients with iron overload. However, in both these experiments, the encapsulation of enzyme or drug was performed through reversed hypotonic lysis of the RBC by dilution in a large volume of hypotonic buffer; a procedure which led to cells depleted of an important part of the intracellular content. Such resealed “ghosts” are of reduced viability in the blood stream and their in vivo life span is shortened to a few days. Furthermore, the final yield of encapsulation of the drug is relatively low. It is likely that the present status of the methodology for encapsulation of drugs into RBC is the main limitation for the use of this very promising therapeutic approach for the delivery of drugs into human patients. De Loach et al33 have proposed an erythrocyte encapsulator dialyzer, based on a dialysis bag device, allowing control of dialyzing conditions such as temperature, homogenous mixing of erythrocytes, osmolarity of the content of the dialysis tubing, for large scale preparation of

Red Blood Cell Loading

11

Table 1. Encapsulation of human hexokinase in human erythrocytes Hexokinase activity of unloaded erythrocytes (IU/ml RBC) Hexokinase activity of hexokinaseloaded erythrocytes (IU/ml RBC) % entrapment % cell recovery Mean cell volume (fl) Mean cell hemoglobin (g/dl)

0.33±0.1 4.77±0.75 19.7±6.2 77.9±8.1 73.3±1.15 27.58±2

Values are mean±S.D. and ranges are obtained in ten different experiments.

lysed and resealed RBC. Such technical developments are of major interest but not yet sufficient to allow the use of this process to produce lysed and resealed RBC for human therapy, taking into account the classical criteria of blood transfusion, i.e., pyrogen free products, sterility, hemocompatibility of plastic compounds or disposable devices. Ropars et al have developed a continous dialysis system (Fig. 8) that allows the processing of a blood unit (450 ml of blood). This apparatus is designed to maintain pyrogen-free and sterile products, as well as hemocompatibility of plastic compounds or disposable devices.40,41 Here we report the general guidelines of the operation; it is elsewhere explained in detail by Ropars et al.42 Briefly, a suspension of washed RBC is introduced continuosly at 4°C through the use of a peristaltic pump in the blood compartment of a dialyzer. During the lysis step, there is rapid exchange between the cytoplasmic compartment of the cells and the external medium through the metastable pores occurring in the membrane, which allows the internalization of a drug present in the RBC suspending medium. The cells are then resealed by increasing temperature and ionic strength. The substance to be encapsulated may be introduced (a) before the arrival of the blood into the dialyzer, (b) inside the dialyzer by separating a swelling compartment and a lysing compartment, or (c) at the output of the dialyzer before collecting the lysed cells for resealing. Mode (a) gives better results than mode (c) for a macromolecule as there is no loss of the substance through the dialyzer and the diffusibility seems to be greatest at the lysis step, when the cells are at their point of disruption. However, a better overall encapsulation yield is obtained following mode (c) as for such a small molecule the pore size will be sufficient to allow a good final equilibrium between the inside and outside of the cells. In our laboratory a new procedure for the encapsulation of non-diffusible drugs into human erythrocytes, starting from as little as 50 ml of autologous blood, was developed. Here, we report briefly the description of this new procedure and of a new apparatus based on two sequential hypotonic dilutions followed by concentration with a hemofilter. This new hypotonic hemolysis method will now be denoted as Preswelled Dilutional with Concentration. The process is easy to perform, is reproducible and can be completed in 2 h, as described in detail elsewhere.43 The method we have developed is based on the following steps: 1. Human erythrocytes are suspended in a hypotonic solution of approx. 180 mOsm/ kg that allows an increase in cell volume with a fixed surface area. The erythrocytes become spheres but are not yet lysed. 2. The preswollen erythrocytes are then suspended in a hypotonic solution (approx. 120 mOsm/kg) where they increase in volume and the pores in the membrane are opened. At this stage the erythrocytes are lysed. 3. The red cell lysate is concentrated to its original volume by pumping the lysed cells through a hemofilter with a cut-off of 30 kDa.

Erythrocyte Engineering for Drug Delivery and Targeting

12

Figure 7. Scheme of the continous-flow method. Reprinted with permission from: Franco RS. American Journal of Hematology 1984, 17:393-400 ∀1984 Wiley-Liss, Inc. a subsidiary of John Wiley & Sons, Inc.

4.

The substance to be encapsulated is introduced into the lysed cell compartment in the form of a solution with an osmolarity close to that of the lysed cells (100-140 mOsm/kg), and the two suspension are maintained in contact for approx. 20-30 min at room temperature to permit equilibrium to be reach. 5. The lysed cells are resealed by adding 5 ml of a solution containig at least 1.6 M KCl and 0.194 M NaCl per 70-80 ml of lysed cells. The resealing process is completed by warming the red cell suspension at 37°C for 30 min. 6. The resealed erythrocytes containing the substance encapsulated are then washed with physiological saline solution and finally collected. The procedure is shown schematically in Figure 9. With the aim of using the procedure described above in human patients, we designed and built a new apparatus that maintains all the products pyrogen-free and sterile and guarantees the hemocompatibility of all plastic parts and disposable devices. The apparatus is shown in Figure 10. This apparatus requires the development of specifically dedicated equipment derived from standard blood-processing machines (Dideco CompactA). Together, the apparatus and the machine, are named “Red Cell Loader”. The sequence of the operations performed by this apparatus is as follows: 1. Approx. 50-60 ml of blood drawn from a donor, collected in a plastic bag (A) containing an anticoagulant solution (ACD, EDTA or heparin), are transferred by the pump into a rotating bowl. The erythrocytes in the bowl are washed with a physiological saline solution to remove plasma, platelets and white cell buffy coat. Then, the washed erythrocytes are pumped and collected into a new plastic bag (B).

Red Blood Cell Loading

13

Figure 8. Schematic diagram of operations for the entrapment of a drug into lysed and resealed erythrocytes. Reprinted with permission from: Ropars C, Avenard G, Chassaigne M. In: Methods in Enzymology, Drug and Enzyme Targeting, 149 (part B):242-248. ∀1987 Academic Press, Inc.

2.

The first hypotonic solution is then transferred into the erythrocyte bag (B) and the suspension is maintained at room temperature for 10 min. The swollen erythrocytes (not yet lysed) are separated from the first hypotonic solution by centrifugation and suspended in the second hypotonic solution. At this stage the erythrocytes are lysed in 15 min at room temperature. 3. The lysed eythrocytes are concentrated in the hemofilter, which is connected to a plastic reservoir maintained at a reduced pressure by a vacuum pump. 4. The lysed and concentrated erythrocytes are finally collected in a bag (B) where the substance to be encapsulated is introduced. The suspension is gently agitated over a 15 min period. Resealing solution is then added and the bag is transferred into a heating system where it is warmed at 37°C for 30 min. 5. The resealed erythrocytes are then pumped into the bowl and washed with 2 Litres of physiological saline. The loaded erythrocytes are finally collected in a disposable plastic bag (C), ready to be infused into the original door or a blood-compatible patient. In order to evaluate the efficiency of the drug-loaded procedure, three different molecules (dexamethasone 21-phosphate, prednisolone 21-phosphate and 125I-Ubiquitin) were encapsulated into human erythrocytes. The results obtained are shown in Table 2. A cell recovery of 3550 % was obtained. Prompted by this new apparatus, we have evaluated the potential use of autologous erythrocytes loaded with the corticosteroid analog dexamethasone 21-phosphate (Dex 21-P) as a slow delivery system for dexamethasone in patients with chronic obstructive pulmonary disease.44

Conclusions The possibility of engineering erythrocytes by encapsulation of macromolecules (i.e., drugs) and the capacity of loaded erythrocytes to act as a drug delivery system, represent a fundamental advancement in the way blood is currently used. Different procedures for the encapsulation

Erythrocyte Engineering for Drug Delivery and Targeting

14

erythrocyte

preswelled erythrocytes

lysed erythrocytes

concentrate red cell lysate

addition of the substance

resealed erythrocytes containing the substance encapsulated hemofilter

Figure 9. Diagram of the procedure used for the encapsulation of substances in human erythrocytes. Reprinted with permission from: Magnani M et al. Biotechnol Appl Biochem 1998; 28:1-6. ∀ 1998 Portland Press Limited.

of drugs in erythrocytes are known. The majority of these are based on the transient opening across the red cell membrane of pores large enough to be traversed by externally placed macromolecules. In this Chapter, four procedures have been selected, the main features of which are summarized in Table 3. In our opinion, the main technique that allows one to prepare loaded RBC for research use, that requires a small amount of erythrocytes, is the dialysis method, especially when a high hematocrit is used. However, if engineered erythrocytes must be used for clinical applications, we propose our new procedure for the encapsulation of non-diffusuble drugs in RBC based on the hypotonic hemolysis method, also called the preswell dilutional with concentration method.

References 1. Magnani M, Rossi L, Bianchi M et al. Improved metabolic properties of hexokinase-overload human erythrocytes. Biochimica et Biophysica Acta 1988;972:1-8. 2. Magnani M., Rossi L, Bianchi M et al. Human red blood cell loading with hexokinase-inactivating antibobies. Acta Hematol 1989; 82:27-34. 3. Magnani M, Rossi L, Bianchi M et al. Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes. Biotechnol Appl Biochem 1989; 11:439-444. 4. Colowick SP, Kaplan NO. Section III. Cellular Carrier. In: Green R, Widder KJ, eds. Methods in Enzymology. San Diego: Academic Press, 1987:217-325. 5. Tsong TY. Electric modification of membrane permeability for drug loading into living cells. In: Green R, Widder KJ, eds. Methods in Enzymology. San Diego: Academic Press, 1987:248-259.

Red Blood Cell Loading

15

Figure 10. Diagram of the apparatus used for the encapsulation of drugs in human erythrocytes. Reprinted with permission from: Magnani M et al. Biotechnol Appl Biochem 1998; 28:1-6. ∀ 1998 Portland Press Limited. 6. Kinosita K Jr, Tsong TT. Hemolysis of human erythrocytes by transient electric field. Proc Natl Acad Sci USA 1977; 74(5):1923-1927. 7. Kinosita K Jr, Tsong TY. Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature (London) 1977; 268(5619):438-441. 8. Kinosita K Jr., Tsong TY. Survival of sucrose-loaded erythrocytes in the circulation. Nature (London) 1978; 272(5650):258-260. 9. Kinosita K Jr., Tsong TY. Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim Biophys Acta 1977; 471(2):227-242. 10. Teissie J, Tsong TY. Evidence of voltage-induced channel opening in Na/K ATPase of human erythrocyte membrane. J Membr Biol 1980; 55(2):133-140. 11. Schekman R, Singer SJ. Clustering and endocytosis of membrane receptors can be induced in mature erythrocytes of neonatal but not adult humans. Proc Natl Acad Sci USA 1976; 73(11):4075-4079.

Erythrocyte Engineering for Drug Delivery and Targeting

16

Table 2. Efficiency of the drug-loading procedure Encapsulation (%) Dexamethasone 21-phosphate

Prednisolone 21-phosphate 30± 3.0

30± 3.5 Values

are

means± S.D.

for

three

125

I-Ubiquitin

30± 2.5

experiments.

12. Ginn FL, Hochstein P, Trump FP. Membrane alterations in hemolysis: Internalization of plasmalemma induced by primaquine. Science 1969; 164(881):843-845. 13. Ben-Bassat I, Bensch KG, Schrier SL. Drug-induced erythrocyte membrane internalization. J Clin Invest 1972; 51(7):1833-1844. 14. Schrier SL., Junga I, Krueger J et al. Requirements of drug-induced endocytosis by intact human erythrocytes. Blood Cells 1978; 4(1-2):339-359. 15. Greenwalt TJ, Lau FO, Swierk EM et al. Studies of erythrocyte membrane loss produced by amphipathic drugs and in vitro storage. Br J Haematol 1978; 39(4):551-557. 16. Murphy MJ Jr. Effects of vitamin A on the erythrocyte membrane surface. Blood 1973; 41(6):893-899. 17. Seeman P. II. Erythrocyte membrane stabilization by steroids and alcohols; a possible model for anesthesia. Biochem Pharmacol 1966; 15(10):1632-1637. 18. Schrier SL. Drug-induced endocytosis and entrapment in red cells and ghosts. Methods Enzymol 1987; 149:260-70. 19. Ihler MG. Entrapment of DNA and fluorescent compounds in erythrocyte carriers by endocytosis. Bibl Haematol 1985; 51:127-133. 20. Franco RS, Wagner K, Weiner M et al. Preparation of low-affinity red cells with dimethylsulfoxide-mediated inositol hexaphosphate incorporation: hemoglobin and ATP recovery using a continousflow method. Am J Hematol 1984; 17:393-400. 21. Franco RS, Baker R, Novick S et al. Effect of inositol hexaphosphate on the transient behaviour of red cells following a DMSO-induced osmotic pulse. J Cell Physiol 1986; 129:221-229. 22. Small WC, Goldstein JH. The effect of the cryoprotectants, dimethylsulfoxide and glycerol on water transport in the human red blood cell. Biochim Biophys Acta. 1982; 720(1):81-86. 23. Franco RS, Weiner M, Wagner K et al. Incorporation of inositol hexaphosphate into red blood cells mediated by dimethyl sulfoxide. Life Sciences 1983; 32(24):2763-2768. 24. Franco RS, Barker R, Mayfield G et al. The in vivo survival of human red cells with low oxygen affinity prepared by the osmotic pulse method of inositol hexaphosphate incorporation. Transfusion 1990; 30(3):196-200. 25. Mosca A, Paleari R, Russo V et al. IHP entrapment into human erythrocytes: Comparison between hypotonic dialysis and DMSO pulse. In: Magnani M, DeLoach JR, eds. The Use of Resealed Erythrocytes as Carriers and Bioreactors. New York: Plenum Press. New York, 1992:19-26. 26. Ihler GM, Glew R, Schnure F. Enzyme loading of erythrocytes. Proc Natl Acad Sci USA 1973; 70(9):2663-2666. 27. Rechsteiner M. Uptake of proteins by red blood cells. Exp Cell Res 1975; 93(2):487-492. 28. De Loach J, Ihler G. A dialysis procedure for loading erythrocytes with enzymes and lipids. Biochim Biophys Acta 1977; 496(1):136-145. 29. Dale GL, Villacorte DG, Beutler E. High-yield entrapment of proteins into erythrocytes. Biochem Med 1977; 18(2):220-225. 30. Baker RF, Gillis NR. Osmotic hemolysis of chemically modified red blood cells. Blood 1969; 33(2):170-178. 31. Baker RF. Entry of ferritin into human red cells during hypotonic haemolysis. Nature (London) 1967; 215(99):424-425. 32. Furusawa M, Yamaizumi M, Nishimura T et al. Use of erythrocyte ghosts for injection of substances into animal cells by cell fusion. Methods Cell Biol 1976; 14:73-80. 33. De Loach JR, Harris RL, Ihler GM. An erythrocyte encapsulator dialyzer used in preparing large quantities of erythrocyte ghosts and encapsulation of a pesticide in erythrocyte ghosts. Anal Biochem 1980; 102(1):220-227.

Red Blood Cell Loading

17

Table 3. Procedures for the encapsulation of agents in erythrocytes Methods

Comments

Electroporation

Suitable for low amount of cells Best suited for low-Mr substances Inducible only by certain drugs

Drug Induced Endocytosis Osmotic Pulse DMSO

Continous-flow Hypotonic Hemolysis Dilutional

Suitable for low amount of cell Require long time Suitable for large amount of cell Require long time Simple and fast Suitable for low-Mr substances Loss of erythrocyte content Low percentage encapsulation

Preswell Dilutional

Simple and fast Good in vivo survival Low percentage encapsulation

Dialysis

Simple High percentage encapsulation Good in vivo survival Good cell recovery Large scale procedures available

Preswell Dilutional with Concentration

Require short time (2 hours) Suitable for clinical use starting from 50 ml blood Good in vivo survival Good cell recovery Good percentage encapsulation Easy to perform by the “Red Cell Loader” machine

34. Sprandel U, Hubbard AR, Chalmers RA. In vitro studies on resealed erythrocyte ghosts as protein carriers. Res Exp Med 1979; 175(3):239-245. 35. De Loach JR, Barton C, Culler K. Preparation of resealed carrier erythrocytes and in vivo survival in dogs. Am J Vet Res 1981; 42(4):667-669. 36. De Loach JR. In vivo survival of [14C]sucrose-loaded porcine carrier erythrocyte. Am J Vet Res 1983; 44(6):1159-1161. 37. Ropars C, Chassaigne M, Villereal MC et al. In: DeLoach JR, Sprandel U, eds. Red Blood Cells as Carriers for Drugs. Basel: Karger, 1985:82-91. 38. Battle AM, Bustons NL, Stella AM et al. Enzyme replacement therapy in porphyrias—IV. First successful human clinical trial of delta-aminolevulinate dehydratase-loaded erythrocyte ghosts. Int J Biochem 1983; 15:1261-1265. 39. Green R, Lamon J, Curran G. Clinical trial of desferrioxamine entrapped in red cell ghosts. Lancet 1980; 2(8190):327-330. 40. Ropars C, Chassaigne M, Villereal MC et al. Resealed red blood cells as a new blood transfusion product. Bibl Haematol (Basel) 1985; 51:82-91.

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Erythrocyte Engineering for Drug Delivery and Targeting

41. Ropars C, Nicolau C, Chassaigne M. French Patent 8,211,749 (1982); Eur Patent 83,401,364-1 (1983). 42. Ropars C, Avenard G, Chassaigne M. Large-scale entrapment of drugs into resealed red blood cells using a continous-flow dialysis system. In: Green R, Widder KJ, eds. Methods in Enzymology. Vol 149, Drug and Enzyme Targeting, Part B. San Diego: Academic Press, Inc., 1987:242-248. 43. Magnani M, Rossi L, D’Ascenzo M. Erythrocyte engineering for drug delivery and targeting. Biotechnol Appl Biochem 1998; 28:1-6. 44. Rossi L, Serafini S, Cenerini L et al. Erythrocyte-mediated delivery of dexamethasone in patients with chronic obstructive pulmonary disease. Biotechnol Appl Biochem 2001; 33:85-89.

CHAPTER 2

Towards Activated Release of Payloads from Carrier Erythrocytes A.M. Rollan and A.P. McHale

A

lthough the potential of erythrocytes as drug delivery vehicles has been suggested for some time, the applicability of this approach has been limited by the lack of a mechanism to achieve active release of payloads from the system. The scope of this chapter will be to review developments, potential applications and challenges associated with activated release of therapeutic agents from erythrocytes using external stimuli.

Introduction By virtue of its natural functions and access to every part of the vasculature, the erythrocyte represents a versatile and convenient means of delivering active agents to almost all regions of the body. Its application range in this context is limited only by the ability to incorporate the relevant agent or payload for any given application. The range of agents which have, thus far, been incorporated into or associated with erythrocytes is vast and includes agents such as therapeutic proteins including enzymes and vaccines,1 nucleic acids,1,2 oligosaccharides,3 cancer chemotherapeutics,4 chemical markers5 and other active agents such as antiviral drugs and metabolic modulators.6 The suggested therapeutic application therefore relates to the agent loaded and the means by which this active agent becomes available to perform its intended function. Based on the range of active agents and payloads that have been incorporated into or associated with erythrocytes, therapeutic application has been suggested in treating cancer,4 circulatory disease,6 metabolic and immunological disorders7 including AIDS8 and in detoxification treatment modalities.9

Current Exploitation of Erythrocytes in Delivery and Targeting It is clear that exploiting carrier erythrocytes in drug delivery and targeting necessitates engineering the cell so that functionality of the payload becomes available to effect therapy. Until relatively recently the suggested applications of the erythrocyte in drug delivery and targeting has been based on: (i) Retention of an active payload inside the erythrocyte carrier and use as a circulating bioreactor.7,10,11 (ii) Slow release of the payload from the carrier during circulation.4 (iii) Fortuitous targeting of engineered erythrocytes to either the liver or spleen.12 (iv) Magnetic targeting of erythrocyte carriers.13 In terms of (i) above the concept involves employing the erythrocyte as a circulating bioreactor and has normally involved the entrapment of enzymatic activities. Candidate enzyme payloads that have received considerable attention include adenosine deaminase in the treatment of severe combined immunodeficiency,7 alcohol and acetaldehyde dehydrogenases for alcohol detoxicification11 and glutamate dehydrogenase for modulation of ammonia levels.10 In most cases the circulating enzyme-containing erythrocyte serves as a bioreactor which sequesters the Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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Erythrocyte Engineering for Drug Delivery and Targeting

substrate and expels the catalytic product. It is therefore evident that the function of the erythrocyte in this case may be deemed passive in terms of delivering enzymatic function and such applications are heavily dependent on the ability of substrate to enter into the erythrocyte and for product to exit from the cell. Although some exciting reports of using such an approach in human subjects have emerged,7 the general applicability of this approach has been limited by a lack of external control on the system. In terms of (ii) above, the erythrocyte carrier is exploited in sustained delivery of a drug. This form of exploitation is dependent on the observation that erythrocyte carriers appear to be limited with respect to retention of low molecular weight molecules such as chemotherapeutic drugs, particularly those that exhibit a relatively high degree of hydrophobicity.1 Again, functionality of the carrier in terms of drug delivery is a passive one and control thereof is limited by the rate at which the payload is released from the carrier. In engineering erythrocytes as carriers for delivery and targeting of active agents and drugs it has been found that even slight perturbation of the erythrocyte membrane signals efficient uptake and processing by the reticuloendothelial system.1,8,12, This leads to sequestration of damaged or modified erythrocytes by the liver and the spleen and it has been suggested that this phenomenon might be exploited in targeting active agents to those organs. Indeed to date, this phenomenon forms the sole basis for targeting of erythrocyte carriers to the reticuloendothelial system. Suggested strategies include modifying the surface of the erythrocyte carrier with glutaraldehyde such that preferential recognition by either the spleen or the liver might be achieved.12 In this case it is believed that reduced elasticity of the glutaraldehyde-engineered carrier during passage through either the spleen or liver promotes sequestration in those organs.12 This results in an elevated deposition of the relevant payload in those organs thereby accomplishing a degree of targeting. It should however be noted that sequestration and uptake of the erythrocyte carrier by erythrophagocytic cells does not necessarily imply that the payload will become available for therapeutic purposes. In a similar approach, it has been found that the same objective may be accomplished by modifying the surface of the carrier with specific cross-linking agents such as bis(sulfosuccinimidyl) suberate and 3,3’dithiobis(sulfosuccinimidylpropionate).14 In the latter case the authors again demonstrated preferential uptake of carrier erythrocytes by the liver and to a lesser degree, by the spleen. It has also been demonstrated that drug-loaded erythrocytes may be targeted to macrophages with a view towards protecting the cells against human immunodeficiency virus (HIV).8 In this case the erythrocytes were loaded with azidothymidine homodinucleotide. Cells were then treated with Zn which results in clustering of band 3 and the clustering was maintained by cross-linking with bis(sulphosuccinimidyl) suberate. The immune system recognised these clusters as ‘non-self ’ and this facilitated recognition by macrophages. Such recognition resulted in delivery of the drug to the macrophages at pharmacologically relevant doses. Although the above approaches are elegant in both design and strategy, their targeting and delivery capabilities result from somewhat fortuitous events and controlled, pre-meditated targeting to other sites would be precluded. Another approach, (iv above) which facilitates a somewhat higher degree of control in terms of targeting, involves the use of external magnetic fields in order to direct carrier erythrocytes to a specific site in the body. This strategy, which involves loading of the carrier erythrocytes with magnetically-responsive particles together with the therapeutic agents, depends on localising the vehicle to a particular target site using external magnetic fields.13 However such an approach does not necessarily imply bioavailability of the payload at the target site since a means of facilitating release of the relevant payload from the vehicle is not incorporated into the system.

The Case For an Activatable Payload Release Mechanism From Carrier Erythrocytes In all of the above mentioned examples employing the erythrocyte as a delivery vehicle, it will be apparent that it is possible to deliver active agents either on the basis of passive systemic

Towards Activated Release of Payloads From Carrier Erythrocytes

21

administration (slow release modalities) or on a targeting basis to selected organs such as the liver and the spleen. However many of these suggested delivery modalities are restricted in terms of targeting to other areas in the body and by a lack of control on release of the active agent or payload at the target site once the vehicle has been delivered. A perusal of the available literature in the context and perspective as that outlined above indicates a need for mechanisms which would therefore (i) accomplish delivery of the erythrocyte vehicle to any pre-defined target area of the body and (ii) facilitate controlled release of the payload from the carrier system at any pre-defined time within the chosen target area. Let us consider the benefits in being able to control release of the active agent or payload. Essentially, there are at least two major areas where this would provide benefit and they include (i) an increased level of control where slow release modalities would provide therapeutic benefit and (ii) providing a means of achieving site-directed deposition or targeting of payload. In realising both of the above benefits it would be necessary to engineer the erythrocyte in such a way that it would release therapeutic agent in response to the application of an external stimulus. Consider the benefits in terms of an increased level of control where slow release would provide therapeutic benefit. Therapeutic benefit would be realised in cases where the naked active entity exhibited a very short half-life in circulation. In this case, when the concentration of drug decreased to non-therapeutic levels in circulation, the external stimulus could be applied to a circulating reservoir of erythrocyte-based carrier containing the therapeutic agent, thereby stimulating release of the therapeutic agent. Such a system would provide for a noninvasive means of maintaining therapeutic levels of the drug in circulation and this would, in turn, provide significant potential benefit in terms of therapeutic regime and cost. How might an activatable erythrocyte carrier provide benefit in terms of targeting payload to a specific site? This is more clearly understood by envisaging the erythrocyte carrier system passing through the microvasculature of a target site. If the target site is subjected to the activating stimulus, then the response of the carrier to the stimulus would result in release of the active payload at that target site. This would, in effect, result in localised elevated concentrations of the active agent and all of the benefits associated with localised delivery would apply. In light of these potential benefits our group began to develop a system which might provide a means of actively releasing payload from a circulating erythrocyte carrier system using an appropriate form of external stimulus.

Photodynamic Activation and Activated Payload Release From a Light-Sensitive Erythrocyte Carrier System Photodynamic activation (PDA) is a term used to describe events that occur following exposure of certain types of chemicals to light. When these compounds are exposed to light at specific wavelengths, events occur which eventually lead to the generation of cytotoxic free radicals and the latter subsequently compromise cell viability.15 This phenomenon has been exploited in the clinic and has evolved into a cancer treatment modality known as photodynamic therapy or PDT. In essence, tumour cells take up the photosensitising chemicals and when the target site is irradiated with low intensity laser light, the generation of free radicals at the tumour site leads to cell death. The cytotoxic effects include compromise of mitochondrial and cell membrane function.15 Peroxidation of membrane lipids results in damage to that membrane which, in turn, becomes highly permeable.16 In terms of activated release of payloads from erythrocyte carriers, a means of compromising the permeability of the membrane would facilitate release of any payload. This suggested that it might be possible to sensitise the erythrocyte membrane with relatively hydrophobic photosensitising agents. Subsequent exposure of the sensitised cells to light would then facilitate compromise of membrane function. If the sensitised cells were loaded with an agent, then release of that agent could be accomplished by exposure to light. In order to test this hypothesis we decided to examine light-mediated release of methotrexate from photosensitised human erythrocytes.17 In these experiments cells were loaded with

22

Erythrocyte Engineering for Drug Delivery and Targeting

Figure 1. Release of methotrexate from photosensitised, methotrexate-loaded erythrocytes following exposure to light (
). Controls consisted of the photosensitised, loaded cells protected from light (). Following exposure of cells to irradiation, samples were centrifuged and the amount of radioactivity in supernatants was determined. Standard deviation from the mean was less that 4%.17 Reprinted from reference 17 with permission from Elsevier Science.

methotrexate (and 3H-labelled methotrexate as a tracer) using electroporation and the loaded erythrocytes were then sensitised with the photosensitiser, hematoporphyrin derivative (HPD). The latter is one of the most commonly used photosensitisers in photodynamic therapy.15,16,17 These loaded and sensitised erythrocytes were then exposed to light from a low power HeNe laser and this resulted in light-stimulated release of methotrexate from the cells (Fig. 1). Control populations of cells, which had been protected from light, failed to release significant quantities of methotrexate. In these studies it was also demonstrated that the released methotrexate retained its cytotoxic activity as demonstrated by treating target populations of cells with lysates from light-treated photosensitive carrier erythrocytes17. Since the activated release mechanism in this case was mediated by free radicals, the latter point was considered important since it demonstrated that the release mechanism did not contribute to payload destruction. In a similar study we subsequently proceeded to incorporate a thrombolytic enzyme, brinase, into erythroyctes. These were then sensitised with HPD. In studies similar to those described above we were again able to demonstrate light-mediated release of the functional enzyme (Fig. 2).18 In that study it was suggested that this system could play a role in the treatment of deep site thrombosis. The hypothesis was based on the presumption that circulating sensitised and brinase-loaded erythrocytes would be incorporated into a forming thrombus. If that thrombus was then exposed to light, the brinase would be released and dissolution of the thrombus would occur. Subsequent in vitro studies demonstrated that exposure of clotted blood, into which brinase-loaded and sensitised erythrocytes had been incorporated, resulted in rapid visual lysis. In more quantitiative terms, this was also assessed by incorporating 125I-labelled fibrinogen into clots together with the brinase-loaded, sensitised erythrocyte carrier..18 Following exposure of the system to light, fibrin hydrolysis was monitored by measuring the amount of soluble 125I-labelled hydrolysis products released from the clot and the results in Figure 3

Towards Activated Release of Payloads From Carrier Erythrocytes

23

Figure 2. Photoactivated release of brinase following irradiation of brinase-loaded, photosensitised erythrocytes. Aliquots (0.2ml) of loaded and photosensitised cells were exposed to 4 min. of irradiation (
) after which brinase release was measured. Control samples () consisted of the loaded, photosensitised system protected from light. (The standard deviation was less than 5% of the mean values.)18 Reprinted from reference 18 with permission from Elsevier Science.

demonstrated that the brinase was highly active following light stimulated release.18 Little or no 125I was released in the absence of light or following treatment of clots which contained loaded, but non-sensitised cells (Fig. 3). These results again demonstrated that functional payload could be released from photosensitised erythrocytes following exposure to light. The above observations with light-dependent release of both methotrexate and brinase from photosensitised erythrocytes suggest that a system employing such a release mechanism might prove useful in the design of an erythrocyte-based delivery and targeting system. This type of system could be envisaged to effect delivery and localised deposition of payloads to any chosen area in the body that would be accessible to the release stimulus, in this case light. Again if one envisages a capillary bed in the target area illuminated by the stimulus, then photosensitised carrier passing through the stimulus would be lysed by PDA. This would lead to an accumulation of payload at that target site and this, in effect, would amount to targeting of that payload to a pre-defined site. The consequences of employing light as a releasing stimulus will be discussed further below.

Electric Field-Mediated Stimulation of Payload Release From HPD-Sensitised Human Erythrocytes Although light stimulation appears to serve as a realistic activation mechanism for release of therapeutic payloads from erythrocyte carriers, we were interested in determining whether or not alternative methods of activation might exist. Since photodynamic activation is based on photon-stimulated electron excitation and energy emission following relaxation in the quadridentate ring of the porphorynin-based HPD, it was felt that electric fields might further enhance this phenomenon. If this was found to be the case then it would provide an alternative

24

Erythrocyte Engineering for Drug Delivery and Targeting

Figure 3. Light-dependent release of radioactivity from clotted blood in which fibrin was labelled by the addition of [125I]-labelled fibrinogen. Radioactivity released from clots containing the loaded, photosensitised system following exposure to (
) and protection from () light was measured as described previously.18 The release of radioactivity from clots containing the loaded erythrocytes in the absence of photosensitiser but exposed to light was also determined (∃). (The standard deviation was less than 4% of the mean values.)18 Reprinted from reference 18 with permission from Elsevier Science.

means of activating human erythrocyte carriers with a view towards payload release. Such an approach, that is, the application of electric fields in vivo is well established and forms the basis of what has become known as electrochemotherapy or electroporative therapy.19,20 This was originally designed to treat tumours that had become resistant to chemotherapeutic agents and such resistance was characterised by the inability of the tumour cells to take up the relevant chemotherapeutic. In electrochemotherapy, the latter was circumvented by the application of electric pulses in vivo thereby electroporating the drug into the target tumour cells.19,20 The application of electric fields for payload release could involve subjecting a target site in the body to short and intense electric pulses. When the sensitised carrier erythrocytes passed through the vasculature at that site, the erythrocytes would actively release their payload. This approach could also benefit from an electroporative or transient permeability induced at the target site thereby facilitating extravasation of payload from microcapillaries at that target site. In order to test this hypothesis, it was decided to subject HPD-sensitised erythrocytes to short and intense electric pulses ( 750V/cm at 25∝F) and to measure the effect of the electric field on cell lysis.21 It was found that exposure of HPD-sensitised erythrocytes to electric fields had a lytic effect on those sensitised cells but failed to stimulate lysis of normal erythrocytes (Fig. 4).21 This effect on the sensitised cells occurred when cells were suspended in either isotonic solutions or autologous plasma. On the basis of these and other experimental observations,21,22 it was suggested that electric fields may provide an alternative stimulus for activated release of payloads from carrier erythrocytes.

Towards Activated Release of Payloads From Carrier Erythrocytes

25

Figure 4. Lysis of photosensitised erythrocytes following exposure to electric pulses in the presence of Hartman’s Dextrose Solution (HDS) (▲) and plasma (). Cells were photosensitised using HPD (100∝g/ ml) as described previously21 and washed preparations were subsequently exposed to a single electric pulse with an electric field strength of 750V/cm. Following exposure to the electric pulses, samples were taken at the indicated times and release of haemoglobin was measured. Control samples (
) consisted of nonphotosensitised erythrocytes which were exposed to the electric field. The results represent the average values from five experiments and the standard deviation was less than 3%.21 Reprinted from reference 21 with permission from Elsevier Science.

Challenges Associated With Activated Release From Erythrocyte Carriers In the approach taken above to achieve activated release of payloads from carrier erythrocytes, a number of limitations and challenges currently apply and these include: (i) The limited penetration of light to target sites deep within the body. Indeed this is a general limitation associated with the practical use of PDT. In earlier work HPD was activated using light in the region of 630nm and this wavelength was chosen because it activated the photosensitiser and exhibited maximum penetration through living tissues.16 In general terms, the limit of penetration through living tissues is approximately 0.5 cm although higher degrees of penetration may be achieved if the intensity of the stimulating light is increased. However increasing the intensity is limited by adverse thermal effects as the intensity is increased and successful PDT involves identifying an optimal balance between light intensity and adverse thermal effects. When deep penetration is required in conventional PDT in the clinic, this is afforded in a semi-invasive manner by delivering light through optical fibers.16 Therefore in terms of activating circulating erythrocyte carriers in a vessel or capillary bed deep within the body, some means of achieving penetration of the stimulus (light) would have to be identified and perhaps fiber optics could play a role in this regard.

26

Erythrocyte Engineering for Drug Delivery and Targeting

Alternatively, lasers emitting at higher wavelengths might provide greater tissue penetration although it should be mentioned that those wavelengths would have to be compatible with photosensitiser activation. In any case the above discussion indicates the need for further developments in this area and our group has been actively identifying more penetrating forms of stimulation to achieve activated release of therapeutic payloads from carrier erythrocytes. (ii) Rate of activation and localised payload deposition. Another challenge associated with activated release of payloads from circulating carrier erythrocytes relates to payload deposition at a target site. This is especially relevant if localised deposition of payload is required. If an active erythrocyte carrier passes through a capillary bed that is being treated with the stimulus (in this case light), then the time taken for the erythrocyte to release its payload will be directly related to the time taken for the activation event to occur. If the cell has passed through the capillary bed before activation has occurred, then deposition of the payload will not occur at the site of stimulation. Therefore if one is to exploit active release of payloads from circulating carrier erythrocytes, the activation event must be extremely rapid or stimulation would have to be initiated at some point up-stream from the proposed site of action. Indeed, we have started to address this question by identifying sub-populations of erythrocytes that exhibit an enhanced response to photodynamic activation.23 In those studies it was shown that when erythrocytes are resolved into younger and older subpopulations, older fractions appear to be more susceptible to photodynamic action in terms of time to activate. These experimental observations suggest that engineering older populations of erythrocytes as photo-responsive carriers might provide advantage in terms of releasing payload more efficiently. Whether or not such an approach would have negative consequences in terms of prolonged survival in circulation remains to be seen. (iii) Retention of payload at a target site. As with any other drug delivery modality which may be designed to release payload at a target site and employing circulation as a route, retention of the payload at the target site represents a considerable challenge. The obvious problem, in terms of localised drug deposition, relates to flushing the payload from the target site, once it has been released from the carrier. This has been shown not to be a major problem for tumour sites since they exhibit a ‘leaky’ vasculature and have very poor drainage.24 In those circumstances, one might expect the payload to diffuse out of the vasculature and into the target cells. How ever, even in those circumstances it would be reasonable to assume that at least some of the payload would be flushed from the site following release from the carrier system. In terms of photodynamic activation, collateral effects on endothelium might provide advantage with respect to permeation of payload into a target tissue. In this context we have demonstrated that, when immobilised photosensitiser is activated with light in the presence of target cells, viability of those cells proximal to the photosensitiser is compromised.25 This suggests that the cytotoxic effect generated during photodynamic activation diffuses away from the activation site and could further suggest a permeating effect on the vasculature. Indeed, if activation could be ‘fine-tuned’ to effect collateral devascularisation of a tumour site this would lead to added therapeutic benefit. Although a number of challenges have been discussed above, the authors are very aware that others exist which will have to be addressed by workers in this field. Amongst these is the rather overly efficient sequestering capabilities of the reticuloendothelial system and its particular attraction for manipulated or engineered erythrocytes. As many others in the past have discovered, even slight modification of the erythrocyte, will promote removal from circulation and indeed others have elegantly exploited this phenomenon as described above.8 Nevertheless, workers should take solace from the fact that it is possible to engineer human erythrocytes and

Towards Activated Release of Payloads From Carrier Erythrocytes

27

replace those engineered cells back into a patient without significant loss of the vehicle.7 We believe that these observations are amongst the most promising in terms of future development. More recently we have suggested that the erythrocyte, under certain circumstances, may play a delivery role in in vivo gene therapy.2 Such an application would, in many cases, necessitate delivery of the payload to specific pre-defined target regions in the body. The design of activated release mechanisms therefore expands the existing applications repertoire and points to a very bright future for the role of engineered erythrocytes in drug delivery.

References 1. Ihler GM, Tsang HC. Erythrocyte carriers. Crit Rev Ther Drug Carrier Syst 1985; 1:155-187. 2. Rollan Haro AM, McHale AP. Nucleic acid delivery. Patent Application (PCT no. WO/07630). 3. DeLoach JR, Wagner GC. Some effects of the trypanocidal drug isometamidium on encapsulation in bovine carrier erythrocytes. Biotechnol Appl Biochem 1988; 10:447-453. 4. Naqi A, DeLoach JR, Andrews K et al. Determination of parameters for enzyme therapy using L-asparaginase entrapped in canine erythrocytes. Biotechnol Appl Biochem 1988; 10:365-372. 5. Alvarez FJ, Jordan, JA, Herraez, A et al. Hypotonically-loaded rat erythrocytes deliver encapsulated substances into peritoneal macrophages. J Biochem 1998: 123:233-239. 6. Bruggemann U, Roux, EC, Hannig, J et al. Low-oxygen-affinity red cells produced in a largevolume, continuous-flow electroporation system. Transfusion 1995; 35:478-486. 7. Bax BE, Bain, MD, Fairbanks LD et al. In vitro and in vivo studies with human carrier erythrocytes loaded with polyethylen glycol-conjugated and native adenosine deaminase. Brit J Haematol 2000; 109:549-554. 8. Fraternale A, Casabianca A, Rossi L et al. Inhibition of murine AIDS by combination of AZT and DDCTP-loaded erythrocytes. In: Sprandel U, Way J L, eds. Erythrocytes as Drug Carriers in Medicine. New York: Plenum Press, 1997:73-80. 9. Way J L, Pei L, Petrikovics I et al. Organophosphorus antagonism by resealed erythrocytes containing recombinant paraoxonase. In: Sprandel U, Way JL, eds. Erythrocytes as Drug Carriers in Medicine. New York: Plenum Press, 1997:89-92. 10. Sanz S, Lizano C, Garin M I et al. Biochemical properties of alcohol dehydrogenase and glutamate dehydrogenase encapsulated into human erythrocytes by hypotonic-dialysis procedure. In: Sprandel U, Way JL, eds. Erythrocytes as Drug Carriers in Medicine. New York: Plenum Press, 1997:101-108. 11. Lizano, C, Sanz S, Sancho P et al. Encapsulation of alcohol dehydrogenase and acetaldehyde dehydrogenase into human erythrocytes by an electroporation procedure. In: Sprandel U, Way JL, eds. Erythrocytes as Drug Carriers in Medicine. New York: Plenum Press, 1997:129-136. 12. DeLoach JR, Barton C. Glutaraldehyde-treated carrier erythrocytes for organ targeting of methotrexate in dogs. Am J Vet Res 1981; 42:1971-1974. 13. Orekova N M, Akchurin R S, Belayaev A A et al. Local prevention of thrombosis in animal arteries by means of magnetic targeting of aspirin-loaded red cells. Thromb Res 1990; 57:611-616. 14. Jordan J A, Alvarez F J, Murciano J C et al. Influence of chemical modification on “in vivo” and “in vitro” mouse carrier erythrocyte survival and recognition. In: Sprandel U, Way J L, eds. Erythrocytes as drug carriers in Medicine. New York: Plenum Press, 1997:109-118. 15. De Rosa F S, Bentley M V. Photodynamic therapy of skin cancer: Sensitizers, clinical studies and future directives. Pharm res 2000; 17:1447-1455. 16. Dougherty T J, Gomer C J, Henderson B W et al. Photodynamic therapy. J Natl Cancer Inst 1998; 90:889-905. 17. Flynn G, McHale L, McHale AP. Methrotrexate-loaded, photosensitized erythrocytes: A photoactivatable carrier/delivery system for use in cancer therapy. Cancer Lett 1994; 82:225-229. 18. Flynn G, Hackett T J, McHale L et al. Encapsulation of the thrombolytic enzyme, brinase, in photosensitized erythrocytes: A novel thrombolytic system based on photodynamic activation. J Photochem Photobiol B: Biology 1994; 26:193-196. 19. Mir L M, Orlowski S. Mechanisms of electrochemotherapy. Adv Drug Deliv Rev 1999; 35:107-118. 20. Mir L M. Therapeutic perspectives of in vivo cell electropermeabilization. Bioelectrochemistry 2001; 53:1-10. 21. Ward T, Rollan A, Flynn G et al. The effects of electric fields on photosensitized erythrocytes: Possible enhancement of photodynamic activation. Cancer Letters 1996; 106:69-74. 22. Ward T, Mooney D, Flynn G et at. Electric field-enhanced activation of hematoporphyrin derivative: Effects on a human tumour cell line. Cancer letters 1997; 113:145-151. 23. Rollan A, McHale AP. Differential response of photosensitized young and old human erythrocytes to photodynamic activation. Cancer Letters 1997; 111:207-213.

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24. Hashhizume H, Baluk, P, Morikawa S et al. Openings between defective endothelial cells explain tumour vessel leakiness. Am J Pathol 2000; 156:1363-1380. 25. McHale AP, McHale ML, Blau W. The effect of hematoporphyrin derivative and human erythrocyte ghost-encapsulated hematoporphyrin derivative on a mouse myeloma cell line. Cancer Biochem Biophys 1988; 10:157-164.

CHAPTER 3

Targeting Drug Loaded Red Blood Cells Mauro Magnani, Luigia Rossi and Giorgio Brandi

T

argeting of drugs can be achieved in a number of ways usually conjugating the drug of interest with specific ligands or by engineering the properties of the carrier system. In this paper we summarize results so far obtained with the use of erythrocytes, as targeting system. Autologous human erythrocytes over conventional drug delivery systems, permit the delivery of high amounts of drug, are totally biocompatible, non-immunogenic and have a longer in vivo life-span. Erythrocytes containing encapsulated drugs can be efficiently targeted to macrophages where a number of intracellular pathogens can be found. Using this system it has been possible to control HIV-1 infection, human herpes virus 1 infectivity and production and a number of co-infections. Furthermore, the same targeting system can also efficiently deliver corticosteroid analogues to macrophages controlling cytokine release and macrophages activation. Based on these and other results, it can be concluded that targeting of erythrocytes is a new option for the selective delivery of drugs to macrophages with clinical benefit for the patients.

Introduction A number of obvious advantages are related to targeting a selected drug to a defined cell or organ. First of all, targeting of drugs usually increases their therapeutic efficacy and decreases their side effects and toxicity. Secondly, drug distribution in the body is usually influenced, with relatively higher concentrations in the targeted cells or tissues and reduced amounts in non-specific districts. Furthermore, when drug targeting is obtained by conjugation of the drug of interest with a specific carrier, the pharmacokinetics and pharmacodynamics of the drug are also affected. Based on these simple considerations and the obvious advantages for patients, a number of different approaches have been developed in recent years in this field. A number of books, meetings and symposia have summarized their results, promises and expectations and will not be discussed in this paper. Readers can instead refer to papers published in the Advanced D rug Delivery R eview, in the Journal of D rug Targeting and in Critical Reviews in Therapeutic D rug Carrier Systems . Drug targeting is usually achieved either by coupling or entrapping the selected drug to or in a carrier system that has a significant affinity for one or more cell types within the body. The most common carrier systems include liposomes, nanoparticles and cell carriers, while soluble carriers are made from antibodies, polysaccharides, bio-degradable polymers, polyamino acids and modified proteins or peptides. Each of these systems has advantages and disadvantages that should be carefully evaluated in selecting the most appropriate system. Under normal conditions the extravasation of particles from the circulation is very limited. However, in some pathological conditions the endothelial layer and the basement membranes can be damaged and thus particles can escape by diffusion or by diapedesis. On the other hand, small peptides can be cleared by the liver while large peptides are filtered by the kidneys and degraded in renal lysosomes. Large proteins and glycoproteins are usually recognized by scavenger receptors or sugar-specific receptors.1 Thus, the drug carrier is an important determinant of both pharmacokinetics and drug disposal. Erythrocyte Engineering for Drug Delivery and Targeting ©2001 Eurekah.com.

, edited by Mauro Magnani.

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Erythrocyte Engineering for Drug Delivery and Targeting

A number of attempts have been made to improve the targeting of drugs by engineering the properties of the carrier system. Examples of this approach include the conjugation of drugs with galactosyl-terminating peptides for liver targeting2, and the use of TAT, VP22, etc. engineered peptides.3,4 In other approaches the targeting of drugs has been optimized by taking advantage of the selective distribution of receptors and thus developing drugs conjugated with the respective ligands.5-9 Although these approaches are very interesting, they allow the delivery of only limited amounts of drug which in many cases are certainly not sufficient to obtain for a therapeutic result. Based on these considerations, we developed a drug targeting system that takes advantage of the main properties of particulate carriers in terms of the amount of drug delivered and the specificity of soluble carriers. Other interesting properties of this carrier are its biocompatibility, non-immunogenicity and extended half-life. This paper will summarize the principles of the methods and some applications.

Red Blood Cells as Drug Delivery System Human red blood cells have a number of properties that make them useful as drug carriers. Erythrocytes are biodegradable, can circulate for long periods of time (months) and have a large capacity; moreover, a high percentage of encapsulation can be obtained. In a different Chapter10 in this book the methods used to encapsulate drugs in red blood cells will be discussed. In principle any drug, including peptides, nucleic acids, etc., can potentially be encapsulated into red blood cells. However, several molecules have been shown to leak rapidly through the red cell membrane due to simple diffusion. Other molecules may be toxic to the red cell itself, thus preventing their use as a carrier system. It is interesting to note that red blood cells are “active” carrier systems, being endowed with a number of enzymatic activities that can be conveniently explored to convert an inactive pro-drug into an active drug.11 This property permits the design of a number of pro-drugs that can be synthesized with charged chemical groups making them non-diffusible or non-toxic. Once these chemical groups have been hydrolyzed by resident red cell enzymes, the pro-drug is converted into an active drug that can diffuse through the red cell membrane and thus released in circulation or at specific sites when red cell targeting is achieved (see below). Thus, knowledge of the biochemistry of red blood cells is a key factor in designing the most appropriate pro-drugs and in predicting the kinetics of conversion into an active drug.

Targeting Drug-Loaded Red Blood Cells Over the years a number of procedures have been developed for the targeting of drugloaded red blood cells. DeLoach and co-workers12 treated red blood cells with glutaraldehyde to target methotrexate to the liver. Using a similar approach, Zocchi et al13 were able to target doxorubicin-loaded red cells to the liver, increasing the therapeutic index of this drug from 1.8 to 4.2 in a murine metastatic model. Unfortunately, treatment of red cells with glutaraldehyde, although very effective in causing in vivo targeting to the liver, is not very reproducible and glutaraldehyde itself can be toxic. An alternative to this approach was developed by Chiarantini et al,14 coupling monoclonal antibodies to the red cell membranes. Both in vitro and in vivo targeting to cytotoxic T-cells was obtained with this system. Immunoerythrocytes obtained by coupling antibodies through a biotin-avidin-biotin bridge were also developed.15 However, although these methods were very effective in vitro, it is likely that in vivo they would elicit an immune response against the coupled proteins and thus could eventually be used only in single treatments. More recently,16 membrane-fusible erythrocyte ghosts were evaluated, but again it was found that, although useful in in vitro studies, it will be difficult to use them in vivo. Other recent approaches have taken advantage of the increasing knowledge we have accumulated on the mechanisms of red blood cell aging and on the process of selective removal of senescent cells from the circulation.17 In fact, mammalian red blood cells have a defined sur-

Targeting Drug Loaded Red Blood Cells

31

vival in circulation that in humans is 120 days. After this time, the senescent red cells are recognized by the phagocytic cells of the reticuloendothelial system and removed from circulation. Thus, the delivery of drugs encapsulated into red blood cells to macrophages is not a problem if the encapsulated drug has a life-span longer than the red cell life-span. However, for many drugs this is not the case, and the carrier red cells should be modified to reduce their in vivo life-span. An important determinant of red cell survival is the membrane phospholipid asymmetry.18 The outer monolayer of the erythrocyte membrane contains mainly phosphatidylcholine and sphingomyelin, while phosphatidylserine and phosphatidylethanolamine are present in the inner leaflet of the bylayer. This asymmetry can be abolished by adding 1 mM Ca2+ during the lysis and resealing steps in the preparation of carrier red cells. As a result, carrier red cells are readily recognized by macrophages and the encapsulated drug targeted to these cells. Unfortunately red cells have an active system that, in the presence of ATP, restores normal phospholipid asymmetry. The most important and physiological mechanism of red cell removal from circulation is immune-mediated. The senescent erythrocytes expose some new antigenic sites on their membrane that are recognized by autologous immunoglobulins and opsonized;19 the opsonized red cells are then recognized by macrophages and phagocytosed. At least one important antigenic site on the membrane of senescent red blood cells is generated by the transmembrane protein band 3,20 which functions as an anion transporter. Paolo Arese in Turin and Philip Low in Lafayette have found that band 3, when present in clusters, becomes an antigenic site that is readily recognized by autologous IgG and complement, promoting red blood cell phagocytosis.21 Subsequently, we discovered that human red blood cells processed by hypotonic hemolysis and resealing to encapsulate drugs could be treated with ZnCl2 to induce band 3 clustering. However, these clusters are reversible (upon removal of Zn2+ ) but can be stabilized by addition of the cross linker BS3 (Fig. 1). Thus, drug-loaded red blood cells can be targeted to macrophages which recognize the treated cells by means of the Fc and C3b receptors on the macrophage membranes. Phagocytosed red cells then release their content within the macrophages.22 It is interesting to note that the extension of band 3 clustering can be controlled by varying the amount of Zn2+ used, and as a consequence it is possible to modulate the in vivo survival of treated cells.23 This method allows a precise estimate of the amount of drugs to be delivered to the macrophage compartment, controlling both the amount of drug encapsulated during the loading procedure and the rate of red cell removal from circulation by modulation of the extent of band 3 clustering.

Drug Targeting to Macrophages by Carrier Red Blood Cells A number of pathogens are known to have a selective macrophage tropism (Table 1) and a drug targeting system that selectively delivers drugs to macrophages should prove beneficial. The targeting of antiretroviral drugs to macrophages will be reviewed elsewhere in this book24 and will not be discussed here. Here we will briefly summarize some results obtained by treating herpes simplex virus I-infected macrophages with drug-loaded red blood cells. Furthermore, other examples will illustrate the possibility of targeting corticosteroid analogues and peptides.

Targeting New Anti-Viral Drugs Macrophages can be easily infected by various herpes viruses including herpes simplex type 1 (HSV-1) and 225 and herpes zoster.26 Moreover, HSV-1/2 infections are common among individuals infected with HIV-1. Furthermore, HIV-1 are able to mutually activate their replication during co-infection of macrophages.27 Keeping this problem in mind, we have designed new antiviral compounds able to inhibit replication of both viruses in macrophages and possibly overcome the low activity of cellular nucleoside-phosphorylating enzymes and/or the viral thymidine kinase from drug-resistant strains of HSV-1. These drugs are listed in Figure 2 and consists of a series of dinucleotides which could act as prodrugs for the production of partially phosphorylated antiviral drugs. These new molecules are not active in the form shown in Fig-

Erythrocyte Engineering for Drug Delivery and Targeting

32

Figure 1. Scheme showing the main steps in the preparation of drug-loaded red blood cells to be targeted to macrophages. Human red cells are first processed to encapsulate the drug of interest. The drug-loaded cells are then treated with ZnCl2 to induce band 3 clustering and BS3 to make these clusters irreversible. Autologous IgG molecules recognize band 3 clusters and opsonize the red cells. Macrophages recognize the opsonized red cells through the Fc and C3b receptors.

ure 2 and cannot be administered free in solution since they are unable to cross the cell membrane. These new drugs can be delivered to macrophages only if encapsulated into a suitable carrier and preferentially by using the red blood cells as described above. In a series of papers29-31 we have shown that: • • • •

the delivery of these new drugs is very effective when using red blood cell carriers the antiviral activity of the drugs is much higher than that of the single molecules administered free in solution a single drug administration is effective for several days as compared to only hours for the parent drugs the administration of these new drugs to macrophages prevents not only viral replication but also the transcription of early and intermediate-phase viral proteins known to induce cellular aggregates.

Targeting Corticosteroid Analogues Glucocorticoid analogues are potent anti-inflammatory and immunosuppressive drugs. Their action is mediated by suppression of cytokine production and superoxide production. The selective delivery of glucocorticoid analogues would certainly be of interest in reducing the possible side effects of these drugs and providing a persistant intracellular concentration (glucocorticoids are membrane-diffusible molecules) within the macrophages.

Targeting Drug Loaded Red Blood Cells

33

Table 1. The most common pathogens known to enter and replicate within macrophages Listeria monocytogenes Brucella spp. Salmonella spp. Mycobacteria spp. Legionella pneumophila Herpes simplex Varicella zoster Cytomegalovirus HIV-1 Hystoplasma capsulatum Cryptococcus neoformans Candida spp. Coccidioides immitis Toxoplasma gondii Trypanosoma cruzi Leishmania spp. Rickettsia spp. Chlanydia spp.

bacteria bacteria bacteria bacteria bacteria virus virus virus virus fungi fungi fungi fungi protozoa protozoa protozoa other pathogens other pathogens

Modified from Murray HW, Seminar in Hematology 25, 1988:101-111.

That this is possible was shown by encapsulating dexamethasone 21-phosphate or prednisolone 21-phosphate in red blood cells.32,33 These analogues are slowly dephosphorylated by red cell enzymes, but targeting is fast enough to release the glucocorticoid analogues within macrophages. The drug delivery system was effective in suppressing TNF-% production from human macrophages upon lipopolysaccharide stimulation,34 and IkB% protein synthesis induced by the glucocorticoid was shown to be the main mechanism for this control. Thus, the use of human red blood cells is very effective in targeting dexamethasone to human macrophages, producing a significant improvement over free drugs in terms of cell-specificity and drug efficacy.

Targeting Peptides Small peptides are usually degraded within red blood cells. Thus, this method for peptide delivery should first be evaluated in vitro to determine the stability of the peptide of interest. Once stable peptides are found, they can be efficiently encapsulated in red blood cells and targeted to macrophages. We have shown that ubiquitin analogues can be conveniently delivered to macrophages by way of red blood cells. Ubiquitin is a 76-amino acid peptide that is essential in marking a protein substrate for ATP-dependent degradation by the proteasome.35 Usually, ubiquitin is conjugated to the target substrate by a complex multienzymatic process that involves E1 (a ubiquitin activation enzyme), E25 (ubiquitin carrier proteins) and E35 (isopeptide ligase). Specifically linked polyubiquitin chains on the target protein are then recognized and degraded by the 26S proteasome. These chains occur mainly through ubiquitin lysine 48. We have also shown that ubiquitin analogue K48R does not form polyubiquitin chains blocking proteolysis. Thus, we have produced a recombinant K48R ubiquitin and encapsulated it into red blood cells. These loaded cells were then targeted to macrophages where, among other functions, ubiquitin is responsible for IkB%-degradation and thus NF-!B activation upon macrophage stimulation.36 Targeting human red cells loaded with ubiquitin K48R

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Erythrocyte Engineering for Drug Delivery and Targeting

Figure 2. Some dinucleotide analogues designed, synthesized and encapsulated into carrier red blood cells. The properties of these antiviral molecules and their efficacy as antiviral agents in macrophages are discussed in Ref. 28, 30, 31.

to macrophages causes stabilization of IkB% and abrogation of expression of genes such as TNF-% that are controlled by NF-!B.37 Thus, the efficient targeting of peptides to macrophages is feasible and functional.

Conclusions The results briefly summarized in this chapter demonstrate that human red blood cells can be efficiently processed for the encapsulation of antiviral agents, anti-inflammatory drugs and peptides. The drug-loaded cells can also be easily and reproducibly modified so as to allow

Targeting Drug Loaded Red Blood Cells

35

them to be recognized and taken up by human macrophages. Thus, this system is able to selectively target the drug encapsulated into the red cells to macrophages. Two important determinants that are able to influence the amount of drug delivered have been identified in the process: the amount of drug encapsulated in the red cells and the extent of band 3 clustering. The first parameter (drug concentration in red cells) can be easily adjusted during the loading procedure. The second parameter (band 3 clustering) depends on the amount of Zn2+ used in the post-encapsulation step. Useful concentrations are in the 0.1-1 mM range. Band 3 clustering is in turn responsible for red cell opsonization by autologous immunoglobulins and complement up to C3b. The amount of bound IgG depends on the extension of band 3 clustering and on the blood cell species.23 In humans, this figure is about 1,500 IgG molecules per red cell. Opsonized red cells are then recognized by the Fc and C3b receptors on macrophages. This recognition is the key factor in determining the specificity of the process. The main advantages of the use of red cells over other carriers are essentially related to the large capacity of this delivery system and its biocompatibility. The encapsulation process can be conveniently performed using autologous blood, thus minimizing the risk of transmission of pathogens. The carrier can accommodate a large variety of different drugs, including macromolecules and oligonucleotides. Mathematical models for this drug delivery delivery in vivo are now available.38-40 In conclusion, the numerous advantages connected with the use of red cells as a drug targeting system are certainly worth further exploration at the clinical level, in that the potential of human blood as a natural resource is certainly not yet fully appreciated.

References 1. Meijer DKF. Drug targeting with glycoproteins and other peptide carriers: An overview. In: Gregoriadis G et al, eds. Targeting of Drugs 4. New York: Plenum Press, 1994:1-30. 2. Di Stefano G, Busi C, Camerino A et al. Coupling of 5-fluoro 2'-deoxyuridine to lactosaminated poly-l-lysine: an approach to a regional, non-invasive chemotherapy of liver micrometastases. Biochem Pharmacol 2001; 61(4):459-465. 3. Schwarts JJ, Zhang S. Peptide-mediated cellular delivery. Curr Opin Mol Ther 2000; 2(2):162-167. 4. Juliano RL, Yoo H. Aspects of the transport and delivery of antisense oligonucleotides. Curr Opin Mol Ther 2000; 2(3):297-303. 5. Vyas SP, Singh A, Sihorkar V. Ligand-receptor-mediated drug delivery: An emerging paradigm in cellular drug targeting. Crit Rev Ther Drug Carrier Syst 2001; 18(1):1-76. 6. Barker SA, Khossravi D. Drug delivery strategies for the new millennium. Drug Discov Today 2001; 6(2):75-77. 7. Russell-Jones GJ. Use of vitamin B12 conjugates to deliver protein drugs by the oral route. Crit Rev Ther Drug Carrier Sys 1998; 15:557-586. 8. Reddy JA, Low PS. Folate-mediated targeting of therapeutic and imaging agents to cancer. Crit Rev Ther Drug Carrier Sys 1998; 15:587-627. 9. Becker A, Hessenius C, Licha K et al. Receptor-targeted optical imaging of tumors with nearinfrared fluorescent ligands. Nature Biotech 2001; 19:327-331. 10. Rossi L, Magnani M. Red blood cell loading: A selection of procedures. In: Magnani M, ed. Erythrocyte Engineering for Drug Delivery and Targeting. Austin: Landes Bioscience, 2001. 11. De Flora A, Zocchi E, Guida L et al. Conversion of encapsulated 5-fluoro-2’-deoxyuridine 5’monophosphate to the antineoplastic drug 5-fluoro-2’-deoxyuridine in human erythrocytes. Proc Natl. Acad Sci USA 1988; 85:3145-3149. 12. DeLoach JR, Barton C. Glutaraldehyde-treated carrier erythrocytes for organ targeting of methotrexate in dogs. Am J Vet Res 1971; 42:1971-1974. 13. Zocchi E, Tonetti M, Polvani C et al. Encapsulation of doxorubicin in liver targeted erythrocytes increase the therapeutic index of the drug in a murine metastatic model. Proc Natl Acad Sci USA 1989; 86:2040-2044. 14. Chiarantini L, Droleskey R, Magnani M et al. In vitro targeting of erythrocytes to cytotoxic Tcells by coupling of Thy-1,2 monoclonal antibody. Biotech Appl Biochem 1992; 15:171-184. 15. Muzykantov VR, Sakharov DV, Domogatsky SP et al. Direct targeting immunoerythrocytes provides local protection of endothelial cells from damage by hydrogen peroxide. Am J Pathol 1987; 128:276-285. 16. Kogure K, Ithoh, Okuda D et al. The delivery of proteins into living cells by use of membrane fusible erythrocyte ghosts. Int J Pharm 2000; 210:117-120.

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17. Magnani M, De Flora A, eds. Red Blood Cell Aging. New York: Plenum Press, 1991:1-883. 18. McEvoy L, Williamson P, Schlegel RA. Membrane phospholipid asymmetry as a determinant of erythrocyte recognition by macrophages. Proc Natl Acad Sci USA 1986; 83:3311-3315. 19. Kay MMB. Mechanism of removal of senescent cells by human macrophages in situ. Proc Natl Acad Sci USA 1975; 72:3521-3525. 20. Kay MMB, Goodman S, Sorensen K et al. The senescent cell antigen is immunologically related to band 3. Proc Natl Acad Sci USA 1983; 80:1631-1635. 21. Turrini F, Arese P, Yuang J et al. Clustering of integral membrane proteins of the human erythrocyte membrane stimulated autologous IgG binding, complement deposition, and phagocytosis. J Biol Chem 1991; 266:23611-23617. 22. Magnani M, Rossi L, Brandi G et al. Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages: In vitro and in vivo studies. Proc Natl Acad Sci USA 1992; 89:6477-6481. 23. Chiarantini L, Rossi L, Fraternale A et al. Modulated red blood cell survival by membrane protein clustering. Mol Cell Biochem 1994; 144:53-59. 24. Fraternale A, Casabianca A, Magnani M. Red blood cells carrier of antiviral agents. In: Magnani M, ed. Erythrocyte Engineering for Drug Delivery and Targeting. Austin: LandesBioscience, 2001. 25. Wu L, Morahan PS, Hendrzak JA et al. Herpes simplex virus type I replication and IL-1 beta gene expression in mouse peritoneal macrophages activated in vivo by an attenuated Salmonella typhimurium vaccine or Corynebacterium parvum . Microbiol Pathog 1994; 16:387-399. 26. Nikkels Af, Debrus S, Sadzot-Delvaux C et al. Comparative immunohistochemical study of herpes simplex and varicella zoster infections. Virchows Arch A Pathol Anat Histopathol 1993; 422:121-126. 27. Heng MC, Heng SY, Allen SG. Co-infection and synergy of human immunodeficiency virus-1 and herpes simplex virus-1. Lancet 1994; 343:255-258. 28. Rossi L, Brandi G, Schiavano GF et al. Macrophage protection against human immunodeficiency virus or herpes simplex virus by red blood cell-mediated delivery of a heterodinucleotide of azidothymidine and acyclovir. AIDS Res Human Retrovir 1998; 14:435-444. 29. Franchetti P, Abu Sheikha G, Cappellacci L et al. Synthesis and biological application of a new heterodinucleotide with both anti-HSV and anti-HIV activity. Nucleosides & Nucleotides 1999; 18:989-990. 30. Franchetti P, Abu Sheikha G, Cappellacci L et al. A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus. Antivir res 2000; 47:149-158. 31. Rossi L, Serafini S, Cappellacci L et al. Erythrocyte-mediated delivery of a new homodinucleotide active against human immunodeficiency virus and herpes simplex virus. J Antimicrob Chem 2001; 47:819-827. 32. Magnani M, D’Ascenzo M, Chiarantini L et al. Targeting dexamethasone to macrophages. Drug Delivery 1995; 2:151-155. 33. D’Ascenzo M, Antonelli A, Chiarantini L et al. Red blood cells as a glucocorticoids delivery system. In: Sprandel V, Way J, eds. Erythrocytes as Drug Carrier in Medecine. 1997; 81-88. 34. Crinelli R, Antonelli A, Bianchi M et al. Selective inhibition of NF-kB activation and TNF-% production in macrophages by red blood cell-mediated delivery of dexamethasone. Blood Cells, Mol Dis 2000; 26:211-222. 35. Peters JM, Harris JR, Finley D, eds. Ubiquitin and the Biology of the Cell. New York: Plenum Press 1998:1-494. 36. Magnani M, Crinelli R, Bianchi M et al.. The ubiquitin-dependent proteolytic system and other potential targets for the modulation of nuclear factor-!B (NF-!B). Current Drug Targets 2000; 1:387-399. 37. Antonelli A, Crinelli R, Bianchi M. et al. Efficient inhibition of macrophage TNF-% production upon targeted delivery of K48R ubiquitin. Brit J Haematol 1999; 104:475-481. 38. Beretta E, Solimano F, Takeuchi Y. A mathematical model for drug administration by using red blood cells phagocytosis. J Math Biol 1996; 35:1-19. 39. Beretta E, Fasano A. Mathematical models for drug administration by using RBC phagocytosis. In: Martelli M, Cooke K, Cumberbatch E et al, eds. Differential Equations and Applications to Biology and to Industry. World Scientific Publishing Co., 1996:23-30. 40. Beretta E, Solimano F, Takeuchi Y. A Mathematical model for a new kind of drug administration by using the phagocytosis of red blood cells. J Math Biol 1996; 35:1-19.

CHAPTER 4

Streptavidin-Mediated Coupling of Therapeutic Proteins to Carrier Erythrocytes Vladimir R.Muzykantov and Juan-Carlos Murciano

R

ed blood cells (RBC) can provide a natural, safe and abundant carrier to prolong the life-time in the bloodstream and thus enhance the efficacy of certain therapeutic agents, while restricting their accessibility to the extravascular compartment and thus reducing side effects. Methods for coupling diverse therapeutic proteins to the RBC surface have been proposed, including coupling via streptavidin-biotin cross-linker. RBC-coupled proteins retain biological activity and effectively interact with either free or immobilized ligands. However, preservation of the biocompatibility of RBC-drug complexes represents a significant challenge. As with other RBC-mediated drug delivery paradigms, complement activation and uptake by macrophages in the reticuloendothelial system (RES, e.g., spleen and liver) represent major pathways for destruction and elimination of RBC carriers modified with biotin and avidin. An extensive biotinylation of RBC and coupling of avidin inactivate complement regulators DAF and CD59 in RBC, thus leading to C3b fixation. This results in phagocytosis and hemolysis, which greatly compromise RBC biocompatibility. However, coupling of streptavidin monovalently to modestly biotinylated RBC obviates these problems and permits stable attachment of 104-105 molecules of antibodies or enzymes to biocompatible RBC. RBC-drug complexes do not fix complement, avoid uptake by macrophages and circulate in a functionally active form in the bloodstream without hemolysis or elimination by reticuloendothelial system. Therefore, the monovalent streptavidin-mediated coupling to RBC allows prolongation of the functional half-life of therapeutic proteins in the circulation by orders of magnitude and markedly alters their pharmacokinetics in rodent models. This approach may be utilized for the site-selective delivery of diverse agents to the intravascular targets (e.g., immunotargeting of drugs to blood and vascular cells), as well as sustained circulation of drugs, which should exert their activity in blood (e.g., RBC carriage of anti-thrombotic enzymes). Further animal and human studies will test the validity, practical feasibility and therapeutic effectiveness of this approach.

Introduction: RBC as Drug Carrier The development of new means for drug delivery is an important biomedical goal. To enhance the specificity, effectiveness and safety of therapeutic or prophylactic interventions, especially with such potent and complex drugs as enzymes and genetic materials, a number of problems must be solved, including, but not limited to: i) prolongation of a drug half-life; ii) restriction of unintended drug uptake by tissues; iii) cell-, tissue-, and organ-specific targeting; and, iv) delivery of drugs to proper target compartments (e.g., extracellular milieu, cell surface, cytosol, nucleus). These challenging issues persist despite marked advances in drug delivery and targeting made over the past 25 years. One means that has been used to prolong a drug half-life and enhance targeting is coupling to a vehicle or carrier, such as synthetic or natural polymers, liposomes, antibodies and plasma proteins. Among other carriers, erythrocytes (red blood cells, RBC, anucleate discoid cells with Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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a diameter of 5 to 7 ∝m, thickness ~2 ∝m and plasma membrane surface area ~160 ∝m2) represent a unique and potentially attractive vehicle for drug delivery and targeting. The total number of RBC in the human body approaches 30 trillion. Therefore, RBC represent the most abundant (>99%) cellular constituent of the blood, have a life span of 100-120 days, travel ~250 km through the cardiovascular system and function as natural carriers for oxygen. RBC interact with injected substances, metabolize more than 40 known drugs (e.g., captopril, sulfanilamide, testosterone, insulin), and partition catecholamines, tacrolimus, antibiotics and other drugs. It has been known for decades, therefore, that RBC can serve as a natural carrier/compartment participating in drug biodistribution, pharmacokinetics and action (for a review see ref. 1). A prolonged life-time in the circulation, availability, considerable volume and surface, high biocompatibility and natural mechanisms for safe elimination and degradation represent attractive features of RBC as a drug carrier. The need for ex-vivo manipulations with RBC, a limited shelf-life and concerns related to the safety of donor matching and blood-born infections are recognized potential downsides of drug carriage by RBC. It should be mentioned, however, that these concerns are of general nature for all types of hemotransfusion therapies. Nevertheless, transfusion of blood and blood products is a very widely used and generally safe therapeutic intervention worldwide, the safest and most effective type of cell transplantation strategy. Use of autologous blood (re-infusion) minimizes the safety concerns. In 1973, Ihler proposed that certain drugs might enjoy prolonged circulation in a protected and active form if they could be incorporated into autologous RBC and re-injected safely in the host.2 Although similar to liposomes in some respects, RBC have dramatically longer halflives, and larger inner volumes and surfaces. Normally, RBC do not travel from the circulation into tissues (except spleen and hepatic sinuses). Thus, RBC can deliver drugs only to intravascular targets in the physiologic setting, although a recent study shows that application of ultrasound may facilitate transport of carrier RBC through the vascular wall.3 Retention of RBCassociated drugs in the bloodstream, however, offers a potential benefit of limited uptake by “non-target” tissues. Therefore, one can expect that RBC carriage will markedly reduce harmful or poorly understood side effects of certain drugs in the tissues. Significant efforts have been invested in order to prove the validity of this paradigm and establish clinically applicable strategies for RBC carriage of drugs. This Chapter will describe one specific approach, namely, coupling of therapeutic proteins to the surface of plasma membrane of the carrier RBC.

Loading of Drugs Inside RBC and Coupling of Therapeutic Proteins to RBC Surface

Initially, RBC carriage has been proposed for enzyme replacement therapy,4 and to deliver enzymes and DNA to target cells.5 Subsequent studies partially fulfilled some of these expectations by reporting that enzymes, DNA and drugs could be loaded into RBC (e.g., using electroinsertion or hypotonic lysis and resealing) with retention of function.6 Predicated on these in vitro studies, several laboratories explored the use of resealed RBC as carriers in laboratory animals. Encapsulation into RBC was demonstrated to prolong the circulation of erythropoietin,7 alcohol oxidase8 and carbonic anhydrase.9 RBC treated with cross-linking agents have been used to deliver encapsulated drugs to hepatic and splenic macrophages.10-13 Microparticles made from RBC ghosts permit delivery of cytotoxic agents to malignant cells.14 Isotope-loaded RBC might be useful as a blood contrast for gamma-scintigraphy, whereas gadolinium-loaded RBC has been explored as a blood pool MRI contrast agent.15 However, in vivo studies have revealed limitations of RBC carriage. First, enzymes loaded into RBC can effectively interact only with membrane-permeable substrates, such as uric acid, methanol or glucose. For example, encapsulation of urokinase inside RBC reduces its fibrinolytic activity by orders of magnitude due to inaccessibility for its large (50kDa) plasma substrate, plasminogen.16 Thus, certain drugs must be released from RBC in order to provide therapeutic effects. Regulation of drug release from RBC (for example, mediated by a

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controlled activation of complement) represents a formidable task that still remains to be completely fulfilled.17 In addition, drugs released from RBC will be removed from therapeutic sites by blood flow and may be eliminated rapidly from the circulation. Secondly, certain drugs and loading schema reduce or even abolish RBC biocompatibility. Drug loading disturbing plasma membrane leads to exposure of charged lipids and cytoskeleton proteins on the cell surface and thus makes modified RBC fragile, leaky and more rigid, i.e., less resistant to shear stress and mechanical damage, as well as more adhesive, due to altered surface charge.18,19 RBC modification may also lead to partial or complete inactivation of regulators of complement in RBC membrane (see below). Such RBC are lysed by complement20,21 and undergo rapid uptake by macrophages.18,22 Destruction and elimination of carrier RBC profoundly compromises drug delivery and may lead to dangerous side effects. For example, generation of pro-inflammatory complement peptides C3a and C5a may cause vascular injury and shock. In addition, uptake of damaged RBC may overload macrophages in the reticuloendothelial system and thus compromise host defenses. 23 Therefore, loss of biocompatibility of the RBC loaded with a drug is a stringent limitation for the strategy. As an alternative to drugs loading in the inner volume of RBC, they can be coupled to RBC surface. Theoretically, coupling of drugs to RBC surface may circumvent issues related to inadequate drug release or to the loss of biocompatibility that results from the inevitable trauma to RBC plasma membrane caused by incorporation of large amounts of drug within RBC to achieve therapeutic levels. Coupling of therapeutic proteins (e.g., antibodies or enzymes) to the RBC surface could be used to facilitate their immunotargeting to intravascular targets and to regulate pathologic situations that often occur intravascularly, such as abnormalities of coagulation and fibrinolysis (for a brief review see ref. 24). Figure 1 schematically illustrates these two strategies. In principle, the combination of loading and coupling approaches is possible. Thus, antibodies attached to RBC surface may serve as affinity carriers (immunoerythrocytes) to achieve site-selective targeting of a drug loaded inside RBC. For example, antibody-directed immunotargeting of RBC was proposed to deliver drugs to other blood cells in circulation.25 A similar approach has been used to deliver cytokines and antigens, in order to facilitate their targeting to blood-accessible immunocytes and boost the immune response.26 Coller and coauthors described a potential substitute for platelet infusion (thromboerythrocytes) comprised of a pro-thrombotic RGD-containing peptide coupled to carrier RBC.27 Numerous previous studies in vitro documented that antibodies and enzymes coupled to RBC retain their functional activity.17,28-31 Importantly, coupling to RBC surface eludes steric restrictions: even enzymes that react with small, membrane-permeable substrate are more active when bound to the RBC surface than when incorporated within the cell.32 This Chapter describes a strategy for coupling of therapeutic proteins to the surface of carrier RBC utilizing streptavidin-biotin cross-linker, with specific focus on the functional activity and biocompatibility of RBC-drug complexes studied in vitro and in animal models.

Destruction and Elimination of Modified RBC by Complement and Phagocytes The biocompatible coupling of drugs or targeting moieties to RBC carrier must produce a stable, non-toxic drug/RBC complex that circulates in the bloodstream as a single, functionally active entity without activation of complement, phagocytes and immune system and enjoys a life-time and final destination similar to those of naïve RBC. However, both modification of RBC plasma membrane and a coupled drug can compromise biocompatibility of RBC carrier. Major mechanisms involved in recognition, destruction and elimination of modified and aged RBC include complement system, anti-RBC immunoglobulins and phagocytes of reticuloendothelial system, first of all splenic and hepatic macrophages. Figure 2 presents a simplified scheme of complement-mediated destruction and elimination of modified RBC. The complement system in humans and many other mammals, including laboratory rodents, consists of about 20 proteins that normally circulate in plasma. Nine complement

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Erythrocyte Engineering for Drug Delivery and Targeting

Figure 1A. Schematic representation of two conceivable scenarios for pharmacokinetics of the therapeutic proteins coupled to the surface of the carrier red blood cells (RBC). Intravascular delivery of drugs with immunoerythrocytes. RBC carrying antibodies have an access and affinity to diverse components of the vascular system and may be used for directed delivery of drugs (antioxidants, genetic materials) to normal and pathologically altered endothelial cells (EC), anti-thrombotic agents to sites of vascular injury, antigens or immunosuppressants to lymphocytes, anti-inflammatory agents to white blood cells (WBC), antiparasitic drugs to macrophages in the liver and spleen. Immunoerythrocytes also can be used for binding, detoxification or/and elimination of circulating pathogens and immune complexes.

components proper (indicated by symbols C1, C2, through C9) act in concert with their plasma cofactors. The cascade reaction of complement may undergo activation via two pathways, classical and alternative. The classical pathway starts when C1q sub-component of C1 binds to Fc portions of immune complexes and forms a tri-molecular complex C1q(C1s/C1r)2, an enzymatically active protease that specifically cleaves the next component, C4 and thus converts it into active protease C4a. In turn, C4a proteolytically activates C3 and converts it to C3b that activates further components. In the alternative pathway, C3b formed by marginal endogenous hydrolysis directly binds to activating particles or cells and becomes associated with their surface in active form. Thus, C3b component plays a central role in both pathways of the complement cascade.33 Unrestricted activation of C3 leads to formation of the membrane attacking complex (MAC, C5-C9) that forms a pore in the plasma membrane and thus causes hypotonic cell lysis (RBC hemolysis). In addition, deposition of C3b on RBC membrane facilitates their recognition and phagocytosis by macrophages (see below). Activation of complement also leads to generation of potent pro-inflammatory peptide mediators, C3a and C5a. Normally, complement activity is under the stringent control of specific regulators both in blood plasma and cell membranes.34,35 Certain regulators of complement, such as Decay Accelerating Factor (DAF, CD55), sialic acids, complement receptor type 1 (CR1, CD35), membrane cofactor protein (MCP, CD 46) homologous restriction factor (HRF), and membrane inhibitor of the reactive lysis (MIRL, CD59) present in the plasma membranes of human cells.

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Figure 1B. Schematic representation of two conceivable scenarios for pharmacokinetics of the therapeutic proteins coupled to the surface of the carrier red blood cells (RBC). Intravascular carriage of therapeutic proteins on RBC surface. Certain therapeutic proteins (TP), such as anti-thrombotic enzymes, undergo rapid elimination, predominantly via hepatic uptake (that reduces the effect towards blood targets) and diffuse through endothelial cells (EC) into normal tissues (that generates side effects). In contrast, RBCcoupled TP would have a prolonged access to the blood targets with a relatively limited uptake by spleen and, at lesser extent, by the liver. This would facilitate therapeutic effect and reduce dose or number of injections. In addition, RBC-coupled TP have no access to normal tissues, hence fewer side effects.

In rodent RBC, two major complement regulators are DAF and CD59, both GPI-anchored glycoproteins. DAF (3x103 copies per RBC) inactivates membrane-associated C3b,36 whereas CD59 (2-4x104 copies per RBC) regulates membrane attack complex.35 In addition to fixation of the complement, modified and aged RBC may fix constitutive plasma immunoglobulins directed against RBC components that are normally absent on the surface of naïve RBC. This leads to activation of the complement via classical pathway, recognition of RBC by macrophages in RES and destruction and elimination of RBC from circulation. Macrophages possess C3b-receptors and Fc-receptors, which bind RBC opsonized by complement and/or immunoglobulins.37 This event leads to RBC phagocytosis via the mechanism that involves activation of signal transduction and reorganization of cytoskeleton. In most organs, such as lungs, tissue macrophages are separated from blood cells by a tight endothelial monolayer, basement membrane and tunica intima of blood vessels. However, endothelial lining is not continuous in the liver and spleen (fenestrated endothelium in sinuses), two major organs of reticuloendothelial system (RES). Thus, splenic and hepatic macrophages have a good access for blood components, including red blood cells, and perform constant surveillance of circulating molecules, particles and cells. Therefore, senescent, damaged or chemically modified RBC undergo rapid elimination by splenic and hepatic macrophages via Fc-receptor and complement-receptors mediated binding and phagocytosis.

Biocompatibility of RBC Modified With Non-Specific Cross-Linkers RBC-carriage strategies, however promising, require a rigorous evaluation to establish their applicability in vivo and must be proven safe and effective in animal experiments and, eventually, in clinical studies. The proposed approach must satisfy the following requirements: i) the RBC-conjugated protein must retain biologic activity; ii) the RBC/protein complex must be stable in the bloodstream; iii) the amount of RBC-bound protein must suffice; and, iv)

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Erythrocyte Engineering for Drug Delivery and Targeting

Figure 2. Schematic representation of complement-mediated elimination of altered RBC. Normal RBC possess plasma membrane proteins inhibiting complement, such as complement receptor type 1 (CR1), decay accelerating factor (DAF), homologous restriction factor (HRF) and CD59. Aging or chemical modification of RBC leads to inactivation of the complement regulators. Thus, inactivation of DAF leads to deposition of active C3b component of complement (depicted as a bomb) on RBC plasma membrane. RBC-bound C3b initiates two mechanisms for RBC elimination. Firstly, macrophages in the reticuloendothelial system, which possess numerous receptors for C3b and other activated components complement, bind and phagocytose RBC opsonized by C3b. Secondly, membrane-bound C3b triggers the cascade of complement reactions resulting in formation of the membrane attacking complex (MAC). When CD59 in RBC plasma membrane is inactivated, MAC forms a pore in RBC membrane and thus causes hypotonic hemolysis.

conjugation should not compromise RBC biocompatibility (ability to escape activation of complement and phagocytosis) and bioavailability (life-time in the circulation and biodistribution in tissues). Conjugation of antibodies, antigens and haptens to RBC has been introduced in the middle of the century to produce tools for immunological reactions of hemagglutination and lysis. In most cases, non-specific cross-linking agents (e.g., glutaraldehyde, bys-sulfosuccinimidyl-suberate or tannic acid) have been used for this purpose. They permitted production of RBC coated by millions of antigen molecules with a relatively prolonged shelf life-time, due to a chemical fixation of RBC plasma membrane. However, the use of non-specific cross-linking agents to conjugate therapeutic proteins to RBC yields relatively low drug/RBC ratios and impaired the function of the RBC-conjugated proteins, in part because of the lack of a spacer between the

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protein and RBC surface. In addition, coupling using those cross-linkers profoundly compromises RBC biocompatibility. Cross-linking of the transmembrane sialoprotein glycophorin (which interacts with spectrin cytoskeleton via band 4.1 in the inner compartment of RBC) and anion transporter band 3 (which interacts with spectrin via ankyrin) cause unintended reorganization of protein and lipid domains on the cell surface.19 Fixation of RBC membrane by glutaraldehyde or tannic acid makes it stiff, rigid and adhesive to plasma opsonins. Thus, certain protocols of RBC modification with cross-linking agents leads to changes in cell shape, binding of autologous IgG, activation of the classical and alternative pathways leading to opsonization by complement, and results in enhanced C3b- and Fc-receptor-mediated phagocytosis by RES macrophages.21,38-41 The resultant activation of complement leads to hemolysis, generation of proinflammatory peptides C3a and C5a, RBC adhesion to nucleated cells and components of the extracellular matrix. In addition, stiffed, rigid and aggregated modified RBC mechanically embolize pulmonary capillaries. All these pathways lead to rapid elimination of carrier RBC from the bloodstream and compromise drug delivery and safety. Therefore, many types of RBC membrane modification lead to their rapid clearance from the bloodstream, thus negating drug delivery to all targets other than macrophages.11,39,42 More specific, safe and effective approaches for coupling of proteins to RBC are acutely required for all other potential therapeutic applications. In the following sections we discuss one of such specific and biocompatible approaches, namely coupling of therapeutic proteins to the carrier RBC using strept(avidin)-biotin cross-linker.

Coupling of Active Therapeutic Proteins to RBC via Streptavidin-Biotin Avidin (a 60 kD positively charged glycoprotein) possesses four high affinity biotin-binding sites.43 Streptavidin (SA, a neutral bacterial analogue of avidin that possesses a pro-adhesive RYD sequence) and neutravidin (a derivative of avidin that lacks sugar moiety, RYD domain and positive charge) also possess four biotin-binding sites and cross-link biotin-containing molecules with even greater specificity than avidin.44 Streptavidin-biotin pair has been utilized in animal and clinical studies for immunoimaging and immunotherapy of tumors, blood clearance and drug targeting.45-52 No harmful effects of streptavidin have been reported in animals or in human recipients to date.48,49,53 Neutravidin, a neutral mammalian analogue that lacks the pro-adhesive RYD domain, may prove to be even safer. These considerations, as well as modular nature of this technique permitting to cross-link practically any given molecule without reduction of activity, motivated interest to utilize this cross-linker as a potentially useful means for a biocompatible coupling of proteins to carrier RBC.28 A variety of biotin derivatives have been shown to be useful either to insert or to covalently couple biotin residues to RBC surface functional groups. For example, biotin hydrazyde couples to RBC sugars (e.g., galactose and syalic acid,54 p-diazobenzoyl biocytin couples to tyrosines and histidines,55 and 3-(N-maleimido-propyonyl)-biocytin couples to sulfhydryl groups.56 Insertion of biotinylated phospholipids in RBC membrane allows effective attachment of avidin.57 Electroinsertion of biotinylated glycophorin (that yields ~104 molecules/RBC, 70% of which have the correct orientation in the RBC membrane) generated biotinylated RBC (bRBC) with a normal half-life in mice.58 However, most studies in the field utilized succinimide esters of biotin providing various length of spacer between biotin residue and modified amino group, an approach introduced for RBC modification two decades ago by Orr, who described a simple and effective method to biotinylate RBC amino groups with N-hydroxysuccinimide biotin ester (BNHS).59 Succinimide biotin esters BNHS, BxNHS, BxxNHS provide increasing length of the spacer (2, 6 and 12 Angstrom, respectively) between biotin residue and modified amino group. RBC modification with biotin esters may be performed both in vitro60 and in vivo.61 RBC modified with BNHS and BxNHS were used to trace normal and damaged RBC in the circulation.60-63

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RBC biotinylation with BNHS or BxNHS permits attachment of biotinylated proteins using avidin, streptavidin or neutravidin cross-linkers alike (indicated thereafter as (strept)avidin in the cases when similar results obtained with all least two analogues, avidin and streptavidin).28,64-66 A simplified scheme illustrating (strept)avidin-mediated attachment of biotinylated therapeutic proteins to RBC is shown in Figure 3. In essence, separately biotinylated RBC and a therapeutic agent are cross-linked by (strept)avidin. The degree of RBC biotinylation (concentration of BxNHS upon biotinylation) is an important parameter of this cross-linking system. Firstly, it affects the effectiveness of coupling of (strept)avidin and biotinylated therapeutic protein. Figure 4 shows that at low input of biotin ester (e.g., in case of human RBC, at BxNHS concentration lower than 1 ∝M), binding of biotinylated protein is not efficient, presumably due to insufficient binding of (strept)avidin. However, extensive biotinylation of RBC (e.g., at BxNHS concentration higher than 100 ∝M) leads to paradoxical suppression of coupling of biotinylated proteins even at high (strept)avidin input. The likely explanation of this phenomenon is that at high density of biotin residues on RBC membrane, (strept)avidin is engaged in multivalent binding. That leads to full occupancy of its biotin-binding sites and precludes binding of biotinylated drugs. In addition, cross-linking of RBC membrane proteins by multivalently bound (strept)avidin greatly compromises RBC biocompatibility (see below). Coupling efficiency of 30-40% can be achieved at optimal extent of biotinylation and (strept)avidin concentration, thus permitting up to 105 molecules of b-antibody or b-enzyme to be attached per b-RBC with retention of biological activity.28,65,66 Importantly, biotin derivatives and (strept)avidin serve as spacers between a therapeutic protein and surface of carrier RBC. Therefore, RBC-coupled proteins enjoy sufficient steric freedom to interact with large and non-soluble ligands. For example, SA/b-RBC carrying biotinylated antibodies (immunoerythrocytes) bind soluble antigens in solution.65 However, not every molecule of RBC-coupled protein can interact effectively with relatively large target molecules. For example, SA/b-RBC possessing 5x104 molecules of biotinylated antibody directed against human IgM bind maximally 4x103 antigen molecules.65 Thus, in this case only ten percent of RBC-bound antibodies can effectively interact with soluble antigen. One explanation is that antigen-binding sites of a fraction of RBC-coupled antibodies are oriented towards the RBC membrane, thus precluding binding of antigens. In addition, extended components of RBC glycocalix may hinder interaction of RBC-coupled antibodies with antigens. It should be mentioned, however, that IgM is a relatively large protein antigen (m.w. >700 kD) and binding of one IgM molecule to RBC-coupled antibody may mask binding sites of adjacent RBC-coupled antibodies. It is conceivable that binding capacity of immunoerythrocytes to smaller soluble antigens (e.g., peptides) may be substantially higher. Conceivably, limitations imposed on immunoerythrocytes interaction with immobilized antigens are more stringent than in case of soluble antigens. Nevertheless, immunoerythrocytes specifically bind to artificial surfaces coated with immobilized antigen28,29 and to cells expressing target antigens.67 Figure 5 shows specific binding of immunoerythrocytes to antigen-coated wells in vitro. Therefore, antibodies coupled to the RBC surface via streptavidin enjoy sufficient freedom to interact with immobilized antigens. Our unpublished observations documented that immunoerythrocytes incubated with antigen-coated surfaces under flow at 37oC change their shape and tend to adhere to the antigen substrate. Most likely, high plasticity of plasma membrane of immunoerythrocytes helps to accommodate such a multivalent binding. In general, immunoerythrocytes carrying more than 5x103 molecules of antibody display sufficient affinity for binding to antigen-coated surfaces and antigen-presenting cells, although this parameter strongly depends on the affinity of carrier antibodies and surface density of target antigens. Model and therapeutic enzymes coupled to SA/b-RBC also enjoy freedom sufficient for conversion of their substrates. For example, SA/b-RBC-coupled peroxidase converted its substrate, a relatively small molecule of ortho-phenylen diamine,29 whereas SA/b-BC-coupled plasminogen activator streptokinase converted its substrate, a 50-KD protein plasminogen into plasmin in vitro.30 Moreover, RBC carrying both collagen antibody and streptokinase were

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Fig.ure 3. Schematic representation of coupling of therapeutic proteins (TP) to carrier RBC using streptavidinbiotin cross-linker. At the first phase, RBC and TP are biotinylated separately by a biotin succinimide ester, for example, BxNHS, to form b-RBC and b-TP. After elimination of biotin excess, streptavidin (SA) binds to b-RBC. When a molar excess of streptavidin is added, formed SA/b-RBC complex possess substantial residual biotin-binding capacity due to non-occupied biotin-binding sites of streptavidin. Therefore, streptavidin tightly bound to b-RBC (SA/b-RBC) provides sites for stable attachment of b-TP and spacer that permits b-TP coupled to SA/b-RBC interact with its ligands or substrates.

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Figure 4. Binding of radiolabeled biotinylated human IgG (a model antibody) to human RBC biotinylated at various concentrations of BxNHS. Amounts of SA added: 50 ∝g (open circles), 25 ∝g (closed circles), 12.5 ∝g (open triangles) or 6 ∝g (closed triangles). Note that at low degree of RBC biotinylation, coupling of bIgG is not effective, presumably due to insufficient attachment of streptavidin. On the other hand, binding of b-IgG also declines at high degree of RBC biotinylation. The likely explanation is that at molar excess of BxNHS, most streptavidin biotin-binding sites are occupied by RBC-coupled biotin residues and unable to accommodate b-IgG. The results of typical experiment. With minor modifications from Muzykantov and Taylor, with permission (Anal Biochem, 1994, 223:142-148).

able to bind to immobilized collagen and degrade fibrin clot formed over the collagen target.30 These data, as well as results published by another group32 indicate that therapeutic proteins coupled to RBC surface via (strept)avidin retain sufficient functional activity and enjoy steric freedom permitting effective and specific interactions with large targets and enzymatic substrates.

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Figure 5. Binding of immunoerythrocytes to immobilized antigen. SA/b20-RBC carrying approximately 4x104 molecules of biotinylated goat antibodies directed against mouse IgG were incubated for 1 hr in the plastic wells coated with mouse IgG (a model antigen) or albumin (BSA). SA/b20-RBC carrying a similar amount of biotinylated goat IgG were used as control. After elimination of non-bound RBC, the number of bound RBC in the wells was determined by hemoglobin absorbance. In this and all following figures, unless specified otherwise, the data are presented as Mean+SD, n=3. With minor modifications from Muzykantov and Murciano, with permission (Biotechnol Appl Biochem, 1996, 24:41-45).

Biocompatibility of RBC Modified With Biotin and (strept)Avidin: In Vitro Studies The results of early studies indicated that RBC biotinylated with 1-1,000 ∝M BHNS or BxNHS display a high stability in vitro. Even after a prolonged incubation at 37oC in physiological buffers or fresh homologous serum or plasma alike, the rate of hemolysis of b-RBC, determined by release of hemoglobin, did not exceed that of naïve RBC of human, rat and

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mouse species.68,69 In addition, b-RBC do not adhere to homologous nucleated cells, for example, fibroblasts, smooth muscle cells and endothelium.70 However, coupling of avidin provokes adhesion of b-RBC to homologous nucleated cells. Experiments in cell cultures revealed that the number of avidin-carrying human RBC bound to fibroblasts, smooth muscle cells, endothelial cells, Kuppfer cells and hepatocytes was 10-30 times higher than that of naïve or biotinylated RBC, whereas b-RBC carrying neutral proteins streptavidin or neutravidin did not bind to nucleated cells.70 Heparin and other polyanions inhibited binding of avidin/b-RBC to the nucleated cells, whereas elimination or heat inactivation of serum did not prevent binding. These results implied that adhesion of avidin-carrying RBC to nucleated cells is mediated by interaction of strongly positively charged avidin with negatively charged surface of nucleated cells. Therefore, this obstacle can be easily overcome by utilization of streptavidin or neutravidin instead of avidin. However, a multivalent attachment of avidin to biotinylated mouse, sheep, rabbit, rat and human RBC caused their rapid hemolysis in fresh autologous serum.69 Neither the cationic charge of avidin, nor the RYD-domain of streptavidin were responsible for activating complement, since both proteins, as well as neutravidin, caused hemolysis. However, serum heating, inhibition of complement by antiserum against C3 component or by chelating of Ca2+ or Mg2+ abolished the hemolysis, thus indicating that (strept)avidin/b-RBC activate complement via the alternative pathway.69,71 Importantly, complement hemolysis of SA/b-RBC depends on the mode of (strept)avidin attachment to b-RBC. Comparison of (strept)avidin-induced hemolysis of RBC biotinylated with BNHS, BxNHS and BxxNHS revealed that at equal (strept)avidin attachment (105 of avidin molecules per RBC), the hemolysis was greater with the longer spacer.69,72 Further, hemolysis depends on the valency of (strept)avidin attachment to b-RBC. Controlled acylation of avidin, partial blocking of biotin-binding sites of (strept)avidin by soluble biotin and reduction of surface density of biotin residues on b-RBC surface all reduce ability of (strept)avidin to bind more than one biotin residue on RBC surface. Such a monovalent attachment of (strept)avidin to b-RBC did not cause complement hemolysis.68,69,72 On the other hand, attachment of (strept)avidin to non-biotinylated RBC utilizing non-specific cross-linking by tannic acid, insertion of a biotinyl-lipid or binding of biotinylated antibody against RBC antigens did not induce hemolysis by complement in vitro.57,73,74 Therefore, methods of (strept)avidin attachment that do not cross-link biotinylated components in the RBC membrane (i.e., monovalent attachment) produce a complement-stable carrier RBC, which are capable of binding of up to 105 molecules of biotinylated protein per RBC without subsequent hemolysis by autologous serum in vitro. Not all these methods, however, can be utilized for the drug delivery in vivo. For example, treatment of RBC with tannic acid reduces RBC plasma membrane plasticity and flexibility, while increasing RBC adhesiveness and thus profoundly compromises their biocompatibility. Insertion of biotinyl-lipids permits attachment of (strept)avidin without complement activation, but mechanical stability of RBC was reduced after this procedure. Western-blotting of avidin-carrying b-RBC with specific antibodies directed against DAF and CD59 revealed both complement regulators in association with high molecular mass avidin-containing complexes.75 Since monovalent attachment of (strept)avidin does not cause complement hemolysis, it is conceivable to postulate that cross-linking and abnormal clustering of biotinylated DAF and CD59 in RBC plasma membrane, but not a simple masking, is responsible for inhibition of these regulators of complement.72,75 Multivalent coupling of avidin caused complete inhibition of DAF and near complete loss of CD59 activity in b-RBC; insertion of physiological amounts of purified CD59 into the membrane of avidin/b-RBC restores resistance to complement hemolysis, whereas insertion of DAF has relatively little effect.75 Therefore, multivalent attachment of (strept)avidin to b-RBC eliminates homologous restriction of the classical and alternative pathways of complement in biotinylated RBC due to cross-linking and inhibiting complement regulating proteins, DAF and CD59. The scheme

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shown in Figure 6 illustrates inactivation of DAF and CD59 in RBC plasma membrane caused by multivalent attachment of (strept)avidin. Ability of (strept)avidin to engage in polyvalent binding to b-RBC depends upon the surface density of biotin residues coupled to the RBC.72 The surface density of biotin on the bRBC membrane may be regulated by variation of the extent of biotinylation of the RBC. In order to further address biocompatibility of streptavidin-carrying b-RBC (SA/b-RBC), rat and human RBC have been biotinylated at various input concentrations of BxNHS (generating rat bn-RBC, where n represents the micromolar input of biotinylating reagent incubated with a 10% RBC suspension), and the properties of these RBC as well as SA/bn-RBC have been studied. For both rat and human RBC, covalent coupling of biotin did not lead to hemolysis in vitro by fresh autologous serum within the entire range of BxNHS inputs used for biotinylation (13,000 ∝M). Attachment of 105 SA molecules to RBC biotinylated at high BxNHS inputs (>200 ∝M BxNHS for rat RBC and >1000 ∝M BxNHS for human RBC) led to their hemolysis by autologous serum in vitro. In contrast, attachment of 105 molecules SA to human or rat b-RBC prepared at BxNHS input concentrations lower than 100 ∝M did not induce their hemolysis by serum in vitro.66 Both moderately biotinylated human RBC (SA/b100-RBC) and modestly biotinylated rat RBC (SA/b20-RBC) bound 5x104 molecules of radiolabeled biotinylated IgG (b-IgG) per RBC. Furthermore, both control rat RBC and b-IgG/SA/b23RBC, as well as human counterparts, display no more than 1% hemolysis after incubation with autologous serum (1 h, 37oC, final serum dilution 1/5).69,72,76 Importantly, complement-stable SA/b20-RBC carrying a model biotinylated antibody specifically bound to antigen-coated surfaces in vitro.66 A monoclonal antibody specific for human C3b, mAb 7C12, has been utilized to study the interaction of complement component C3b with human b-RBC and SA/b-RBC exposed to serum. Figure 7A demonstrates that extensive biotinylation of human RBC with 3000 ∝M BxNHS followed by incubation in fresh serum leads to binding of C3b to the RBC. Both human b3000-RBC and SA/b3000-RBC bound C3b to a similar extent. However, although human SA/b3000-RBC were lysed by complement in vitro, b3000-RBC were stable in serum (Fig. 7B). Thus, extensive biotinylation of RBC leads to a non-lytic fixation of C3b. This can be explained by the fact that extensive biotinylation of RBC caused substantial inhibition of DAF (that controls C3b), but not CD59 (that controls membrane attacking complex).75 C3b plays a central role in both the classic and alternative pathway.33 On the other hand, hepatic and splenic macrophages in the liver have a number of receptors for C3 activation and breakdown products, and it is known that these receptors play a key role in the clearance of complement-opsonized particles from the circulation.37 To address potential consequences of non-lytic fixation of C3b by extensively biotinylated rat RBC (b700-RBC), their interaction with macrophages have been studied in vitro.77 Figure 8 shows that after treatment with serum, b700-RBC, but not control RBC or RBC biotinylated at low level, bound to peritoneal macrophages. Inactivation of complement by heating or chelating of divalent cations abrogated binding of b700-RBC to macrophages. Figure 9 shows that binding of b700-RBC to macrophages at 37oC led to their phagocytosis that could be inhibited by cytochalasin D, an agent disrupting actin cytoskeleton. Therefore, although complement does not induce lysis of rat b700-RBC in serum in vitro, partial inactivation of DAF leads to C3b fixation that apparently mediate their uptake by tissue macrophages. Nevertheless, a modest biotinylation provides serum-stable SA/b-RBC, which do not fix complement and do not bind to macrophages in vitro.

Biocompatibility of RBC Modified with Biotin and Streptavidin: In Vivo Studies Several groups demonstrated that modest biotinylation of RBC per se does not affect their life span or biocompatibility in vivo.60,76,78,79 Hoffmann-Fezer and co-authors have reported that BNHS and BxNHS may be injected intravenously in active form to biotinylate RBC circulating in the bloodstream, a procedure potentially useful for RBC survival/metabolism

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Figure 6. Mechanism of streptavidin-induced hemolysis of biotinylated RBC by autologous complement activated via the alternative pathway. A. Naïve and modestly biotinylated RBC inactivate C3b and membrane attacking complex due to activities of DAF and CD59 anchored in RBC plasma membrane. B. Monovalent coupling of streptavidin to modestly biotinylated RBC may lead to partial masking of DAF and CD59, but does not cause their complete blocking or abnormal clusterization of DAF and CD59. Hence, neither fixation of complement nor hemolysis prevails. C. Polyvalently bound streptavidin reorganizes membrane clusters in RBC plasma membrane and cross-links biotinylated DAF and CD59. This leads to formation of plasma membrane domains lacking DAF or/and CD59. These domains serve as sites for deposition of C3b, activation of complement cascade and formation of MAC that leads to hemolysis.

studies as well as for drug delivery.61,80,81 Biotinylated RBC have been used safely to study circulation of normal, aged or oxidized RBC in diverse animal species and in humans.60,82-85 Figure 10 shows the rate of elimination from the bloodstream of 51Cr-labeled b-RBC after iv injection in intact anesthetized rats. Rat RBC biotinylated at 700 ∝M BxNHS (b700-RBC, stable in serum in vitro) were eliminated from the bloodstream immediately after injection. Approximately 20% of the recovered radioactivity in blood was detected in the plasma, indicating partial hemolysis of b700-RBC in vivo. Figure 11 shows tissue distribution of bn-RBC one hour after injection in rats. Hepatic uptake of b700-RBC-associated radioactivity (a total of ca. 80% of the injected radioactivity, rat liver weight is 10 grams) was substantially higher than that of RBC, b23-RBC and b70-RBC. Splenic uptake of b700-RBC also was higher than that of RBC, b23-RBC and b70-RBC. Therefore, a high level of biotinylation of RBC induces their uptake by liver and spleen in the absence of SA. Based on in vitro results shown in the previous

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Figure 7. Effect of human RBC biotinylation with different input of BxNHS and binding of streptavidin on fixation of C3b and lysis in fresh homologous serum. Panel A: Fixation of C3b. Extensive biotinylation of human RBC at BxNHS concentrations 1000 ∝M and higher followed by opsonization in serum leads to binding of C3b to the RBC. Therefore, both human b3000-RBC and SA/b3000-RBC bound C3b to a similar extent. Panel B: Lysis by fresh human serum. Although human SA/b3000-RBC were hemolysed by complement in vitro, b3000-RBC were stable in serum. Note that modestly biotinylated SA/b100-RBC did not fix C3b and were stable in serum. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

section, it is conceivable that rat b700-RBC fix C3b without hemolysis and undergo C3bmediated phagocytosis by hepatic and spleenic macrophages. Lower levels of RBC biotinylation (b 70-RBC and b20-RBC) did not induce significant alterations in their lifespan and biodistribution, as compared with control RBC. However, attachment of streptavidin imposes further limitations on the biocompatibility of b-RBC. Serum-labile rat SA/b700-RBC and SA/b240-RBC undergo extremely rapid elimination from the bloodstream (Fig. 12). Of the radioactivity remaining in the circulation, about 75% was detected in plasma after injection of SA/b240-RBC, implying that complement-mediated hemolysis plays a major role in the elimination of these RBC from the bloodstream. Direct hemolysis by complement seems to be a major mechanism for the elimination of these RBC in vivo. SA/b240-RBC are lysed by complement in vitro. On the other hand, although rat SA/b70RBC were also rapidly cleared from the bloodstream, in vitro these RBC demonstrated resistance to hemolysis by complement.79 Hemolysis of rat SA/ b70-RBC in vivo did not exceed 40% based on the distribution of radioactivity between plasma and blood cells, implying that both hemolysis by complement and uptake by macrophages contribute to the clearance of SA/ b70-RBC from the bloodstream. However, the high level of the hepatic and splenic uptake of SA/b70-RBC (Fig. 13) suggests that uptake by liver macrophages with receptors for C3b (or perhaps by splenic receptors for C3bi or C3dg)86 plays a major role in their elimination.

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Figure 8. In vitro binding of biotinylated rat RBC to isolated peritoneal macrophages. Control RBC (left bars) or b700-RBC (right bars) were incubated with adherent macrophages for 2 hr at 37oC. Fresh rat serum (closed bars) or albumin-containing PBS (open bars) was added to the wells as incubation media. After elimination of non-bound RBC, amount of RBC in the wells was determined by hemoglobin absorbance. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

In contrast with SA/ b240-RBC and SA/b70-RBC, serum-stable SA/b23-RBC circulated in the bloodstream for at least 1 hour without detectable lysis and with only mildly increased elimination. Figure 13 shows that hepatic uptake of radioactivity associated with SA/b240-RBC and SA/b70-RBC was dramatically higher than that associated with SA/b23-RBC and RBC. Splenic uptake was enhanced for all preparations of streptavidin-coated RBC, including the SA/b23-RBC. In view of the fact that the rat spleen weight is less than 1 gram, no more than 10% of injected radioactivity was accumulated in the spleen 1 hour after injection of SA/ b23-RBC.

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Figure 9. Phagocytosis of extensively biotinylated RBC. Electron transmission microscopy images of rat b700-RBC incubated with elicited rat peritoneal macrophages in the presence of fresh serum. Panel A: Phagocytic vacuoles containing RBC (arrows). Panel B: Higher magnification of the vacuoles, arrows show RBC membrane.

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Figure 10. Blood clearance of biotinylated 51Cr-RBC in rats. A: Blood level of radiolabeled RBC after intravenous injection. B: Distribution of blood radioactivity between plasma and cell pellet. Note that b700RBC (closed triangles) are cleared from the bloodstream almost instantly (A) and major fraction of residual blood radioactivity was found in plasma (B), thus indicating hemolysis. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

Therefore, a monovalent attachment of streptavidin to modestly biotinylated (i.e., b20-RBC and lower extent of biotinylation) does not compromise their biocompatibility and circulation in the bloodstream in intact animals. This result implied that SA/b20-RBC, or, perhaps, SA/ b10-RBC can be used for carriage of biotinylated therapeutic proteins.

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Figure 11. Tissue distribution of biotinylated 51Cr-RBC in rats one hour after intravenous injection. Note high uptake of b700-RBC by liver and spleen. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

Prolonged Circulation of Therapeutic Proteins Coupled to RBC

In order to explore this paradigm, the in vivo fate of b-IgG/SA/b23-RBC carrying 3x104 molecules of biotinylated IgG per SA/b23-RBC labeled with 51Cr has been examined after intravenous injection in intact rats.79 Figure 14 shows that b-IgG/SA/b23-RBC circulate in rats for at least several hours without marked elimination. One day after injection, 60% of the injected radioactivity was still associated with circulating blood cells. Less than 1.5% of the blood 51Chromium was found in the plasma, indicating there was very little hemolysis of bIgG/SA/b23-RBC in the circulation. In contrast, complement-susceptible b-IgG/SA/b700-RBC were lysed and eliminated from the bloodstream within minutes after injection. Figure 15 shows that the distribution of b-IgG/SA/b23-RBC in rat tissues is similar to that of control RBC. The only exception is splenic uptake of b-IgG/SA/b23-RBC, which is slightly (nonsignificantly) higher than that of control RBC. Therefore, carriage of IgG by SA/b23-RBC did not compromise their biocompatibility. To evaluate whether immunoerythrocytes circulate in the bloodstream as a complex possessing attached b-IgG, b-IgG/SA/b23-RBC containing 51Cr-labeled b23-RBC and 125I-labeled b-IgG have been injected in rats. Only 25-30% of 125Iodine could be found in the plasma several hours after injection of such a complex, whereas 100% of 125Iodine circulate in plasma after injection of non-conjugated 125I-labeled b-IgG. Therefore, a major fraction of immunoerythrocytes circulates as b-IgG/SA/b23-RBC complexes carrying about 70% of initial amount of attached b-IgG. Noteworthy, conjugation with SA/b23-RBC prolongs circulation time of 125I-labeled b-IgG (Fig. 16). Three hours after injection, blood level of 125Iodine was 60+7% of injected dose for 125 I-b-IgG/SA/51Cr-b23-RBC vs 25+8% for non-conjugated 125I-labeled b-IgG (M+SD, n=3, p<0.05). Therefore, conjugation with carrier RBC prolongs circulation time of b-IgG,

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Figure 12. Blood clearance of streptavidin-coated biotinylated 51Cr-RBC in rats. A: Blood level of radiolabeled RBC after intravenous injection. B: Distribution of blood radioactivity between plasma and cell pellet. Note that attachment of streptavidin causes rapid elimination and intravascular hemolysis of b240RBC (closed triangles) and b70-RBC (open triangles). With minor modifications from Muzykantov and coauthors, with permission (Anal Biochem, 1996, 241:109-119).

probably due to reduction of its filtration in tissues. About 30% of b-IgG, however, detached from SA/b23-RBC during the first hours after injection; one day after injection, about 50% of RBC-conjugated b-IgG detaches from the carrier b-RBC. Figure 16C shows distribution of both radiolabels in rats 60 min after iv injection. During first hour after injection, both labels

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Figure 13. Tissue distribution of streptavidin-coated biotinylated 51Cr-RBC in rats one hour after intravenous injection. Note high uptake of SA/b240-RBC and SA/b70-RBC by liver and spleen. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

display the same level in the blood. This supports the conclusion that majority of immunoerythrocytes circulate as an intact complexes. One example of therapeutic proteins amenable to RBC carriage would be anti-thrombotic enzymes because their activity must be limited to the bloodstream. Thus, RBC carriage of fibrinolytics, such as clinically applicable tissue-type plasminogen activator (tPA) and urokinase (uPA), could prolong their life-time in circulation. The preliminary results strongly support this hypothesis. Approximately 4x104 molecules of either tPA or urokinase, both biotinylated, have been coupled to SA/b10-RBC. The resulting b-tPA/SA/b10-RBC and b-uPA/ SA/b10-RBC, both possessing high fibrinolytic activity and stable in serum in vitro, were injected in rats. Firstly, coupling of plasminogen activators did not compromise biocompatibility of the carrier RBC. Blood level and tissue distribution of 51Cr-RBC carrying tPA or urokinase were similar to those of control RBC (Fig. 17). Slightly enhanced blood clearance and splenic uptake of urokinase-carrying RBC have been detected; most likely this result reflects the fact that urokinase binds to the vascular cells and components of vascular extracellular matrix via kringle domain and other pro-adhesive parts of the molecule. However, the most striking result was obtained when the rates of blood clearance of free and RBC-coupled plasminogen activators have been compared after intravenous injection in intact rats (Fig. 18). As soon as ten minutes after intravenous injection, non-conjugated control 125Ilabeled tPA and urokinase practically disappeared from blood. In a sharp contrast, biotinylated tPA and urokinase coupled to modestly biotinylated RBC SA/b10-RBC enjoyed a prolonged circulation. These data clearly indicate that a coupling of plasminogen activators via monovalent streptavidin to b-RBC dramatically prolongs their life-time and bioavailability in the bloodstream of intact animals.

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Figure 14. Circulation of complement-stable immunoerythrocytes after intravenous injection in rats. A: Blood level of radiolabeled RBC carrying approx. 4x104 molecules of b-IgG per cell after intravenous injection. B: Distribution of blood radioactivity between plasma and cell pellet. Note that b-IgG/SA/b2351 Cr-RBC circulate in rats for at least several hours after intravenous injection without marked elimination or hemolysis. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

Conclusion and Perspectives Red blood cells represent an attractive potential carrier for intravascular administration of therapeutic proteins for which prolonged action restricted to the blood is required. Streptavidinbiotin cross-linker provides a versatile, modular technology for the biocompatible coupling of

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Figure 15. Tissue distribution of complement-stable immunoerythrocytes in rats one hour after intravenous injection. Note that b-IgG/SA/b23-51Cr-RBC displays tissue distribution similar to that of control 51CrRBC. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

proteins to the carrier RBC. The scheme in Figure 19 illustrates our working model for mechanisms by which RBC modified with biotin and streptavidin are cleared from the bloodstream. Extensive biotinylation of RBC (700 ∝M and more biotin ester in the reaction mixture) leads to DAF inactivation and fixation of C3b. Active CD59 in the b-RBC membrane can prevent RBC hemolysis by complement in vitro. However, deposition of C3b opsonizes b700-RBC and leads to the C3b-mediated uptake of b700-RBC by macrophages in the liver and spleen. Thus, non-lytic (“frustrated”) activation of complement and C3b-mediated phagocytosis underlies the mechanism for elimination of b700-RBC from the circulation. Moderate biotinylation of RBC (70-200 ∝M biotin ester) does not markedly alter the biocompatibility of b-RBC. These cells are resistant to hemolysis by complement both in vitro and in vivo and circulate in the bloodstream for a prolonged time. However, attachment of SA induces cross-linking of biotinylated DAF and CD59, thus inactivating both regulators of complement. This leads to both opsonization of SA/b-RBC by fixed C3b (due to DAF inactivation) and to hemolysis by MAC (due to CD59 inactivation). In vitro these SA/bn-RBC demonstrate intermediate stability in serum: SA/b240-RBC are susceptible to hemolysis, while SA/b70-RBC are resistant to hemolysis. In vivo, however, both SA/b240-RBC and SA/b70-RBC undergo rapid elimination. Probably, both hemolysis by complement and macrophage uptake of C3b-opsonized SA/b-RBC contribute to this process. In addition to these complementmediated mechanisms, alternative opsonins recognizing cross-linking and/or clusterization of RBC membrane proteins (band 3, e.g.11) might participate in elimination of SA/b-RBC. Nevertheless, a modest biotinylation of RBC permits production of complement-stable streptavidincarrying b-RBC (e.g., b10-RBC), which are capable of binding therapeutic proteins and prolonged circulation in vivo. This point is illustrated by a prolonged circulation of RBC-carried model (biotinylated IgG) and therapeutic (biotinylated plasminogen activators) protein cargoes in intact animals. In certain cases, binding of therapeutic proteins to components of the vascular wall may induce partial elimination of the carrier RBC from circulation; urokinasemediated uptake of RBC in spleen illustrates this point.

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Figure 16. Effect of carriage by complement-stable SA/b23-RBC on blood clearance and tissue uptake of biotinylated IgG. A. Blood level of radiolabeled 125I-IgG after intravenous injection. B: Distribution of 125IIgG between plasma and cell pellet. C. Tissue distribution of 51Chromium (open bars, traces RBC) and 125 Iodine (hatched bars, traces IgG) one hour after injection of 125I-b-IgG/SA/b23-51Cr-RBC in rats. With minor modifications from Muzykantov and co-authors, with permission (Anal Biochem, 1996, 241:109-119).

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Figure 17. Blood clearance (A) and tissue distribution one hour after intravenous injection in rats (B) of RBC carrying plasminogen activators. Note that in vivo fate of 51Cr-labeled SA/b10-RBC carrying 3x104 molecules of tissue type plasminogen activator (tPA, triangles and closed bars) or urokinase (uPA, squares and hatched bars) does not differ significantly from that of control 51Cr-labeled RBC (circles and open bars), except the RBC/uPA display enhanced spleenic uptake.

The transfusion of RBC is the most common and best-tolerated form of tissue transplantation, with over 14x106 units of blood donated for transfusion in USA in 1993. Nevertheless, the use of donor RBC for drug delivery is hindered by the need for antigen matching, as well as concern for transmission of HIV, hepatitis and other infectious organisms. Re-injection of autologous RBC is safer in terms of transmission of infectious diseases and potential immune reactions (e.g., RBC radiolabeled ex vivo is useful for radionuclide ventriculography in nuclear medicine departments). Re-injection of autologous blood has become the standard of care for otherwise healthy donors undergoing elective procedures for which transfusion is usually required. This practice extends the applicability of our paradigm to a number of common clinical circumstances associated with thrombosis. For example, in the case of patients undergoing autologous blood donation for hip replacement surgery, a drug (e.g., anti-thrombotic proteins) can be coupled to autologous RBC ex vivo and administered safely and effectively with little or no additional risk to the recipient. Nevertheless, the need for ex vivo manipulation with RBC before re-injection, however accurate and safe, may restrict the feasibility of our strategy. Importantly, RBC possess 2-5,000 copies of a unique transmembrane complement receptor, CR1, that binds C3b87 and via this mechanism delivers C3b-containing immune complexes to macrophages without damage to RBC.88,89 Capitalizing on this privileged function of CR1, Dr. R.Taylor pioneered a strategy to eliminate pathogens from blood utilizing anti-CR1 conjugated with specific antibodies (heteropolymers). Studies from his laboratory performed in animal models including primates have demonstrated that: i) after intravenous injection, heteropolymers bind to normal RBC in vivo and circulate for a prolonged time coupled to RBC without a significant reduction in RBC survival or increased tissue uptake; ii) circulating pathogens (or a model ligands) bind to RBC-associated heteropolymers in vitro and in vivo without detectable uptake or destruction of the carrier RBC.65,90-92 A recent study from another laboratory implies that this strategy is applicable to various proteins conjugated with anti-CR1.93 Anti-CR1 heteropolymers can be injected directly to the bloodstream and, having unlimited access to much higher number of RBC, may ultimately have the effect of delivering higher total amounts of drug. Although each RBC would be expected to carry less drug in this setting, the total number of drug-carrying RBC may be much greater and drug distribution throughout the circulation would be more homogenous. Therefore, utilization of anti-CR1/enzyme heteropolymers, if shown to exhibit a reasonable shelf-life, may permit ex vivo manipulations of RBC to be excluded, thereby eliminating the attendant concerns about safety and limitations based on real-time product availability.

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Figure 18. Prolonged circulation of RBC-carried tissue type plasminogen activator (Panel A, tPA) and urokinase (Panel B, uPA) after intravenous injection in rats. Biotinylated radiolabeled plasminogen activators were injected in rats in a form of free protein (circles) or coupled to SA/b10-RBC. Note that RBC carriage dramatically prolongs life-time of plasminogen activators in vivo.

Certain RBC-coupled proteins may be immunogenic. However, mice did not develop an immune response to biotinylated human glycophorin electroinserted into murine RBC.58 Further, the immune response in humans exposed to RBC-associated L-asparaginase and free enzyme was comparable.94 Thus, there is precedent for the concept that the immune response elicited by a single administration of RBC-coupled proteins is likely to be limited and tolerable. Nevertheless, to limit this problem further and permit prolonged repetitive administration, it is possible to camouflage RBC/protein complexes using polyethylene glycol (PEG). PEG polymer (m.w. 5,000) hinders interaction of proteins or liposomes with immunocompetent cells. Covalent modification of proteins with PEG prolongs their half-life in the circulation dramatically and diminished the immune response.95,96 Numerous laboratories have successfully utilized PEG derivatization since that time to produce stealth enzymes and liposomes.97,98 Recently, several laboratories have extended this concept by showing that coating of heterologous RBC with PEG diminishes immune response (“stealth RBC”).99 Coating with PEG also improves rheological properties of RBC significantly by reducing spontaneous aggregation and lowering shear viscosity.100 Thus, coating with PEG may be tested as an additional means to improve the safety of RBC-coupled proteins. Prolonged circulation of therapeutic agents may cause potentially harmful local or/and systemic side effects. However, the activity of RBC/tPA complexes will be limited to blood, with minimal or no access to extravascular compartment. This may reduce harmful effects of longcirculating drugs. For example, RBC-coupled proteases (such as plasminogen activators) are less likely to cause degradation of extracellular matrix and cell detachment. Localization of RBC/tPA complexes in blood makes them maximally amenable to antidotes. It is also possible to eliminate RBC-coupled drugs rapidly in the case of bleeding through exchange transfusion. The strategies for RBC-mediated drug delivery are universal and could be applied to optimize the therapeutic profile of a wide variety of drugs such as anti-thrombotic agents, antigens, cytokine antagonists and other drugs.

Acknowledgements Authors would like to acknowledge that their interest to the studies of RBC as carriers for drug delivery was inspired by participation in the research project initiated two decades ago by Drs. G.P. Samokhin, M.D. Smirnov and V.N. Smirnov in Cardiology Research Center, Moscow, USSR; as well as Drs. J. Luque and M. Pinilla in the department of Biochemistry, Alcalá University, Spain. We thank Dr. A. Zaltzman (University of Wales, Cardiff ), Dr. E. Atochina

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Figure 19. Biocompatibility of the carrier RBC after streptavidin-mediated coupling of therapeutic proteins (TP). See explanations for the scheme in the text.

(University of Pennsylvania, Philadelphia), Dr. R.P. Taylor (University of Virginia, Charlottesville) and Dr. A. Herraez (University of Alcala, Madrid), for valuable experimental contributions to our previous studies, some of which are analyzed in this Chapter. Authors express their deep gratitude to Dr. Douglas B. Cines (Department of Pathology, University of Pennsylvania) for friendly mentorship, numerous stimulating discussions, editorial help and constant support to our studies of RBC-mediated drug delivery. This work is supported in part by research grants from National Institutes of Health (RO-1 from NHLBI), American Heart Association (BugherStroke Award) and PENN Research Foundation to VRM, as well as by Research Fellowships from NATO and Spanish Government to JCM.

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CHAPTER 5

Vaccination Strategy Using Red Blood Cells as Antigen Delivery System Laura Chiarantini

T

he use of adjuvants is usually required to induce strong immunological responses to protein antigen. However, serious side effects preclude their use in human vaccines. Currently, the only adjuvant that is licensed for use in human vaccines is alum. Red blood cells (RBC) can be used as an antigen delivery system for enhancement of immunotargeting for vaccination purposes. The method for coupling antigen on the autologous RBC membrane is based on biotin-avidin-biotin bridges. Several potentially attractive features are reviewed. The use of RBC-based immunization strategies do not require adjuvants. RBC are naturally removed by macrophages that are able to process and present antigens to MHC. RBC-based immunization does not have any side effects and at the mean time assures a prolonged exposure to the antigen. The appreciable titre of anti-avidin antibodies could be advantageous for the opsonization of antigen carrying RBC facilitating their recognition by macrophages. This antigen delivery system is also able to induce an immunogenic response in mice with an amount of antigen 10-1250 times lower with respect to conventional administration. Furthermore, the antigen carrying RBC appears to be an effective tool for antigen delivery into dendritic cells, and INF-& could be used advantageously to augment the ability of RBC to induce Th1 responses. The studies reviewed herein have extended the immunotargeting approach to adjuvantindependent immunization and support further exploitation of this new strategy as a viable alternative in the design of new vaccines and new delivery systems.

Introduction Vaccination is one of the medical success stories of the 20th century; nevertheless, there are many diseases for which no prophylactic regimens are available. A major hindrance that has prevented the development of effective mass immunization programs is the inability to induce an appropriate, protective, immune response. Such responses can be extremely difficult to elicit, especially when employing recombinant, soluble protein subunits. This difficulty is due to the inability of these antigens to access the machinery of the appropriate antigen-processing pathway. Following progress that has led to an improved understanding of the mechanisms underlying this processing, as well as the realization that delivery systems can quantitatively and qualitatively affect the resulting immune response, the last decade has witnessed an intense research effort in this field. The capacity to identify the nature and form of antigen epitopes in proteins now allows the specific design of molecules able to promote relevant and protective immune responses. Such entities, although ideal in term of specificity and purity, may not achieve their goals through failure to reach relevant cells of the immune system simply due to dilution, elimination by host enzymes or lack of specific targeting. Furthermore, a plethora of adjuvants has been developed with the aim of enhancing immune responses to new immunogens with respect to antigen processing, the nature and role of cytokines and the importance of T-cell subsets in infection. Erythrocyte Engineering for Drug Delivery and Targeting ©2002 Eurekah.com.

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Adjuvants can be grouped according to their physical characteristics and mode of action. They include particulate adjuvants, oil and emulsion-based adjuvants, adjuvants providing controlled antigen delivery, or based on specific targeting of antigens and gel-type adjuvants. As adjuvant technology develops it is becoming increasingly clear that differing approaches may be combined, and an adjuvant/delivery system should be designed to provide slow release of a targeted antigen. Although there will probably be no universally ideal vaccine immunologically effective against a wide range of infectious diseases, several factors that are required can be identified, including: 1. the presence of one or more antigens able to promote long-lasting protection after a single dose; 2. efficiency of contact with the immune system; 3. promotion of specific immune responses and immunological memory; 4. stability, non-toxicity and suitability for use in humans. In addition, vaccines against viral diseases should stimulate immune responses at mucosal surfaces, induce the appropriate isotype of antibody and elicit cell-mediated immunity, particularly cytotoxic T-lymphocyte (CTL) responses. Among the vaccines currently available, it is generally accepted that live, attenuated products are more effective than inactivated whole virions or viral subunit proteins. It is therefore not surprising that the introduction into the host of sub-viral components such as highly purified viral proteins, antigenic determinants derived by recombinant DNA technology or synthetic peptides elicits sub-optimal or inappropriate immune responses. An exciting way of circumventing these difficulties may possibly be direct injection of viral nucleic acids into the host in the form of plasmid-encoding protective viral antigen.1 However, the new proteins developed through modern technology are relatively poorly immunogenic and require some form of assistance to optimise their immunising capabilities. A number of strategies have been devised to enhance the immune response to these sub-viral elements. 1. Use of adjuvants (e.g. bacterial products and derivatives, saponin derivatives).2 2. Use of a delivery system (e.g. biodegradable microparticles, erythrocytes, bacterial toxins).3-5 3. Direct targeting to cells of the immune system (e.g. fusion with cytokines, complement fragment).6,7 4. Use of a part of an attenuated defective non-replicating microbial vector (e.g., Vaccinia virus, Salmonella spp.),8,9 accompanied by a cell-surface modifier.10 Currently, the only adjuvant that is licensed for use in human vaccines is alum. Nevertheless, alum is certainly less than ideal in many situations. For example, it does not elicit a strong humoral response to all antigens and generally fails to induce cell-mediated immunity.11 Complete Freund’s adjuvant (CFA) has long been recognized as a powerful immunologic adjuvant; however, serious side effects preclude its use in human vaccines.12 In our laboratory, we have explored an adjuvant-independent antigen (Ag) delivery system that involves the targeting of the antigen to the monocyte-macrophage compartment. A biotinylated model protein Ag-conjugated to intact red blood cells (RBC) by an avidin bridge4,13 effectively delivered this Ag to the immune system cells. RBC modification by the avidin-biotin complex was proposed in late 1970s as a method for the study of the RBC membrane proteins.14 Since then, the avidin-biotin-modified RBC have found a wide application both in vitro and in vivo.

Immobilization of Antigen on Red Blood Cells In the last decades several methods have been proposed to couple proteins to the external membrane of erythrocytes, including methods based on: 1. the formation of covalent bonds between aldehyde groups of oxidized RBC and amino or carboxyl groups of protein in the presence of isocyanide,15 2. the use of chromium chloride as bifunctional reagent,16,17 3. disulfide bond formation,18

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4. electroinsertion,19 5. biotin/avidin interaction.20,21 Magnani and co-workers compared different procedures of biotinylation of RBC from different mammalian species.21 The basic procedure is shown in Figure 1. In this study several biotin derivatives were tested and the best was found to be NHS-biotin, which is able to react with membrane amino groups that include amino sugar and protein. The extent of biotinylation of RBC from several mammalian species is shown in Figure 2. Biotinylation by NHS-biotin is a reproducible procedure, which yields a homogeneous population of biotinylated RBC (B-RBC) easily detectable by flow cytometry using a streptavidin FITC. The rabbit B-RBC have a normal circulation, while avidin strongly reduced RBC circulation time. Studies on mice showed a recruitment from circulation preferentially by the liver and spleen.21 The biocompatibility of modified RBC and their prolonged and safe circulation in the body are important for in vivo applications. The unique properties of the avidin-biotin complex, particularly the high-affinity polyvalent interaction between avidin and biotin, have been attracting the attention of researchers who are attempting to employ various avidin-biotin complex modified entities (molecules, liposomes and cells) in vivo.22,23 The immunological properties of avidin probably promote its extremely rapid clearance from the blood stream after an intravenuous administration, with the liver and spleen being the main clearing tissues.

Immunological Response to RBC Coupled With Proteins In preliminary experiments several proteins were used as antigens to exclude the possibility that the results obtained were antigen-dependent.4 Studies in vivo using RBC as an antigen delivery system for bovine serum albumin (BSA), uricase and hexokinase showed results similar to those obtained with classical immunization protocols utilizing Freund’s adjuvant.4 This delivery system takes advantage of the fact that RBC are naturally removed from circulation by macrophages, which are known to be able to present antigens with MHC.24 Even in the case of antigens with low immunogenicity, such as ubiquitin, this method of delivery provides an immunological response that is considerably higher than that obtained by the administration of free antigen.21

Immunological Response to RBC Coupled With Recombinant Proteins Herpex simplex viruses 1 and 2 (HSV-1 and HSV-2) cause human diseases which are often associated with significant morbidity and mortality and which are continuing to grow in importance throughout the world. Of the 11 known glycoproteins of HSV, gB and gD have been extensively analysed for their immunotherapeutic and prophylactic potential.25,26 Several positive results have been obtained in different animal models by immunization with recombinant forms of gB and gD associated with immunological adjuvants. Although all the adjuvants used (MTP, ISCOMs and complete or incomplete Freund’s adjuvant) have shown a strong efficacy in enhancing an antibody response to the tested antigen, to date, most of them cannot be used in human or veterinary vaccines because of unacceptable side effects. Chiarantini and co-workers27 described the results obtained in a murine model utilizing autologous RBC to which the antigen gB1 (gB1s-RBC) was linked via an avidin bridge, demonstrating that gB1s-RBC can elicit an immunological response similar to that obtained with the same antigen in Freund’s adjuvant, and much better than that obtained with alum. Furthermore, the animal group treated with gB1s-RBC received a dose of antigen (0.15 ∝g total) ten times smaller than the control group (1.5∝g total). Serum antibodies elicited in animals vaccinated with the gB1s-RBC formulation showed a neutralization activity significantly higher than that observed in the control groups. In fact, mice immunized with this new vaccine formulation, as well as mice treated with gB1 in Freund’s adjuvant, achieved a total protection against lethal HSV-1 challenge and a high degree of prevention against the latent infection.

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Figure 1. Antigen coupling to red blood cells. Antigen and RBC are separately biotinylated. Avidin serves as a bridge for the coupling of biotinylated antigen. B, biotin; A, avidin. Modified from Chiarantini L. 1996.13

Immunological Response to RBC Coupled with Surface Particles of Virus Feline immunodeficiency virus (FIV) is extensively used for the development of effective anti-human immunodeficiency vaccines. Chiarantini and co-workers chose to immunize cats with homologous RBC coated with enriched native surface antigens of FIV particles by means of a biotin-avidin-biotin bridge (FIV-RBC).28 For immunogen preparation, the proteins exposed on the intact virions were biotinylated by treating freshly harvested, purified whole FIV with impermeant biotin derivatives which react with exposed NH2 groups. The virions were then gently disrupted by using Triton X-100 and the biotinylated proteins were selectively bound to RBC by an avidin bridge. Cats receiving only 14 ∝g of FIV antigens developed clearly evident anti FIV-humoral and cell-mediated immune responses. The results have also shown that, following the primary immunization, the cats were at least partially protected from an ex vivo FIV challenge. Finally, the results obtained corroborated observations that vaccineinduced protection against FIV tends to be short-lived and may be difficult to restimulate.

Antigen Delivery System for Human Dendritic Cells Dendritic cells (DC) can represent an important target for vaccine development against viral infections. Due to their potent immunostimulatory capacity, DC have become the centrepiece of many vaccine regimens. Immature DC capture, process, and present Ags to CD4(+) lymphocytes, which, in turn, activate immature DC through CD40. The resulting mature DC loose phagocytic capacity, while acquiring the ability to efficiently stimulate CD8(+) lymphocytes. The capacity of DC to induce CD4+ and CD8+ T cell responses is enhanced when antigens are provided as particulates.29 Dendritic cells reside in unperturbed tissues in an immature form, where they are suitable for capturing and accumulating antigens and are essential for initiation of immune responses.

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Figure 2. Biotinylation of RBC from several mammalian species. Modified from Magnani M. 1994.21

A variety of danger signals, including microorganisms, dying cells or pro-inflammatory cytokines induce terminal differentiation, also known as maturation of DC.30,31 Mature DC migrate to secondary lymphoid organs and acquire a potent T cell-stimulating capacity. Maturation of DC is strengthened by T cell-derived signals such as CD 40 ligands (CD40L) and INF-&.30,32 Mature DC express higher levels of antigen-presenting and costimulatory molecules and release large amounts of interleukin-12, thereby preferentially stimulating Th1 responses. Given their central role in the immune system, DC represent an important target for new vaccines against viral infections. Various approaches have been developed to ensure efficient access of exogenous antigens to the MHC class I for processing and to induce CD8+ T cell responses.33,34 The use of either particulate antigens coupled to latex beads or encapsulated in liposomes or of recombinant bacteria or apoptotic cells has been demonstrated to elicit both CD4+ and CD8+ cell responses.33,35,36 Red blood cells are an interesting tool for antigen delivery because they can be easily obtained and conjugated to protein antigens via biotin-avidin bridges.21 The capacity of RBC to deliver the HIV regulatory protein Tat into DC was recently investigated. RBC appear to be an effective tool for Tat delivery into DC, and INF-& could be used advantageously to augment the ability of RBC to induce Th1 responses (data submitted for publication).

Conclusions The results reviewed in this paper suggest that autologous RBC can be used as a new delivery system for enhancement of immunotargeting for vaccination purposes.4,13,21,27,28 With this system we were able to show that this kind of antigen construct has several potentially attractive features for vaccine development: 1. RBC-based immunization strategies do not require adjuvants or potentially toxic agents. The data thus far obtained demonstrate that this delivery system is effective in eliciting a significant humoral response comparable with that obtained with conventional adjuvants. The bases for this adjuvant effect are not yet fully understood; however, it must be taken into consideration that erythrocytes are naturally removed

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from circulation by macrophages and that these cells are able to process and present antigens associated to MHC molecules to the immune system.24 2. an important advantage of antigens coupled to RBC is that they do not have any side-effects in the host tissue; thus, even toxins could be potentially delivered with this system. 3. the antigen-carrying RBC are slowly removed from circulation over a period of 8-10 days or longer;21 thus, this system should be considered equivalent to a continuous daily boostering. 4. this antigen delivery system can induce low but appreciable titres of anti-avidin antibodies.4,27 In our opinion this can be considered an advantage in terms of targeting the antigen-carrying RBC to macrophages,37 suggesting that the antibodies raised after a first immunization against the coupled antigen and avidin are able to recognize the carrying antigen RBC when administered the second time. This opsonization by autologous antibodies and by complement deposition is able to facilitate their recognition by macrophages that have receptors for both Fc and complement. 5. this antigen delivery system is able to induce an immunogenic response in mice with an amount of antigen 10-1250 times lower than that of the conventional administration. This could represent a further advantage, at least in terms of costs when recombinant proteins have to be used. 6. the antigen carrying RBC appear to be an effective tool for antigen delivery into DC, and INF-& could be used advantageously to augment the ability of RBC to induce Th1 responses. In conclusion, the studies reviewed herein have extended the immunotargeting approach to adjuvant-independent immunization and support further exploitation of this new strategy as a viable alternative in the design of new vaccines and new delivery systems.

Acknowledgements This work was supported by “Progetto AIDS I.S.S. 1999” and “P.F. Biotechnology” CNR Italy.

References 1. Ulmer JB. An update on the state of the art of DNA vaccines. Curr Opin Drug Discov Devel 2001;(2):192-197. 2. Gupta RK, Siber GR. Adjuvants for human vaccines—Current status, problems and future prospects. Vaccine 1995; 13(14):1263-1276. 3. Kissel T, Koneberg R, Hilbert AK et al. Microencapsulation of antigents using biodegradable polyesters: Facts and fantasies. Behring Inst Mitt 1997; (98)"172-183. 4. Magnani M, Chiarantini L, Vittoria E et al. Red blood cells as an antigen delivery system. Biotechnol Appl Biochem 1992; 16:188-194. 5. Freytag LC, Clements JD. Bacterial toxins as mucosal adjuvants. Curr Top Microbiol Immunol 1999; 236:215-236. 6. Forni G, Boggio K. Cytokine gene-engineered vaccines. Curr Opin Mol Ther 1999; 1(1):34-38. 7. Ross GD, Vetvicka V, Yan J et al. Therapeutic intervention with complement and beta-glucan in cancer. Immunopharmacology 1999; 42(1-3):61-74. 8. Smith GL, Symons JA, Khanna A et al. Vaccinia virus immune invasion. Immunol Rev 1997; 159:137-154. 9. Bumann D, Hueck C, Aebischer T et al. Recombinant live Salmonella spp. for human vaccination against heterologous pathogens. FEMS Immunol Med Microbiol 2000; 27(4):357-364. 10. Rhodes J, Zheng B, Morrison CA. Galactose oxidation as a potent vaccine adjuvant strategy. Efficacy in murine models and in protection against a bovine parasitic infection. Ann NY Acad Sci 1995; 754:169-186. 11. Warren HS, Vogel FR, Chedid LA. Current status of immunological adjuvants. Ann Rev Immunol 1986; 4:369-388. 12. Claassen E, de Leeuw W, de Greeve P et al. Freund's complex adjuvant: An effective but desagreeable formula. Res Immunol 1992; 143(5):478-483. 13. Chiarantini L, Magnani M. Immobilization of enzymes and proteins on red blood cells. In: Birckeustaff GF, ed. Methods in Biotechnology. Totowa: Humana Press, 1996:143-152.

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14. Kahane I, Polliack A, Rachmilewitz EA et al. Distribution of sialic acids on the red blood cell membrane in beta thalassaemia. Nature 1978; 16, 271(5646):674-675. 15. Drevin H, Richter W. Covalent coupling of proteins to erythrocytes by isocyanide. A new, sensitive and mild technique for identification and estimation of antibodies by passive hemaggluntination. J Immunol Methods 1985; 77(1):9-14. 16. Steinitz M, Tamir S. The coating of erythrocytes with detergent-solibilized molecules: A general method for improving coupling of antigens and antibodies. J Immunol Methods 1985; 76(1):27-38. 17. Gold ER, Fudenberg HH. Chromic chloride: A coupling reagent for passive hemagglutination reactions. J Immunol 1967; 99:859-867. 18. Jou YH, Bankert RB. Coupling of protein antigens to erythrocytes through disulfide bond formation: Preparation of stable and sensitive target cells for immune hemolysis. Proc Natl Acad Sci USA 1981; 78(4):2493-2496. 19. Nicolau C, Mouniemne Y, Tosi PF. Electroinsertion of proteins in the plasma membrane of red blood cells. Anal Biochem 1993; 214(1):1-10. 20. Muzykantov VR, Zaltsman AB, Smirnov MD et al. Target-sensitive immunoerythrocytes: Interaction of biotinylated red blood cells with immobilized avidin induces their lysis by complement. Biochim Biophys Acta 1996; 1279(2):137-143. 21. Magnani M, Chiarantini L, Mancini U. Preparation of characterization of biotinylated red blood cells. Biotechnol Appl Biochem 1994; 20:335-345. 22. Taylor R, Reist C, Sutherland W et al. In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor. J Immunol 1992; 148:2462-2468. 23. Suzuki T, Dale GL. Biotinylated erythrocytes: In vivo survival and in vitro recovery. Blood 1987; 70(3):791-795. 24. Kovacsovics-Bankowski M, Clark K, Benacerraf B et al. Efficiently MCH I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci USA 1993; 90:4942-4946. 25. Manservigi R, Grossi MP, Gualandri R et al. Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector. J Virol 1990; 64(1):431-436. 26. Inglis SC. Challeneges and progress in developing herpesvirus vaccines. Trends Biotechnol 1995; 13(4):135-142. 27. Chiarantini L, Argnani R, Zucchini S et al. Red blood celld as delivery system for recombinant HSV-1 glycoprotein B: Immunogenisity and protection in mice. Vaccine 1997; 15:276-280. 28. Chiarantini L, Matteucci D, Pistello M et al. AIDS vaccination studies using and ex vivo feline immundeficiency virus model: Homologous erythrocytes as a delivery system for preferential immunization with putative protective agents. Clin Diagn Lab Immunol 1998; 5:235-241. 29. Bonini C, Lee SP, Riddell SR et al. Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4(+) and CD8(+) T cells. J Immunol 2001; 166(8):5250-5257. 30. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-252. 31. Gallucci S, Matzinger P. Danger signals: SOS to the immune system. Curr Opin Immunol 2001; 13:114-119. 32. Katajima T, Caceres-Dittmar G, Tapia FJ et al. T-cell mediated terminal maturation of dendritic cells. J Immunol 1996; 157:2340-2347. 33. Jondal M, Schrimbeck R, Reimann J. MHC class-I-restricted CTL responses to exogenous antigens. Immunity 1996; 5:295-302. 34. Jenne L, Schuler G, Steinkasserer A. Viral vectors for dendritic cell-based immunotherapy. Trends Immunol 2001; 22:102-107. 35. Ignatius R, Mahnke K, Rivera M et al. Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8+ T-cell responses in vivo. Blood 2000; 96:3505-3513. 36. Inaba K, Turley S, Yamaide F et al. Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med 1998; 188:2163-2173. 37. Friedman A, Zerubavel R, Gitler et al. Molecular events in the processing of avidin by antigenpresenting cells (APC). II. Identical processing by APC of H-2 high- and low-responder mouse strains. Immunogenetics 1983; 18(3):277-290.

CHAPTER 6

Engineered Nanoerythrocytes as a Novel Drug Delivery System Sanjay Jain and N. K. Jain

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ngineered nanoerythrocytes (nEs) as a novel controlled drug delivery system is a topic of interest to the pharmacist working in industry, research and development, academia, drug control administrators and professionals. This Chapter presents an overview of the concept of controlled drug delivery, advantages, the erythrocyte membrane, requirements for encapsulation, isolation of erythrocytes and preparation of erythrocyte ghosts; preparation, loading of nanoerythrocytes and their optimization; inside out red cell membrane vesicles, in vitro characterization, routes of administration, stability studies, in vivo studies and toxicity; immunological considerations, targeting potential and applications; and advances, conclusion and future prospects of engineered nEs. This novel drug delivery system is endowed with several exclusive advantages and hence holds potential for further research and clinical applications.

Introduction Drug delivery is now entering quite an exciting and challenging era. Significant high costs involved in the development of new drug molecules have compelled scientists all over the world to search for alternative ways of administering the existing drug molecules with enhanced effectiveness. Apart from this, conventional drug delivery generally has a limited level of precision and specificity for pharmacodynamic activity and is accompanied by profound undesirable side effects, lack of efficacy, inconvenience due to multiple dosing etc. Improper drug distribution inside the biological system not only causes distress to other body tissues but also demands more therapeutic molecules to elicit the appropriate response. Frequent and repeated dosing is required to maintain a therapeutic drug level due to drug instability in the biological milieu. Drugs have often been observed to be ineffective due to their lack of interaction with receptors and the development of resistance in the case of microorganisms. In these perspectives, treatment of various diseases reflects a compromise between the beneficial and hazardous effects. Thus improvement of drug delivery is of utmost importance for their better utilization. Current research in drug delivery is aimed at maximizing the therapeutic efficacy of drugs and minimizing side effects. Targeted or site-specific drug delivery is but a means of achieving this objective. The concept of designing a delivery system to achieve drug targeting originated from the perception of Paul Ehrlich, who imagined drug delivery as a “magic bullet”, describing the targeted drug delivery system as an event where a drug carrier complex delivers drug(s) exclusively to the preselected target cells. Gregoriadis described targeting with the help of novel drug delivery systems as ‘old drugs in new clothes’.1 Among the various carriers used for targeting drugs to various body tissues, the cellular carriers meet several criteria desirable in clinical applications, among the most important being biocompatibility of carrier and its degradation products. Leukocytes, platelets, erythrocytes and nanoerythrocytes etc. have been proposed as cellular carrier systems. Among these, the erythrocytes have been the most investigated and have been found to possess great potential in Erythrocyte Engineering for Drug Delivery and Targeting ©2002 Eurekah.com.

, edited by Mauro Magnani.

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drug delivery. In its infancy, erythrocyte encapsulation attracted many scientists who were enamored with the idea of RBC encapsulation. In the past three decades many claims have been made about RBC as useful carriers for enzymes and other exogenous agents. Attempts towards encapsulation of substances in erythrocytes and other carrier systems began in early 1970’s. The term carrier RBC was first introduced in 1979. Such erythrocytes, which contain no or little hemoglobin, are called “ghosts”. Jain & Jain2,3 have extensively reviewed the topic of engineered erythrocytes. Recently, a derivative of erythrocytes ghosts, engineered nanoerythrocytes (nanovesicular carriers, reverse biomembrane vesicles, red cell membrane vesicles, nanoerythrosomes, etc.), have been proposed as a new carrier, which could be useful as drug carrier. Nanoerythrosomes (nEryt)4 is a patented carrier system.

Advantages Nanoerythrocytes (nEs) have many advantages. They are natural products of the body; biodegradable in nature; isolation is easy and large amount of drug can be loaded in a small volume of cells; non-immunogenic in action and can be targeted to diseased tissue/organ; prolong the systemic activity of drug while residing for a longer time in the body; protect the premature degradation, inactivation and excretion of proteins and enzymes; act as a carrier for a number of drugs; target the drugs within the reticuloendothelial system (RES) as well as nonRES organs/sites; biocompatible; no possibility of triggered immunological response; drug is chemically bonded with the protein of the erythrocyte membrane; less prone to aggregation and fusion; and flexibility of membrane allows them to escape RES for longer periods.5,6

The Erythrocyte and Erythrocyte Membrane The erythrocyte membrane’s relative simplicity, availability and ease of isolation have made it the most extensively studied and best understood biological membrane. It is therefore a model for the more complex membranes of other cell types. A mature mammalian erythrocyte is devoid of organelles and carries out few metabolic processes; it is essentially a membranous bag of hemoglobin. Erythrocyte membranes can therefore be obtained by osmotic lysis, which causes the cell contents to leak out. The resultant membranous particles are known as ‘erythrocyte ghosts’ because, upon return to physiological conditions, they reseal to form particles that retain their original shape. Indeed, by transferring sealed ghosts to another medium their contents can be made to differ from the external solution. The erythrocyte membrane has a more or less typical plasma membrane composition of about one-half protein, somewhat less lipid, and the remainder carbohydrate. The fluidity and flexibility imparted to an erythrocyte by its membrane skeleton has important physiological consequences. A slurry of solid particles of a size and concentration equal to that of red cells in blood has the flow characteristics approximating that of sand. Consequently, in order for blood to flow at all, much less for its erythrocytes to squeeze through capillary blood vessel smaller in diameter than they are, erythrocyte membranes, with their membrane skeletons, must be fluid-like and easily deformable.7

Requirements for Encapsulation Nanoerythrosomes are patented nano-vesicles (Dignocure Inc., Canada) derived from red blood cell membranes through a process of hemodialysis through filters of defined pore size.8 nEs vesicles have the ability to be loaded with a diverse array of biologically active agents including proteins. The nanoerythrocyte’s membrane is a most versatile natural membrane structure, which is composed of proteins, phospholipids and cholesterol. The presence of membrane is particularly advantageous since it permits the conjugation, using simple and wellknown chemistry of polyethylene glycols and proteins, of for example, monoclonal antibodies. Additionally, natural membrane stability allows the insertion of recombinant ligands providing another method for incorporating targeting moieties into the nanoerythrosomes. A wide variety of biologically active substances can be loaded with nanoerythrosomes. Generally, the molecules should be polar or hydrophilic but non-polar molecules have also been successfully entrapped. The erythrocyte membrane contains approximately 60% protein and 40% lipid by

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dry weight. A wide variety of drugs having hydroxyl, amino and/or carboxyl groups are good candidate for drug conjugation.5

Isolation of Erythrocytes, Preparation of Erythrocyte Ghosts and Nanoerythrocytes Separation and Washing of Erythrocytes: Blood samples can be collected from animals by cardiac puncture (rats, mice etc.) / vein puncture (rabbit, human etc.) into a syringe containing heparin sodium (100 I.U./ml in 0.9% saline). The freshly collected blood is centrifuged in a refrigerated centrifuge at 1000 rpm for 10 minutes at 4±1°C. The plasma and buffy coats are discarded and sedimented erythrocytes are washed with washing buffer (pH 7.4). These washed cells are stored at 4±1°C (Fig. 1). Preparation of Erythrocyte Ghosts: The hypotonic osmotic lysis method described by DeLoach et al9 can be used for the preparation of ghost suspension. The cells are lysed and washed several times with hypotonic saline solution. After each wash, the solution is centrifuged at 1000 rpm for 10 minutes at 4±1°C in a refrigerated centrifuge and the supernatants are aspirated and discarded. The ghost suspension is finally obtained when supernatant becomes colorless. Packed ghost cell suspension is diluted with 0.9% saline to obtain 50% hematocrit and stored at 4±1°C until used. Preparation and Loading of Nanoerythrocytes: The nanoerythrocytes can be prepared using extrusion, sonication and electrical breakdown methods. In the extrusion method erythrocyte ghosts are passed through polycarbonate membrane filter, which causes them to break into smaller vesicles, nanoerythrocytes; in the sonication method erythrocyte ghosts are converted into small vesicles using a dismembrator; and the electrical breakdown method is used to convert ghosts into small vesicles under the influence of electrical potential. Of the three methods nEs prepared by extrusion method yield vesicles of more uniform size. It is a quicker and cheaper method in comparison to sonication and electrical breakdown. Furthermore, in the sonication and electrical breakdown methods, heat generated during preparation if not controlled, may also modify the membrane. Drugs are conjugated to nEs with the help of a crosslinker. The nanoerythrocyte concentration is determined by quantitation of protein by a reported method.10 Methotrexate (MTX) was loaded on nanoerythrocytes by three methods, namely extrusion [E], sonication [S] and electrical breakdown [EB]. Jain and Jain11,12 have developed an electrical breakdown method. Various process variables that could affect the preparation and properties of the nEs were established, identified and optimized. The MTX loaded nanoerythrocytes were characterized in vitro for various pharmaceutical and physiochemical attributes.13-19 A brief review of this work is presented below. Extrusion Method: The extrusion method reported by Lejeune et al20 and Desilets et al21 was used with little modification for conjugation of drug with nEs. The erythroycte ghost suspension (50% Hct) was extruded through a polycarbonate membrane filter (0.4 ∝m; Millipore, USA) attached to an adapter. nEs were obtained by eight consecutive extrusions and the final preparation was stored at 4±1°C in a refrigerator. Extrusion was conducted under nitrogen pressure (Fig. 2). The yield as determined by protein recovery was 80%. Drug Nanoerythrocyte Conjugation: MTX was conjugated to nEs membrane using gluteraldehyde (G) as a crosslinker; 2,500 ∝g of drug was added to 2ml of nEs preparation (50% Hct) in the presence of 0.03% gluteraldehyde (in 0.9% saline) in a final volume of 3ml. The mixture was incubated for 45 min. at 4±1°C and then the reaction was terminated by addition of 1ml of 15% glycine solution in 0.9% saline. The suspension was centrifuged at 20,000 x g for 20 min. at 4±1 °C. The nanoerythrocytes-glutaraldehyde-methotrexate complex (nEs-G-MTX) were washed four times with 5 ml of PBS until no free MTX was detected in the supernatant. The MTX-G-nEs complex was separated from free MTX by centrifugation. Finally the preparation was stored at 4±1°C in a refrigerator until used. All stages were accomplished under aseptic conditions. To check the membrane binding of MTX on nEs, samples were treated identically except that crosslinker was not used.

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Figure 1. Electron microphotograph of normal erythrocytes (x 1500).

The optimized parameters under the extrusion method were drug concentration (2.0 mg/ ml of MTX), pore size of membrane (0.4 ∝m), type and concentration of crosslinker (gluteraldehyde; 0.030%), incubation period (45 min.), and temperature (37°C). Nearly 1.02.5% of MTX was found to be associated with the cells when estimated in the control experiment, which could be ascribed to the adsorption of the drug on nEs. The formulation was prepared using optimum parameters. The maximum drug conjugation using optimized variables was found to be 210ng/∝g protein. Sonication Method: The procedure reported by Al-Achi and coworkers22-24 and Jain and Jain25 was used for the preparation of nEs. The erythrocyte ghost suspension (50% Hct, in 0.9% saline) was sonicated at an energy level of 50W for 5 minutes using a dismembrator (Sico, India). During the experiment, the temperature was maintained at 4±1°C and finally nEs were stored at 4±1°C in a refrigerator. Drug-nanoerythrocyte conjugation was affected as described under the extrusion method. The optimised parameters were drug concentration (2.5 mg/ml), sonication (50W for 3min.), type and concentration of crosslinker (gluteraldehyde; 0.030%), incubation period (45 min.), and temperature (37°C). The maximum drug conjugation using optimized variables was found to be 175 ng/∝g protein. Electrical Breakdown Method: The erythrocyte ghost suspension (50% Hct in 0.9% saline) was subjected to variable electrical voltage for 100∝s. During the experiment the temperature was maintained at 37°C and finally nEs were stored at 4±1°C in a refrigerator. MTX-G-nEs were separated by centrifugation. Drug was conjugated to nanoerythrocytes as described under the extrusion method. The optimized parameters under the electrical breakdown method were: drug concentration (2.0mg/ml), voltage (2KV/cm and 200∝s), type and concentration of crosslinker (gluteraldehyde, 0.030%), incubation time (45 min.), and temperature (37°C). The maximum drug conjugation using optimized variables was found to be 160ng/∝g protein.

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Figure 2. Mechanism for formation of nanoerythrosomes using extrusion method.

Inside-Out Red Cell Membraneous Vesicles The plasma membrane navigates with two distinctly different compartments. Investigation of the biochemical specialization across this membrane has been limited by the inaccessibility of its inner surface to direct examination. This problem was approached by promoting the budding of red cell plasma membrane ghosts into their cytoplasmic vesicles whose outer faces are the cytoplasmic space thereby generating inside-out vesicles whose outer faces are the

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Figure 3. Proposed course of energization for the formation of inside-out and right-side out nanoerythrocytes.

cytoplasmic aspects of the parent membranes. Conversely, normally oriented vesicles are formed when a surface membrane buds into the extracellular space.26 Ginn et al27 have presented methods for the preparation and purification of inside-out and right-side out red cell membrane vesicles suitable for the direct comparative analysis of the membrane’s two faces (Fig. 3). A new derivative of erythrocyte ghosts, reverse biomembrane vesicles, was developed and loaded with doxorubicin hydrochloride.28 This formulation is based on budding of membrane into ghost interiors (endocytosis) leading to accumulation of small vesicles within each parent

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ghost. The stability studies were performed at 4°C by assessing the effect on vesicle size and leakage after 7, 15, and 21 days. The stability showed negligible leakage and no appreciable change in vesicle size after storage for several days indicating good stability of vesicles. The formulation and process variables were optimized to obtain vesicles of 1.2 to 1.8 ∝m. The amount of doxorubicin entrapped in vesicles was 0.75 mg/ml of packed vesicles. The in vitro release profile showed 52.86% of drug release in 16hr. A membrane orientation study revealed inversion of membrane surface because only 10% of sialic acid was accessible on trypsin treatment. The plasma clearance data revealed an increase in half-life and bioavailability of drug. Tissue concentration data suggest that liver and spleen remain the major organs for clearance of these drug loaded vesicles, which suggest preferential uptake by the RES.

In Vitro Characterization The optimized nEs-G-MTX formulations were prepared using the optimized parameters. These optimized formulations were subjected to in vitro characterization for evaluation of their suitability as drug carriers. Percent recovery/protein content: Nanoerythrocyte concentration is determined by quantitation of the protein content by a method reported by Lowery et al10 The mean particle size was found to be 130±26, 170±30 and 190±25 nm, respectively for methotrexateglutaradehyde-nanoerythrocytes (extrusion method) (MTX-G-nEs[E]), methotrexateglutaradehyde-nanoerythrocytes (sonication method) (MTX-G-nEs[S]), and methotrexateglutaradehyde-nanoerythrocytes (electrical breakdown method) (MTX-G-nEs[EB]). The percent recoveries of nEs were found to be nearly 80.8%, 76.6% and 76.42% in case of extrusion, sonication and electrical breakdown methods, respectively. Morphological examination: The shape and size of MTX-G-nEs are determined using SEM and TEM after proper dilution. Mean values and standard deviation for overall particles are determined after staining with uranyl acetate. Fig. 4 shows morphology of ghost cells. The electron microscopic examination of MTX-G-nEs(E) (Fig. 5) revealed that these are spheroid closed vesicles. The same observations were made invariably at all drug concentration, the control nEs also exhibited similar characteristics. TEM microphotographs (Figs. 6,7) represent samples of MTX-G-nEs[S] and MTX-G-nEs[EB] respectively. MTX-G-nEs[E] are more uniform in size in comparison to MTX-G-nEs[S] and MTX-G-nEs[EB]. The size of nanoerythrocytes were determined by electron microscopy after staining with uranyl acetate (1%). Scanning/Transmission Electron Microscopy of nEs:: Ghosts/erythrocyte-vesicles were diluted with saline water by mixing two parts of ghosts or erythrocyte-vesicles suspension with 25 parts of saline buffer. The formulation was dehydrated and dried to critical point by using elevated concentrations of alcohol. One drop of the diluted mixture was placed on a silvercoated copper grid, then coated with gold. The material was tested on a scanning electron microscope. Ghosts/erythrocyte-vesicles are diluted with distilled water by mixing two parts of ghost or erythrocyte-vesicular suspension with 25 parts of saline buffer. One drop of the diluted mixture was placed on a coated copper grid and negatively stained with 1% uranyl acetate (pH 7.0). The material was examined on a transmission electron microscope. Percent drug conjugation: MTX-G-nEs (0.2 ml) were deproteinized using acetonitrile after centrifugation at 20,000 x g for 15 min. The clear supernatant is withdrawn and percent drug conjugation is estimated. Centrifugal stress: For centrifugal stress study, MTX-G-nEs (10% Hct) were centrifuged at variable rpm in a refrigerated centrifuge at 4±1° C for 15 min. Drug leakage in supernatant solution is estimated. The formulations are stable against centrifugal stress, as only 2.62 % , 2.92 % and 3.20% of drug could be released after centrifugation at 7500 rpm for 15 minutes from MTX-G-nEs(E), MTX-G-nEs(S) and MTX-G-nEs(EB), respectively. Centrifugal stress is the reliable parameter for the in vitro evaluation of nEs with respect to shelf-life, in vivo survival and the effect of the encapsulated substances. Turbulence shock: The formulations were passed through a 25 gauge hypodermic needle at the rate of 8-10 ml/minute and drug leakage in supernatant is estimated after a fixed number of

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Figure 4. Electron microphotograph of a ghost cell (x 10,000).

passes. MTX-G-nEs(E), MTX-G-nEs(S) and MTX-G-nEs (EB) were found to withstand turbulence shock as only 0.882%, 0.991% and 1.66% of drug could be released after 15 passes through 25 gauge needle. It is a measure of simulating destruction of loaded nEs during injection. Viscosity and relative density: A rotatory viscometer (Brookfield Synchro-Lectic LVT, USA) was used to determine the viscosity of the formulation. The viscosity of MTX-G-nEs(E), MTXG-nEs(S) and MTX-G-nEs(EB) was found to be slightly higher than viscosity of nEs (36.2, 34.0, 36.6, and 29.2 cps respectively). The relative density of the formulation was determined using relative density bottle. The relative density of MTX-G-nEs(E), MTX-G-nEs(S) and MTXG-nEs(EB) was found to be slightly higher than relative density of nEs (1.222, 1.118, 1.114, and 1.095a respectively). Sedimentation volume: Sedimentation volume of unity in each case revealed very good stability of the formulation. It has been reported29 that polystyrene latex particles of size less than 500 nm do not settle even upon one month storage. In vitro release: In vitro release studies were conducted using dialysis tubing (Sigma, USA). The in vitro drug release of MTX-G-nEs[E], MTX-G-nEs[S] and MTX-G-nEs[EB] was found to be 16.1, 22.2 and 25.1%, respectively after 8 hr. following first order release. Antineoplastic activity: The antineoplastic activity of both free and conjugated MTX was determined on leukemia cell line (L-1210). These studies were performed according to the method reported.30 Cell viability was measured by the tryphen blue extrusion test, which is based on the ability of tryphen blue to stain dead cells. A drop of culture was added on haemocytometer and stained, non-stained and the total number of cells were counted. MTXG-nEs have higher cytotoxic and neoplastic activity than free drug as seen in in vitro studies on leukemia cells (L-1210). In vitro leukemia cell (L-1210) toxicity studies demonstrated that toxicity of nanoerythrosomes increased in proportion to the concentration of the drug. The percent inhibition (ratio of difference between number of viable cells and number of viable cells after treatment to number of viable cells without treatment) was 90.6, 89.6, 89.2, and 87.6% for MTX-G-nEs[E], MTX-G-nEs[S], MTX-G-nEs[EB] and free MTX respectively after 24 hours.

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Figure 5. TEM of MTX loaded nanoerythrocytes (Extrusion method) (x 10,000).

Mechanism of action: An attempt was made to evaluate drug-cell line interaction. Cell line (Leukemia, L-1210) and formulation were incubated and studied by fluoroscence microscopy. Using microscopic studies it was observed that the MTX-G-nEs neither diffused through the cell membrane nor entered the cell by endogenesis. The MTX-G-nEs are rapidly absorbed on the cell membrane. Free MTX was then slowly released by hydrolysis of the gluteraldehyde linking arms, producing a high concentration of free drug in the cell vicinity over a prolonged period of time. The cytotoxicity of MTX conjugated to nEs is mediated by attachment of nEs complex to the cell membrane, slow hydrolysis of the linking arm, protection of a high concentration of drug around the cell periphery that will penetrate into the cells over a long period of time. The results are nearly similar to Moorjani et al.31 Further, they had studied localization of daunorubicin (DNR) in P388D1 cells exposed to 5 ∝g/ml of free and nEryt conjugated drug after exposure of 30 min., 1hr. and 4hr. The results indicate that cytotoxic activity of nErytDNR complex is much more as compared to free drug. Others: The spectroscopic based characterization of nEryt has been reported by workers at Canada.8

Routes of Administration The in vivo evaluation of drug-carrier nanoerythrocyte preparation is normally conducted in laboratory animals, which include mice, rats and rabbits. The carrier cells are normally injected intravenously or intraarterially; however, the intraperitional and subcutaneous routes can also be utilized.

Stability Studies Effect of aging: The formulations [MTX-G-nEs(S), MTX-G-nEs(E) and MTX-G-nEs(EB)] were kept at variable temperature and change in particle size, sedimentation volume and relative

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Figure 6. TEM of MTX loaded nanoerythrocytes (Sonication method) (x 10,000).

turbidity were monitored periodically. Stability of these formulations were also assessed at 4±1°C, R.T. and 37±1°C over time. Formulations exhibited little change in relative turbidity at 4±1°C but more at R.T. and 37±1°C. The effect of temperature on sedimentation volume with aging was negligible. Effect of mice serum: Mice serum (0.1 ml) was added to 1.0 ml of the formulation and incubated at 37±1°C for 24 hr. The change in formulation before and 24 hr. after addition of mouse serum were measured and compared. In addition to particle stability of these formulations over time at different temperatures, these were also assessed after incubation at 37±1°C in the presence and absence of serum in order to have an idea about in vivo stability. These formulations exhibited better stability in the presence of serum as revealed from turbidity measurement. Effect of centrifugation: Each formulation was taken in centrifuge tubes and centrifuged at different rpm for 20 minutes and sedimentation volume was noted. After centrifugation at 2500 to 10000 rpm for 15 minutes, sedimentation volume of these formulations remained at unity. This indicated stability of formulation upon centrifugal stress. Separation of formulations in powder form: The MTX-G-nEs were collected by centrifugation and dried in a vacuum (at 200 mm of Hg) for 10 hr. The MTX-G-nEs suspension was filled in vials and lyophilized at -40°C to 0.01 torr using a laboratory lyophilizer (Sico, India). The dried powder thus obtained was filled in amber colored glass vials, sealed and stored at 4°C in a refrigerator. The MTX loaded nEs were easily transformed into dry powder by vacuum drying and lyophilization. The morphological examination of the dry vesicles under electron microscopy (Fig. 8) exhibited no alteration in the vesicle morphology as compared to the vesicles not subjected to drying. The drug content of lyophilized erythrocyte powder was monitored for drug content every month, for 4 months. Drug recoveries of 99.32% to 98.23% were recorded. The drug content of stored cells estimated periodically for 4 months remained constant. Following 1, 2 and 4 months of storage of the lyophilized powder, the cells were suspended in PBS (5% Hct). In vitro release profiles of MTX after 1, 2 and 4 months of storage were recorded. The dried formulation ( loaded) exhibited release of 12.10 to 13.42% of drug upon storage for 4 months. Thus no significant variation could be detected in release of conjugated MTX from freshly prepared formulations which exhibited the release of 12.10 to 16.36% of MTX.

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Figure 7. TEM of MTX loaded nanoerythrocytes (Electrical breakdown method) (x 10,000).

Thus a major problem encountered with the nEs carrier, i.e., shelf-stability, could be resolved by storing them in a powder form. Further the shelf life of the carrier may be enhanced to months as compared to normal nEs. Furthermore, MTX-G-nEs as powder could be considered as and may be highly promising as a stable carrier for MTX to be used for sustained delivery and localization especially in different organs on the basis of their particle size. However, extensive studies are required in order to investigate the biochemical changes of drug loaded nEs upon storage for prolonged periods of time, the effect of storage on in vivo disposition of nEs etc.

In Vivo Studies and Toxicity The formulated products with promising in vitro performance were further evaluated for their in vivo performance on albino mice. Pharmacokinetic profile:The albino mice of either sex (average weight 20-25 g) were divided into five groups each comprising six mice. The first group was administered drug solution equivalent to 200 ∝g of MTX (calculated at dose level of 8 mg/kg). The second to fourth groups were administered with a formulation [MTX-G-nEs(S), MTX-G-nEs(E) and MTXG-nEs(EB) respectively]. The fifth group was a control. The blood samples were collected at different time intervals from retro-orbital plexus using a heparinized syringe; the samples were centrifuged and plasma was collected. The samples were analyzed for drug content by polarography.32 Tissue distribution studies: Albino mice of either sex weighing about 20-25 g were used to study the organ distribution of MTX. The mice were divided into five groups each containing six mice. The animals of the first group were given drug solution (equivalent to 200 ∝g of MTX) through the caudal vein. The animals of the second, third and fourth groups were given MTX-G-nEs(E), MTX-G-nEs(S) and MTX-G-nEs(EB) formulations (equivalent to 200 ∝g MTX respectively) through the caudal vein. The fifth group was a control. After 1 and 24 hr. mice of each group were scarified. The body organs (liver, spleen, kidney, bone marrow, and plasma) were removed. The body organs were washed to remove adhering debris and dried

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Figure 8. SEM micophotograph of MTX loaded lyophilized nEs (x 10,000).

with tissue paper. The organs were homogenized and drug was extracted using an acetic acid : ethyl acetate mixture (1:2). The samples were centrifuged and supernatants were analyzed. Biochemical tests of liver function: The method of Reitman and Frankel33 was used for the estimation of serum glutamate pyruvate transaminase (SGPT) while the method reported by King and King34 was used for the estimation of serum alkaline phosphatase (SALP). All the estimations were done on an autoanalyser [RA-50, Technicon,USA]. The blood MTX levels following the administration of MTX in free and MTX-G-nEs form were recorded. At 1 hr. post administration, the free methotrexate level in blood was reduced to 50%, and only 1.5% of the administered dose was present at 24 hr post dosing. Blood levels of MTX-G-nEs administration were clearly sustained as compared to free drug. This establishes the efficacy of nEs in prolonging the release of entrapped drug and also the structural integrity of the nEs. If the nEs had maintained their integrity in vivo, they would have been excreted rapidly. The observation of the organ distribution and blood level study led to the conclusion that upon in vivo administration, the nEs were localized preferentially in the liver, spleen and bone marrow by virtue of phagocytic uptake by the RES. They maintained their structural integrity and continued to release the entrapped drug over a prolonged period of time. In order to evaluate the systemic availability, organ specificity and in vivo performance of developed erythrocyte systems, tissue distribution of drug following I.V. administration of MTX-G-nEs was estimated in various organs. The tissue distribution of unmetabolised MTX (represented as percent of dose injected) was recorded. After 1hr. of administration of drug solution 1.47, 3.24 and 1.10 ∝g/g of drug was found in liver, kidney and spleen, respectively, which declined by 24 hr. From bone marrow 0.22% of the MTX was recovered after 1 hr. The drug level in bone marrow also declined after 24 hr. when less than 1 ∝g/g of the drug was recovered in all the organs except spleen. Following administration of MTX-G-nEs, higher hepatic, splenic, kidney and bone marrow localization of MTX was noted.

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MTX-G-nEs are reported to be cleared from circulation by RES recognition,5 by phagocytosis in the liver, by the Kupffer cells resulting in sustained drug release from these cells. The drug is made available to the parenchymatous cells and hepatocytes and metabolized following slow release from the Kupffer cells. The nEs administration thus provides an intrahepatic slow release system of MTX with possible modification of metabolism kinetics. The administration of the nEs carrier system resulted in an elevation in the level of unmetabolised MTX, suggesting the improvement in prophylactic and therapeutic efficacy of the drug. The efficacy may be related to the greater amount of unmetabolised drug available for activity against the cancer. In order to assess the selectivity of the erythrocytes for the RES, the drug localization index was determined at various times.5 The values were found to be greater than 1, indicating high specificity and selectivity of the erythrocyte carrier for the liver tissue. The value of the index increases with time, which could be due to the slow release of drug in liver. The tissue drug concentration data indicate the rapid clearance of MTX solution by splenocytes after 1 hr followed by decrease after 24 hr., while the spleenic clearance of MTXG-nEs after 24 hr. was found to be increased indicating higher circulation life of MTX-G-nEs. On the basis of in vivo studies it is clear that the nEs carrier resulted in a higher concentration of the MTX in the RES (liver, spleen and bone marrow), with slower elimination of drug as compared to the free drug administration. The higher concentration achieved in the RES and for prolonged time(s) may result in improvement in therapeutic efficacy of MTX against the liver tumors as more drug would be available for a longer time, thus aiding in cure of the disease by completely destroying the cancer cells. MTX-G-nEs can be utilized for treatment of all forms of tumors of the RES as well as for cure. The organ localization of the drugs may result in lowering of toxic manifestations. Biochemical tests of liver function indicated no significant change in values as compared to normal cells suggesting no detrimental effect of treated cells on liver.

Acute and Hematological Toxicity Studies

The method reported by Udupa30 was used for these studies. Acute toxicity in terms of body weight was determined in albino mice. Hematological toxicity of free and formulated MTX was determined by injecting (I.V.) 8.0 mg/kg of free and formulated MTX to albino mice. The peripheral and bone marrow counts of WBC were carried out at definite time intervals post injection. The study of acute toxicity revealed improving weight loss of animals 5 days after receiving MTX-G-nEs (E) in comparison to MTX solution. The WBC suppressive characteristics of MTX-solution and MTX-G-nEs were recorded. The peripheral and bone marrow WBCs responded similarly to MTX solution and MTX-G-nEs at a dose of 8.0mg/Kg. The drug induced decrease in WBC population was observed first in the bone marrow, followed by a decrease in the peripheral count. The least peripheral WBC count was seen on day 3 for both MTX-solution and MTX-G-nEs. Both peripheral and bone marrow WBC count reached almost normal values on day 9. Thus, it is concluded that drug-loaded nanoerythrocytes can be successfully used as drug carriers for controlled and targeted delivery of methotrexate.

Immunological Considerations Autologous erythrocytes are not immunogenic. However, there is concern that the lysis procedure utilized for drug entrapment might elicit some cryptic antigens. Desnick et al35 examined this phenomenon and found no immunological response against resealed erythrocytes. Further they examined the immunological response in mice to bovine ∋-glucuroxidasebearing autologous erythrocytes. The repeated I.V. administration of cells entrapping ∋glucuroxidase did not elicit any detectable antibody against ∋-glucuroxidase in control mice and those previously sensitized to ∋-glucuroxidase. There was no difference in tissue distribution or survival of administered ∋-glucuroxidase between the control and sensitized animals. Similar results can be expected in case of drug-loaded nEs, as these are derivative of erythrocyte ghost.

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Targeting Potential and Applications of Nanoerythrocytes nEs have been proposed for a variety of applications in medicine. Most of technologies developed by Diagnocure Inc., Canada involve an antibody as vehicle for delivery of nanoerythrosomes, which are tiny spheres loaded with general application drugs or toxic agents used specifically to destroy selected carrier cells.36 Lejune et al20,37 reported that doxorubicinnanoerythrosome conjugates have higher antineoplastic activity than the free drug on CDF1, leukemia tumors. They have reported covalent binding of daunorubicin to proteins using various crosslinkers. Moorjani et al31 reported the mechanism of action of drug loaded nanoerythrosomes. Al-Achi and coworkers22-24 have successfully reported erythrocyte membrane vesicular delivery of insulin and doxorubicin. Jain and Jain5,12,38 have reported nanoerythrosome-based delivery of mitomycin-C, hydroxyurea and 6-mercaptopurine. Mishra and Jain28 have reported reverse biomembrane vesicles bearing doxorubicin. Khopade et al39 have reported magnetic cellular nanovesicles for delivery of a bioactive agent.

Advances, Conclusion and Future Prospects In the future, nanoerythrosome technology will remain an active arena for research. A company that is developing products for human use is currently testing the commercial medical applications of nEryt in Canada. The coming years represent a critical time in this field as commercial applications are explored. In near future, nEs based delivery system with their ability to provide controlled and site specific drug delivery may revolutionize disease management. Diagnocure Inc. has recently been granted a patent from US patent office for a nErytbased prolonged release system that it developed to reduce the destructive adverse effects experienced by cancer patients on chemotherapy. According to the company, this system allows the delivery to affected parts, instead of affecting both healthy and affected cells. In animal testing, they have proven to be valuable in detecting pathology. Nanoerythrosomes coupled with antibodies can increase test sensitivities. The system is projected to be available in 3 to 5 years.36 To this end Diagnocure Inc., Canada has an exclusive agreement with Seragen/Ligand Pharmaceuticals for the exploitation of recombinant proteins derived in part from the transmembrane domain of diphtheria toxin. The International Society for the Use of Resealed Erythrocytes (ISURE) through its biannual meetings provides an excellent forum for exchange of information to the scientists in this exciting and rewarding field of research.

References 1. Gregoriadis G. Targeting of Liposomes: Study of influencing factors. In: Gregoriadis G, Senior S, Trauet A, eds. Targeting of Drugs. New York: Plenum Press, 1982: 155-184. 2. Jain S, Jain, NK. Engineered erythrocytes as a drug delivery system. Ind J Pharm Sci 1997; 59(6):275-281. 3. Jain S, Jain NK. Preparation, characterization and pharmaceutical potential of enginnered erythrocytes. Die Pharmazie 1998; 53(1):5-14. 4. Thoppil SO, Gandhi AK. Nanoerythrosome technology: A new drug delivery system. Express Pharma Pulse 1999; 5(49):14-30. 5. Jain S, Jain NK. Engineered nanoerythrosomes as a novel drug carrier. In: Jain NK, ed. Advances in Novel Drug Delivery. New Delhi: CBS publisher, 2001:332-364. 6. Widder KJ, Senyei AE, Ranney DF. In vitro release of biologically active adriamycin by magnetically responsive albumin microsphere. Cancer Res 1980; 40:3512-3517. 7. Voet D, Voet JG. Lipids and membranes. In: Voet D, Voet JG, eds. Biochemistry. New York: John Wiley and Sons, 1990:291-295. 8. Diagnocure.com/eryl/prod/tech.html 9. DeLoach JR, Barton C. Circulatory carrier erythrocytes: Slow release vehicle for an antileukemic drug, cytosine arabinose. Am J Vet Res 1982; 43(12):2210-2212. 10. Lowery OH, Rosebrough NJ, Farr AL, Randall RI. Protein measurement with folin reagent. J Biochem 1991; 193:265-275. 11. Jain S, Jain NK. Nanoerythrosomes: A new derivative of erythrocyte ghost as carrier for 6-mercaptopurine. Proc Ind Pharm Cong, India 1996:94.

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12. Jain S, Jain NK. Nanoerythrosomes as carrier for anticancer drug. 24th Intl Conf Contr Rel Soc, Sweden 1997:857-858. 13. Jain S, Jain SK, Dixit VK. Magnetically guided engineered erythrocytes bearing isoniazid: Prepration, characterization and evaluation. Drug Dev Ind Pharm 1997; 23:999-1006. 14. Jain S, Jain NK. Resealed erythrocytes as drug carrier. In: Jain NK, ed. Controlled and Novel Drug Delivery. New Delhi: CBS Publishers, 1997:256-291. 15. Jain S, Jain NK. Crosslinked engineered erythrocytes as a carrier for meglumine antimonate. Proc 26th Intl Conf Contr Rel Soc, Sweden 1999:575-576. 16. Jain S, Jain NK. Nanovesicular carrier for delivery of mitomycin-C. Proc 3rd Intl conf Cellular Engineering, Itlay 1997:121. 17. Mishra PR, Jain S, Jain NK. Engineered human erythrocytes as carrier for ciprofloxacin. Drug Delivery 1996; 3(4):239-244. 18. Jain S, Jain SK, Dixit VK. Erythrocytes based delivery of isoniazid: Preparation and characterization. Indian Drugs 1995; 32(10):471-475. 19. Magnani M, Casobianca A, Fraternate A et al. Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection. Proc Natl Acad Sci USA 1996; 93(9):4403-8. 20. Lejeune A, Moorjani M, Gicquard C, Lacroix J, Poyet P, Gauderault R. Nanoerythrosomes, a new derivative of erythrocyte ghost: Preparation and antineoplastic potential as drug carrier of daunorubicin. Anticancer Res 1996; 14:915-918. 21. Desilets J, Lejeune A, Mercer J et al. Nanoerythrosomes, a new derivative of erythrocyte ghost IV. Fate of reinjected nanoerythrosomes. Anticancer Res 2001; 21:1741-1748. 22. Al-Achi A, Boroujerdi M. Adsorption isotherm for doxorubicin on erythrocyte membrane. Drug Dev Ind Pharm 1990; 16(8):1325-1328. 23. Al-Achi A, Greenwood R. Human insulin binding to erythrocyte membrane. Drug Dev Ind Pharm 1993; 19(6):673-684. 24. Al-Achi A, Greenwood R. Intraduodenal administration of biocarrier-insulin system. Drug Dev Ind Pharm 1993; 19(11):1303-1315. 25. Jain S, Jain NK. Engineered cellular nanostructure bearing methotrexate for tumor targeting. Proc 51st Ind Pharm Cong, Indore, India 1999:76. 26. Steck TL, Weinstein RS, Straus JH, Wallach DFH. Inside-out red cell membrane vesicles: Purification and preparation. Science 1970; 168(10):255-257. 27. Gin FL, Hochsteuin P, Trump BF. Membrane alteration in hemolysis: Internalization of plasmalemma induced by primaquine. Nature 1969; 221:843-845. 28. Mishra PR, Jain NK. Reverse biomembrane vesicles for effective controlled delivery of doxorubicin hydrochloride. Drug Delivery 2000; 7:155-159. 29. Vonderhoff JW, El-Aasssar M. Theory of Colloids. In: Liberman HA, Rieger MM, Banker GS, eds. Pharmaceutical Dosage Forms: Disperse Systems. Vol I. New York: Marcel Dekkar Inc., 1988:93-98. 30. Udupa N. Targeted drug delivery for treating cancer and inflammation, In: Udupa N, ed. Novel Drug Delivary Systems: Manipal Experience. Udupi: Honna-Padma Press, 1995:294-320. 31. Moorjani M, Lejune A, Gicquadid C, Lacroir J, Poyet B, Gaudreault RC. Nanoerythrosomes, a new derivative of erythrocyte ghost II: Identification of mechanism of action. Anticancer Res 1997; 16: 2831-34 32. Dias AR, Dhake JK. Polarographic determination of methotrexate. The Royal Society of Chemistry’s (Anal Div), International Symposium on Electrolysis, Cardiff, UK 1994. 33. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyurvic transaminases. Am J Clin Path 1957; 28:53-56. 34. King PRN, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine. J Clin Path 1954; 7:322-324. 35. Desnick RJ, Fiddler MB, Donglas SD, Hudson LDS. Immunological response in mice to bovine b-glucuroxidase bearing autoiogous erythrocytes. Adv Expt Med Biol 1998; 101:753-754. 36. Woolley BH. Delivery system receives patent. Therapeutic Letter 1997; 3(8):2-3. 37. Lejeune A, Poyet P, Gaudreault RC et al. Nanoerythrosomes, a new derivative of erythrocyte ghost III: Is phagocytosis involved in the mechanism of action? Anticancer Res 1997; 17:3599-3604. 38. Jain S. Nanoerythromes bearing methotrexate for tumor targeting: prepration, characterization and performance evaluation. Proc Seminar on role of science in sustainable development, India 2000:6. 39. Khopade AJ, Jain S, Jain NK. Magnetically guided red cell vesicles as carrier. 24th Intl Conf Contr Rel Soc, Las Vegas, USA 1997:875-876.

CHAPTER 7

Red Blood Cells as Carriers of Antiviral Agents A. Fraternale, A. Casabianca and M. Magnani

I

n an attempt to overcome the problems of nucleoside analogue toxicity and of their lim ited efficacy and short plasma half-life we have investigated the possibility of using eryth rocytes both as a slow delivery system and as a targeting system for the release of drugs at specific sites. Herein we report some successful examples of using loaded erythrocytes as a delivery system for antiviral agents.

Introduction Erythrocytes possess the singular ability to swell and to become leaky when placed in hypoosmotic solutions. Extracellular substances can enter the red cell at this stage, after which membrane resealing can be performed using hyper-osmotic solutions. Resealed erythrocytes show normal shape, membrane permeability and biochemical characteristics; moreover, they can survive in circulation with a nearly normal life span.1-4 The procedure of encapsulation based on hypotonic dialysis, isotonic resealing and reannealing has also been used to load erythrocytes with antiviral drugs. Three different experimental conditions can develop: if the encapsulated drug is diffusible it can permeate the erythrocyte membrane and be released slowly into the circulation; if the drug is not diffusible it may be metabolized by erythrocyte enzymes into diffusible molecules; if the encapsulated drug is neither diffusible nor metabolized, it remains entrapped in the carrier cells and can be targeted to selective organs or cells by appropriate manipulations of the erythrocyte.

Red Blood Cells for a Slow Release of Antiviral Drugs Herein we report the use of erythrocytes to slowly deliver two nucleoside analogues currently used in the treatment of HIV infection: 2',3'-dideoxycytidine (ddCyd) and 3'-azido3’-deoxythymidine (AZT). Both drugs are potent anti-HIV-1 inhibitors but, due to their short plasma half-life, frequent administrations of the drug are needed to maintain therapeutically useful drug levels and can lead to the peak concentrations that are toxic. Encapsulation of AZT and ddCyd into erythrocytes would solve these problems, but unfortunately both AZT and ddCyd diffuse across the erythrocyte membrane and thus they cannot be encapsulated in these forms.5,6 For this reason we synthesized phosphorylated derivatives of ddCyd (2',3'dideoxycytidine-5'-phosphate, ddCMP) and of AZT (3'-azido-3'-deoxythymidine-5'-monophosphate, AZT-MP) as prodrugs and encapsulated them in human erythrocytes. We found that they are dephosphorylated by endogenous pyrimidine nucleotidases and subsequently released by the cells as ddCyd and AZT, respectively.

Human Erythrocytes as Bioreactors for the Release of ddCyd DDCMP was synthesized and loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing at concentrations of up to 4 ∝moles/ml packed erythrocytes.7 Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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The results obtained show that the appearance of ddCyd in the medium is accompanied by a disappearance of ddCMP in erythrocytes (not shown) and that the efflux of ddCyd is a linear function of ddCyd concentration. This strategy has two main advantages: one is represented by the fact that the prodrug is confined inside the erythrocytes, thus strongly reducing its toxicity; the other one is represented by the fact that the release of the drug happens slowly, reducing the toxic effects of ddCyd and ensuring long-lasting plasma concentrations.

Human Erythrocytes as Bioreactors for the Release of AZT AZT-MP was synthesized with the aim of checking whether it represents a prodrug of AZT.8 AZT-MP was encapsulated in human erythrocytes by a procedure of hypotonic hemolysis and isotonic resealing and found to be dephosphorylated to the corresponding nucleoside (AZT), which was subsequently released outside the erythrocytes. Small amounts of AZT-MP were released as well (Fig. 1). The results obtained suggest that even if the rate of AZT-MP dephosphorylation in the erythrocytes is too high, the red blood cells can be used to encapsulate other phosphorylated derivatives to obtain a still longer delivery of AZT.

Red Blood Cells for Targeting of Antiviral Drugs The main target cells for HIV-1 infectivity and propagation are macrophages and CD4+ T lymphocytes. Macrophage and microglia cells in the central nervous system of patients with AIDS dementia complex were identified as one of the first nonlymphocyte cell lineages that supported virus replication .10,11 Moreover, over the last few years it has been widely demonstrated that the combination antiretroviral therapy currently available fails to eliminate HIV-1 from infected individuals, indicating the existence of a refractory reservoir(s) of virus in these subjects .12-14 During the past few years, the persistence of a latent reservoir of HIV-1 in resting CD4+ T cells in HAART-treated individuals was considered to be a major impediment to the long-term control of HIV-1 infection.13,15,16 However, recent findings indicate that, although latent HIV in the resting CD4+ T-cell compartment is a potentially important source of re-emerging virus, it is not the sole and often not the main source of viral rebound after discontinuation of therapy in the majority of patients, indicating that other viral reservoirs, such as tissue macrophages, must be seriously considered.17-19 For these reasons, our attention turned to the protection of the macrophage compartment. For this purpose, erythrocytes subjected to the procedure of encapsulation by hypotonic hemolysis and isotonic resealing were treated with agents that promote band 3 clusterization, resulting in the formation of a new antigenic site that is readily recognized by autologous IgG and complement. This treatment increases phagocytosis of drugloaded erythrocytes, thus allowing the delivery of the encapsulated drug to the phagocytic cells. The first molecule to be targeted to macrophages was 2',3'-dideoxycytidine-5'-triphosphate (ddCTP). This choice was based on the consideration that macrophages have a reduced ability to phosphorylate several antiviral nucleoside analogues and thus the delivery of drugs in phosphorylated form could be advantageous. Although in order to protect macrophages an antiviral drug should be administered in a phosphorylated form for the reason given above, it should not be administered as a nucleoside 5'-triphosphate, but rather as a close precursor of it to avoid toxicity. Therefore, we synthesized an AZT homodinucleotide (AZTp2AZT) in which the pyrophosphate linkage would provide a target for enzymatic hydrolysis in the macrophages rather than in the carrier erythrocytes. The antiviral efficacy of both molecules was evaluated both in vitro and in vivo in a murine model of immunodeficiency. The animal model used consists of C57BL/6 mice infected with the retroviral complex LP-BM5 which develop severe immunodeficiency (i.e., murine AIDS).20,21

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Figure 1. AZT release from AZT-MP-loaded erythrocytes. Human red blood cells were loaded with AZTMP by a procedure of hypotonic dyalisis and isotonic resealing to a final concentration of 2 mM.9 These cells were then resuspended in physiological saline solution containing 5 mM glucose and incubated at 37°C at a 20% hematocrit. At the time intervals indicated, AZT and AZT-MP released in the medium were measured as described in ref. 8. AZT-MP (filled symbols) and AZT (open symbols) released in the medium. Reprinted with permission from: Magnani M, Giovine M, Fraternale A et al. In: Drug Delivery, Red blood cells as a delivery system for AZT, 2:57-61. © 1995 Academic Press, Inc.

Erythrocytes for Targeting of ddCTP The first experiments to evaluate the antiviral efficacy of ddCTP encapsulated in erythrocytes were performed in murine and human macrophages infected with LP-BM5 and HIV-1 respectively. Their efficacy was evaluated in terms of inhibition of proviral DNA and the results obtained are reported in Table 1; it is worth noting that ddCTP-loaded red blood cells were more efficient in protecting both human and murine macrophages than was free dideoxycytidine (ddC). Administration of ddCTP-loaded erythrocytes to LP-BM5-infected mice at 10-day intervals over a period of 3 months caused a reduction in all the features of disease such as lymphadenopathy, splenomegaly and hypergammaglobulinemia. Table 2 reports the results obtained with the combination of ddCTP-loaded erythrocytes and a classic antiretroviral drug: that is, AZT administered in drinking water. The combined treatment resulted in a further reduction of lymphadenopathy (a further 27% with respect to the single treatment of AZT) and splenomegaly (a further 28% with respect to the single treatment of AZT) but not of IgG levels. Also the proviral DNA content in spleen and lymph nodes was positively influenced by the combination of the two drugs (Table 2).

Erythrocytes for Targeting of AZTp2AZT

A new AZT analogue, the AZT homodinucleotide di(thymidine-3’-azido-2’,3’-dideoxyD-riboside)-5’-5’-p1-p2-pyrophosphate (AZTp2AZT), was synthesized and encapsulated in erythrocytes that had been properly modified to increase their recognition by macrophages.24

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Table 1. Inhibition of proviral DNA content in human and murine macrophages infected with HIV-1 and LP-BM5 respectively by ddC and ddCTP-loaded erythrocytes

% Inhibition

I+ddC I+ddCTP

vs.

Infected (I)

HIV-1

LP-BM5

77±4 93±3

56±4 90±3

Values are the means ± SD for 3 experiments. Macrophages were cultured and treated with 1∝M ddC or ddCTP at a final concentration of 1 ∝mole/ml erythrocytes as described in ref. 22. PCR detection of HIV-1 and LP-BM5 proviral DNA contents were performed as described in ref. 22.

Stability experiments on the compound were performed in human and murine erythrocytes and the results obtained showed that as much as 90% of AZTp2AZT was still present after incubation of the loaded erythrocytes under isotonic conditions for 24 h at 37°C in both species. In macrophages AZTp2AZT decreased, becoming undetectable 3 days after onset of phagocytosis of the loaded erythrocytes, and was converted to AZT-5'-triphosphate. The addition of AZTp2AZT-loaded erythrocytes to human macrophages infected with HIV-1 caused a significant reduction in both p24 production and proviral DNA content (Table 3). The treatment of C57BL/6 mice infected with LP-BM5 with weekly administration of AZTp2AZT encapsulated into autologous erythrocytes was found to reduce typical signs of the disease and the proviral DNA content in lymph nodes, spleen and brain (Table 4). The same treatment combined with oral AZT did not produce additive effects with respect to single treatments, except in the reduction of proviral DNA content in brain tissue (67%).

Red Blood Cells as Carriers of Antiretroviral and Antiherpetic Drugs Macrophages are not only important targets and reservoirs for HIV-1: they can also be easily infected by various herpes viruses, including herpes simplex virus type 1 and 2 (HSV-1 and HSV-2).26 In addition, HSV-1 is able to activate and increase HIV replication, thereby accelerating the progression of the disease.27,28 For these reasons, strategies able to inhibit replication of both viruses in macrophages are needed. A new antiviral agent (Bis-PMEA) consisting of two 9-(2-phosphonylmethoxyethyl) adenine (PMEA) molecules bound by a phosphate bridge was synthesized. The efficacy of PMEA in inhibiting both HIV and herpes viruses both in vitro and in vivo had already been demonstrated.29,30 However its efficacy is limited by the low cellular uptake caused by the negative charge of the phosphonate moiety. In order to increase the selective delivery of PMEA to macrophages, Bis-PMEA was synthesized and encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages. Bis-PMEA is stable inside the erythrocytes and in macrophages. Bis-PMEA and PMEA were found at high concentrations 10 days after the onset of phagocytosis of Bis-PMEA-loaded erythrocytes (not shown). Anti-HIV and anti-HSV-1 activity of Bis-PMEA encapsulated in erythrocytes was evaluated in human macrophages and was compared with antiretroviral activity of Bis-PMEA and PMEA given as free drugs in culture medium. The results obtained show that by administering Bis-PMEA-loaded RBC, 95% inhibition of HIV replication was obtained. Bis-PMEA and

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Table 2. Inhibition of splenomegaly, lymphadenopathy, hypergammaglobulinemia and proviral DNA content in LPBM5-infected C57BL/6 mice by AZT plus ddCTP encapsulated in erythrocytes

% Inhibition Splenomegaly

Lymphadenopathy

Hypergammaglobulinemia

Proviral DNA

Spleen

Lymph nodes

I+AZT

50±10

65±15

60±15

70±10

78±6

I+AZT +ddCTP

78±22

92±6

50±18

77±9

83±8

Values are means ± SD for 5 animals and were obtained 13 weeks postinfection. The percentage of inhibition refers to the infected (I) mice. All mice were infected with a single intraperitoneal administration of LP-BM5 virus and treated with 250 ∝g/ml of AZT in drinking water or AZT (250 ∝g/ ml) in drinking water plus ddCTP-loaded erythrocytes (1.7 ± 0.6 ∝moles ddCTP/ml erythrocytes) as described in ref. 23. Proviral DNA content was assessed by semiquantitative PCR (sqPCR) as described in ref. 23.

PMEA (both at the concentration of 1.0 ∝M) given as free drugs for the same time as the loaded RBC, inhibited HIV replication by 60% and 40% respectively. Another molecule able to protect macrophages from HIV and HSV-1 replication is a new heterodinucleotide (AZTp2ACV) consisting of both an antiretroviral and an antiherpetic drug, bound by a pyrophosphate bridge. This molecule was encapsulated in erythrocytes that were thereafter selectively targeted to macrophage cells, where metabolic activation of the drug occurred. The addition of AZTp2ACV-loaded erythrocytes to human macrophages provided effective and almost complete in vitro protection from HIV-1 and HSV-1 replication.31

Conclusions The use of properly engineered erythrocytes as a delivery system for antiviral agents has two main advantages: the drug is never free in circulation thus reducing toxicity, and the drug halflife in circulation becomes a function of the carrier survival. As a consequence, the pharmacokinetic patterns and therapeutic performances of drugs are significantly improved. Herein we have reported two examples (AZT-MP and ddCMP) of pro-drugs susceptible to metabolic conversion by endogenous erythrocyte enzymes to membrane-releasable active drugs (AZT and ddCyd respectively). The results obtained suggest that erythrocytes can be used to slowly release drugs, providing a new approach to the problem of the short plasma half-life of some molecules. Furthermore, engineered erythrocytes (either carriers or bioreactors) can be used for the release of drugs at specific sites. In particular, the natural phagocytic ability of macrophages renders these cells ideal compartments for erythrocyte-based delivery of drugs. In this Chapter we have reported some successful examples of this strategy. The efficacy of anti-HIV-1 compounds encapsulated and targeted to macrophages was tested in vitro and in vivo: the active form of ddCyd, ddCTP and a new AZT homodinucleotide, AZTp2AZT. We found that both

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Table 3. Inhibition of HIV-1 proviral DNA content and p24 production in macrophages by AZT and AZTp2AZT encapsulated in erythrocytes

% Inhibition vs. Infected (I) p24

Proviral DNA

4±3

70±1

I+AZTp2AZT

80±5

95±2

I+AZT

58±9

-

I+UL RBC

Values are the mean ± SD for 3 experiments. Macrophages were cultured and treated with unloaded erythrocytes (UL RBC), AZTp2AZTloaded erythrocytes at a final concentration of 0.4 ± 0.03 ∝moles/ml erythrocytes or 0.1 ∝M AZT as described in ref. 24. PCR detection of HIV-1 proviral DNA content wasperformed as described in ref. 24.

compounds were more effective in protecting macrophage cells by different antiretroviral infection than were their respective free nucleosides. These data are very interesting given the increasing importance assumed by macrophages as HIV-1 refractory reservoirs.16,17,19 For this reason, in our opinion new strategies aiming at protection of the macrophage compartment are becoming more and more necessary. Moreover, it should be remembered that macrophages are important hosts not only for HIV-1 but also for HSV-1, and that both viruses reciprocally enhance their replication in AIDS patients with non-genital herpes simplex lesions.32 We have briefly discussed the example of two new molecules (Bis-PMEA and AZTp2ACV) that, when encapsulated in erythrocytes and targeted to macrophages, are able to efficiently protect macrophages against de novo HIV and HSV infections. Very interestingly, preliminary experiments have shown that administration of AZTp2ACV-loaded erythrocytes can also protect macrophages against HSV-1 strains that are Acyclovir-resistant. This result is particularly important as severe infections with Acyclovir-resistant HSV have been documented in AIDS patients.33 Furthermore, we have demonstrated that additive antiretroviral effects can be obtained if the delivery system used to protect macrophages is combined with drugs that protect other cell compartments (i.e., lymphocytes). The data presented herein show that loaded red blood cells represent a new and unconventional therapeutic approach offering significant protection of macrophages against retroviral infections.

Acknowledgements This work was supported by Progetto AIDS I.S.S. 1999 and P. F. Biotechnology C.N.R. Italy

References 1. Ihler GM, Tsang HCW. Hypotonic hemolysis method for entrapment of agents in resealed erythrocytes. In: Green R, Widder KJ, eds. Method in Enzymology. Vol 149. Drug and Enzyme Targeting, Part B. San Diego: San Diego Academic Press, 1987:221-229. 2. Dale G. High-efficiency entrapment of enzymes in resealed red cell ghosts by dialysis. In: Green R, Widder KJ, eds. Method in Enzymology. Vol 149. Drug and Enzyme Targeting, Part B. San Diego: San Diego Academic Press, 1987:229-234.

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Table 4. Inhibition of splenomegaly, lymphadenopathy, hypergammaglobulinemia and proviral DNA content in LP-BM5-infected C57BL/6 mice by AZTp2AZT-loaded erythrocytes % of Inhibition Lymphadenopathy

Hypergammaglobulinemia

Proviral DNA

Spleen

I+AZTp2AZT

26±3

51±6

0

41±15

Lymph nodes Brain

38±10

36±8

Values are means ± SD of 10 animals and were obtained 10 weeks postinfection. The percentage of inhibition refers to the infected (I) mice. All mice were infected by two intraperitoneal administrations of LP-BM5 virus and treated with AZTp2AZT-loaded erythrocytes (1.06 ± 0.45 ∝moles /ml erythrocytes) as described in ref. 25. Proviral DNA content was assessed by semiquantitative PCR (sqPCR) as described in ref. 23.

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3. DeLoach JR. Dialysis method for entrapment of proteins into resealed red blood cells. In: Green R, Widder KJ, eds. Method in Enzymology. Vol 149. Drug and Enzyme Targeting, Part B. San Diego: San Diego Academic Press, 1987:235-242. 4. Ropars C, Avenard G, Chassaigne M. Large-scale entrapment of drugs into resealed red blood cells using a continuous-flow dialysis system. In: Green R, Widder KJ, eds. Method in Enzymology. Vol 149. Drug and Enzyme Targeting, Part B. San Diego: San Diego Academic Press, 1987:242-248. 5. Zimmerman TP, Mahony W, Prus KL. 3’Azido-3’-deoxythimidine an unusual nucleoside homologue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion. J Biol Chem 1987; 262:5748-5754. 6. Magnani M, Bianchi M, Rossi L et al. 2’, 3’-dideoxycytidine permeation of the human erythrocyte membranes. Biochem International 1989; 19:227-234. 7. Magnani M, Bianchi M, Rossi L et al. Human red blood cells as bioreactors for the release of 2’,3’-dideoxycytidine, an inhibitor of HIV infectivity. Biochem Bioph Res Comm 1989; 164:446-452. 8. Magnani M, Giovine M, Fraternale A et al. Red blood cells as a delivery system for AZT. Drug Delivery 1995; 2:57-61. 9. Magnani M, Rossi L, Brandi G et al. Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages: in vitro and in vivo studies. Proc Natl Acad Sci USA 1992; 89:6477-6481. 10. Koenig S, Gendelman HE, Orenstein JM et al. Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 1986; 233:1089-1093. 11. Glass JD, Johnson RT. Human immunodeficiency virus and the brain. Annu Res Neurosci 1996; 19:1-26. 12. Chun TW, Fauci AS. Latent reservoirs of HIV: obstacle to the eradication of virus. Proc Natl Acad Sci USA 1999; 96:10958-10961. 13. Finzi D, Blankson J, Siliciano JD et al. Latent infection of CD4+ T cells provide a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999; 5:512-517. 14. Siliciano RF. Latency and reservoirs for HIV-1. AIDS 1999; 13:549-558. 15. Chun TW, Stuyver L, Mizell SB et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA 1997; 94:13193-13197. 16. Schranger LK, D’Souza MP. Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy. JAMA 1998, 280:67-71. 17. Davey RT, Niranjan B, Yoder C et al. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc Natl Acad Sci 1999; 96:15109-15114. 18. Chun TW, Davey RT, Engel D et al. Re-emergence of HIV after stopping therapy. Nature 1999; 401:874-875. 19. Igarashi T, Brown CR, Endo Y et al. Macrophages are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus HIV type 1 chimera (SHIV): Implications for HIV-1 infections of human. Proc Natl Acad Sci USA 2001; 98:658-663. 20. Jolicoeur P. Murine acquired immunodeficiency syndrome (MAIDS): An animal model to study the AIDS pathogenesis. FASEB J 1991; 5:2398-2405. 21. Morse HC III, Chattopadhyay M, Makino M et al. Retrovirus-induced immunodeficiency in the mouse: MAIDS as a model for AIDS. AIDS 1992; 6:1752-1757. 22. Magnani M, Casabianca A, Rossi L et al. Inhibition of HIV-1 and LP-BM5 replication in macrophages by dideoxycytidine 5'-triphosphate. Antiviral Chem Chemother 1995; 6:312-319. 23. Fraternale A, Casabianca A, Rossi L et al. Inhibition of murine AIDS by combination of AZT and dideoxycytidine 5'-triphosphate. J Acquir Immune Defic Syndr Hum Retrovirol 1996; 12:164-173. 24. Magnani M, Casabianca A, Fraternale A et al. Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection. Proc Natl Acad Sci USA 1996; 93:4403-4408. 25. Fraternale A, Tonelli A, Casabianca A et al. Role of macrophage protection in the development of murine AIDS. J Acquir Immune Defic Syndr 1999; 21:81-89. 26. Morahan WL, Hendrzak JA, Eisenstein TK. Herpes simplex virus type 1 replication and IL-1 beta gene expression in mouse peritoneal macrophages activated in vivo by an attenuated Salmonella typhimurium vaccine or Corynebacterium parvum. Microbiol Pathog 1994; 16:387-399. 27. Golden MP, Kim S, Hammer SM et al. Activation of human immunodeficiency virus by simplex virus. J Infect Dis 1992; 166:494-499.

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28. Vlach J, Pitha PN. Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence. J Virol 1992; 66:3616-3623. 29. Balzarini J, Naesens L, Slachmuylders J et al. 9-(2-phosphonylmethoxyethyladenine (PMEA) effectively inhibits retrovirus replications in vitro and simian immunodeficiency virus infection in rhesus monkeys. AIDS 1991; 5:21-28. 30. De Clercq E, Holy A, Rosenberg I. Efficacy of phosphonylmethoxyalkyl derivativeses of adenine in experimental herpes simplex virus and vaccinia virus infection in vivo. Antimicrobiol Ag Chemoter 1989; 33:185-191. 31. Franchetti P, Sheikha GA, Cappellacci L et al. A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus. Antiv Res 2000; 47:149-158. 32. Heng MC, Heng SY, Allen SG. Co-infection and synergy of human immunodeficiency virus-1 and herpes simplex virus-1. Lancet 1994; 343:255-258. 33. Erlich KS, Mills J, Chatis P et al. Acyclovir-resistant herpes simplex virus infection in patients with the acquired immunodeficiency syndrome. N Engl J Med 1989; 320:293-296.

CHAPTER 8

Erythrocytes as Carriers of Anthracycline Antibiotics In Vitro and In Vivo Victor M. Vitvitsky

Introduction

M

odern pharmacology largely relies on the use of drugs that selectively affect their target cells and tissues. The selectivity of antibacterial drugs of new generations is quite high. It comes as no surprise because most of them are specific inhibitors of metabolic pathways vitally important in bacteria but absent from multicellular organisms. The selectivity of antitumor drugs is usually relatively low, because tumor cell metabolism is qualitatively similar to that of normal cells. A natural consequence of the low selectivity of antitumor drugs is their high toxicity. At present, it is a general view that the use of various vehicles to carry drugs in the organism is a promising way to enhance their efficacy while reducing their toxicity. The carrier is able to prevent premature drug degradation, inactivation, or elimination from the organism, as well as to abolish or reduce undesirable immune responses to drug administration. Retarded release of the drug may prolong its persistence at a necessary level in the organism. One of the most important results expected of the use of drug carriers is their selective delivery to the target cells or organs. In one way or another, carriers may profoundly alter the pharmacokinetics of the drug, diminishing the required dose or a peak concentration, which, in its turn, results in reduced toxicity and increased efficacy. Various macromolecules, microparticles, microcapsules, or even cells are suitable for the use as drug vehicles. All the aforesaid are fully pertinent to anthracycline antibiotics.

Anthracycline Antibiotics Anthracycline antibiotics are potent antitumor drugs active against a wide range of malignancies. The first identified anthracyclines, daunorubicin and doxorubicin, were isolated from Streptomyces spp. in the early 1960s. They are among the most utilized antitumor drugs ever developed.1,2 Chemically, all anthracyclines consist of an aglycone ring coupled with amino sugar (Fig. 1). Administered intravenously, daunorubicin or doxorubicin are rapidly eliminated from the bloodstream. Two phases may be distinguished in the kinetics of daunorubicin and doxorubicin clearance from plasma. The antibiotic concentration declines an order of magnitude or more in some 30-60 min after its administration and then to less than 1% of the initial (peak) concentration over the subsequent 12-24 h.3-8 Anthracycline antibiotics are highly toxic. Their side effects include myelosuppression, tissue necrosis (if the antibiotics are accidentally injected subcutaneously), alopecia, lesions of the gastrointestinal tract, and cumulative-dose-dependent cardiotoxicity.1,2 Many studies aimed at improving the efficacy and reducing the toxicity of anthracycline antibiotics solve the problem by using various carriers.

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Figure 1. Structure of daunorubicin (R = CH3), and doxorubicin (R = CH2OH).

Carrier Macromolecules The possibility of using various macromolecules as carriers for anthracycline antibiotics has been examined in a number of studies. While retaining a high antitumor activity, complexes of anthracycline antibiotics with DNA exhibit lower nonspecific toxicity than their free forms.9,10 Anthracyclines have been successfully used in the form of their copolymers with albumin11 or N-(2-hydroxypropyl)methacrylamide (HPMA).12,13 Anthracycline antibiotics can be selectively delivered to tumor cells being conjugated to antibodies against their surface antigens.14,15 As demonstrated in animal experiments and with cell cultures, conjugation of anthracycline antibiotics to a-fetoprotein or epidermal growth factor increased their antitumor potency even in the case of resistant tumors.16-18

Liposomes At present, liposomes are among most popular drug vehicles. Liposomes are tiny vesicles formed by polar group-containing lipids 19,20. Liposomes as carriers of anthracycline antibiotics have been a subject of a great number of studies 21. As a result, liposome formulations of daunorubicin (DaunoXome) and doxorubicin (Doxil) are now commercially available. The pharmacokinetics of the liposomal forms of anthracycline antibiotics differ from that of their free forms in higher peak concentrations and longer circulation times of the drugs.22-26 The kinetics of DaunoXome and Doxil clearance from plasma is close to monoexponential. The

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half-life of DaunoXome in patient plasma is on the order of a few hours.22,23 In Doxil, polyethylene glycol-coated liposomes are used. The immune system poorly recognizes such liposomes; therefore, the plasma half-life of Doxil is tens of hours.24-26 The concentration of free antibiotics in plasma remains at a very low level because their release from liposomes is very slow. It is no surprise that when compared with their free forms administered at equal doses, DaunoXome and Doxil exhibit less toxicity.22-26 The liposomal forms of the antibiotics are selectively accumulated in the foci of tumor growth.24,25,27,28 It is not fully clear what is the mechanism of this selectivity. If coated with marker (e.g., antibodies) liposomes can be used to selectively deliver their encapsulated drugs to target cells and organs.29,30 Daunoxome and Doxil have been approved in the USA and a number of European countries for treatment of Kaposi’s sarcoma. Clinical trials are now underway to evaluate their use in therapy for hematological diseases and solid tumors.

Carrier Erythrocytes Erythrocytes are promising cells for use as vehicles for drug delivery. First, these cells are readily available in great quantities. Second, procedures of erythrocyte loading (through encapsulation or binding) are simple and quite efficient.31,32 Third, their undoubted merits are ideal biocompatibility and the long-term (about 100 days) circulation in the organism. Fourth, erythrocyte degradation in the organism is a natural process, which does not cause any side effects. Fifth, a possibility of using autologous erythrocytes reduces the risk of transmitting infection. Sixth, relatively simple treatment makes it possible to produce carrier erythrocytes that are selectively trapped by organs of the reticuloendothelial system33-36 and by cells of the immune system.37-39 A lot of studies are known in which erythrocytes are used to transport a wide variety of drugs. One of the most promising applications of carrier erythrocyte is transport of enzymatic preparations that metabolize cell membrane-permeating substrates. This approach is promising in correcting enzymopathies,40 eliminating toxic substances from the blood41 in chemotherapy for oncological diseases,42,43 etc. Also of interest is the possibility of using erythrocytes as bioreactors able to convert the loaded inactive drug precursors into active forms.44,45 There are many studies reporting on loading the erythrocytes with various anticancer drugs or their precursors, such as methotrexate,46,47 L-asparaginase,42,43 carboplatin,4 fludarabine phosphate44 and others. Erythrocytes as carriers for anthracycline antibiotics are also extensively studied.

Incorporation of Anthracycline Antibiotics Into Erythrocytes

A common method for loading drugs into erythrocytes is reversible osmotic lysis.31,32 This method includes two steps. First, erythrocytes swell in a hypotonic drug-containing medium at 4°C. Pores are formed in their cell membranes through which the drug enters the cell. The second step is resealing the cell membrane by incubating the cells at 37°C in an isotonic medium. This method was used to load mouse erythrocytes with daunorubicin or doxorubicin35,49 and human and dog erythrocytes with doxorubicin.50-52 With this method, the daunorubicin concentration in erythrocytes was as high as 4 mg/l cells; the doxorubicin concentration ranged from 0.33 to 1.6 mg/l cells. In human erythrocytes, the major fraction of the entrapped doxorubicin (about 73%) was bound to the cell membrane.51 Doxorubicin, when loaded into erythrocytes, is metabolized relatively slowly. The major product of its metabolism is doxorubicinol.51,52 Yet another method for loading daunorubicin into mouse and human erythrocytes has been described that is based on the use of amphotericin B, a polyene antibiotic that increases cell membrane permeability.53 With this method, the daunorubicin concentration reported in erythrocytes was 0.2 mg/l cells. Whatever the method, the loaded antibiotic appreciably leaks from erythrocytes.35,49,51-53 At a hematocrit of 5-10% and 37°C, up to 70% of the loaded antibiotic is lost in 1 to 2 h.35,53 The doxorubicin leakage from erythrocytes is higher in plasma than in saline media,52 presumably because plasma proteins and lipoproteins bind the antibiotic. As demonstrated by many

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researchers, immobilization of daunorubicin or doxorubicin in erythrocytes with glutaraldehyde treatment decreases their leakage rate several times.35,52,54 The use of reversible osmotic lysis or amphotericin B to load anthracycline antibiotics into erythrocytes is expedient if their membrane is poorly permeable for these drugs. However, it is not the case. Cytostatic effects of anthracycline antibiotics result from their binding to nucleic acids, which leads to inhibition of protein synthesis in cells. Obviously, if the membrane were impermeable to these antibiotics, they would not be potent cytostatics. A relatively fast leakage of anthracycline antibiotics from loaded erythrocytes also suggests that they permeate easily through the cell membrane. Moreover, as demonstrated in a number of studies, intact erythrocytes can directly bind anthracycline antibiotics contained in an isotonic incubation medium.52,55-60 Analysis of the kinetics of daunorubicin or doxorubicin binding with mouse or human erythrocytes incubated at a hematocrit of about 50% showed that about 80% of the antibiotic added was bound at 37°C in 30-60 min.56-58,60 The binding was inhibited at lower temperatures and almost stopped at 4°C. Presumably, it was the low rate of anthracycline antibiotic binding at low temperatures that had led to a belief in the impermeability of the erythrocyte membrane to these antibiotics. No doubt, the easiest way to obtain anthracycline antibiotic-loaded erythrocytes is to allow erythrocytes to bind the antibiotic in an isotonic medium. Its intracellular concentration can reach a few milligrams per milliliter cells.58-60 The erythrocyte-bound antibiotics may be immobilized intracellularly with glutaraldehyde.52,57,59,61 In studies of the role played by glutaraldehyde in anthracycline antibiotic immobilization in erythrocytes, membrane permeability to the antibiotics was suggested to remain unchanged by glutaraldehyde treatment. The treated cells retained the ability to reversibly bind antibiotics from the extracellular medium.62,63 Moreover, the rate of antibiotic binding was higher in treated (versus intact) cells. Therefore, it is likely that glutaraldehyde immobilizes antibiotics within cells by covalently linking them to some cellular components. In fact, glutaraldehyde is able to form chemical bonds between substances containing amino groups.64 Molecules of anthracycline antibiotics contain one amino group; therefore, they can form chemical bonds via glutaraldehyde to different amino-group-containing erythrocyte components such as proteins (including hemoglobin), cell membrane phospholipids, etc.

Antitumor Activity of Anthracycline-Antibiotic-Loaded Erythrocytes The antitumor activity of anthracycline-antibiotic-loaded erythrocytes was demonstrated in a number of experimental studies. Daunorubicin-loaded erythrocytes were therapeutically more efficacious against mouse L1210 leukemia, as compared with the free form of the antibiotic. The survival time of mice transplanted with L1210 cells intraperitoneally and treated with intraperitoneally injected erythrocytes loaded with daunorubicin was prolonged by 14-26% compared with control mice treated with the free form of the antibiotic at equivalent doses.49,53 The effect was enhanced threefold if the loaded erythrocytes were injected along with wheat germ agglutinin, causing tumor cells to agglutinate the erythrocytes.49 In P388-bearing mice, the survival time did not depend significantly on whether they were treated with daunorubicinloaded erythrocytes or the free form of the antibiotic.54,56 A high antitumor activity of daunorubicin-loaded erythrocytes was demonstrated in mice with Rausher virus-induced erythroblastic leukemia.61 The weight loss and mortality rate data indicated that erythrocyte-bound daunorubicin was significantly less toxic than its free form. Immobilization of daunorubicin in erythrocytes with glutaraldehyde affected neither its antitumor efficiency nor toxicity.61 The erythrocytes could be frozen without loss of antitumor activity, indicating the feasibility of their long-term storage.61 In mice injected intravenously with doxorubicin-loaded glutaraldehyde-treated erythrocytes, the largest fraction of the antibiotic was detected in liver and lungs.35 As both liver and lungs are preferred sites where metastases develop, the delivery of the antibiotic mainly to these organs is of practical importance. In fact, compared with the free form of doxorubicin, doxorubicin-loaded glutaraldehyde-treated erythrocytes turned out more efficacious in prevention of liver and lung metastases in a murine metastatic model.65,66 With the antibiotic in the free

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form, the dose required that metastatic growth be completely inhibited was 12 mg/kg. This dose was only 1.5 mg/kg with the antibiotic immobilizes in erythrocytes. The therapeutic index defined as the ratio of the dose at which 10% of the animals died to the dose at which metastatic growth was inhibited by 90% was greater for the antibiotic entrapped into erythrocytes (4.2 vs. 1.8 for the antibiotic in the free form). In in vitro experiments, a possibility was demonstrated of targeted delivery of anthracycline antibiotics using antibody-coated erythrocytes.67 Doxorubicin was targeted to cytotoxic T lymphocytes using anti-Thy-1.2-antibody-coated erythrocytes. Their cytostatic effect was higher than that of erythrocytes coated with nonspecific antibodies.

Lesions in Erythrocytes Induced by Anthracycline Antibiotics A problem of great concern with erythrocytes carrying anthracycline antibiotics is damage they cause to cells. Erythrocyte lesions produced by interaction with anthracycline antibiotics include oxidation of cellular components,68,69 abnormal cell morphology,55,70 impaired cell membrane ultrastructure,70 increased cell membrane permeability,55 inhibition of ATPases,69 K+ leakage,69,71 reduced cell deformability,71 swelling,55,71 increased osmotic fragility,52,72 hemolysis,56-59,71 and others. Obviously, the most severe lesion is hemolysis. Therefore, a study was made of how the rate of hemolysis depended on the daunorubicin or doxorubicin concentration, temperature of incubation, and duration of erythrocyte incubation with the antibiotic.56-59,71 In the range of temperatures from 4 to 21°C and at antibiotic concentrations less than 1 mg/ml cells, the rise in extracellular hemoglobin was insignificant. The rate of hemolysis increased dramatically at 37°C. If daunorubicin concentration was 5 mg/ml cells, the rate of hemoglobin release at 37°C was up to 5-10% of the total hemoglobin content in the cell per hour.56,71 Concomitantly, K+ leaked at a rate of about 8% per hour.71 In an hour at 37°C, doxorubicin loaded to concentrations of 2 and 9 mg/ml cells destroyed 30 and 65% of the erythrocytes, respectively.59 Considerable hemoglobin and K+ leakage from erythrocytes loaded with anthracycline antibiotics makes their storage unlikely. If they were subjected to glutaraldehyde treatment, hemoglobin leakage decreased profoundly.57,61 Erythrocyte deformability was found to markedly depend on the concentration of anthracycline antibiotics.71 The deformability of loaded erythrocytes was assessed from their ability to pass through membrane filters with pores 3 mm in diameter.73 The deformability index was defined as the 1% erythrocyte suspension-to-suspending medium ratio of filtration rates. With antibiotic concentrations less than or equal to 0.4 mg/ml cells, the deformability index remained at a normal level over a few hours. At about 1.0 mg/ml cells, the deformability index reduced to zero in a few minutes. The deformability index reflects the ability of erythrocytes to pass through small capillaries in tissues. Therefore, varying the antibiotic concentration in loaded erythrocyte may be a way to intentionally obtain normally or poorly circulating cells. The latter would lodge in capillaries of organs of the reticuloendothelial system and thereby preferentially increase the antibiotic concentration there. It is of interest that the ATP concentration of erythrocytes incubated with anthracycline antibiotics remained unaffected.71 All these data lead to a conclusion that the antibiotic concentration in the loaded erythrocytes should not exceed 0.4 mg/ml to avoid their substantial destruction and to preserve their deformability. Under these circumstances ordinary random-donor packed erythrocyte units containing about 200 ml of these cells allow up to 80 mg of the antibiotic to be infused.

Erythrocytes as Carriers of Anthracycline Antibiotics in Veterinary and Clinical Practice By now, several cases of the use of erythrocytes loaded with anthracycline antibiotics in veterinary and clinical practice have been reported in the literature. In four cases, both doxorubicin and L-asparaginase were loaded into erythrocytes using the hypotonic osmotic technique and used to treat dogs with lymphosarcoma.74 Without assessing the antibiotic incorporation,

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the therapeutic efficacy cannot be judged with confidence. However, some dogs achieved remission, and no short-term toxicities related to doxorubicin were observed after transfusion of antibiotic-loaded erythrocytes. In yet other three cases of dog lymphosarcoma treated with antibiotic-loaded erythrocytes, the loading technique included doxorubicin binding to erythrocytes and its immobilization by glutaraldehyde treatment.75 As demonstrated by pharmacokinetic analysis, administration of such erythrocytes resulted in considerably decreased peak concentrations of the antibiotic in plasma and significantly prolonged times of its circulation (compared with administration of the free form of the drug).75-77 The plasma concentration of doxorubicin remained above 10% of its peak concentration over six days of observation. In the course of treatment, the dogs received two to four injections of doxorubicin-loaded erythrocytes (30 mg/m2 body surface per injection). In all cases, the antitumor activity of the erythrocyte-bound doxorubicin was high, the acute toxicity minimal, and no signs of cardiotoxicity were observed. Unfortunately, substantial, unanticipated, chronic, nonregenerative myelosuppression developed later in all the dogs, which eventually led to their death. Doxorubicin binding to erythrocytes followed by its immobilization with glutaraldehyde was used to prepare loaded erythrocytes for treatment of a patient with refractory colorectal cancer metastatic to the liver.59,78 The erythrocyte-bound doxorubicin was administered at a dose of 10 mg/m2 body surface into the hepatic artery. Labeling the erythrocytes with 99mTc revealed that the loaded erythrocytes were preferentially accumulated in the liver and only about 10% of them circulated. The patient received a total of six infusions at two-week-intervals. Although no objective response to the treatment was achieved, repeated liver ultrasonic observations indicated stabilization of the disease. After each infusion of loaded erythrocytes, the rapid onset of shaking chills followed by high fever was observed. Two weeks after the sixth infusion, the patient developed grade IV pancytopenia and died six days later from Gramnegative septic shock. In this patient, glutaraldehyde treatment of loaded erythrocytes was used because the results of previous animal studies indicated that preferential delivery of the drug to the liver was expected of such a treatment.33,35 As mentioned above, glutaraldehyde-treated carrier erythrocytes were highly efficacious in treatment of mice with experimental metastases 65,66 . However, a more recent study did not confirm that glutaraldehyde-treated erythrocytes lodged mostly in the liver of patients with liver metastases. The organ distribution of glutaraldehyde-treated erythrocytes obtained in that study varied greatly.79 It is not inconceivable that the development of myelosuppression in the cases mentioned above has been a result of doxorubicin modification caused by its immobilization in erythrocytes with glutaraldehyde. Specific activities, toxicities, and therapeutic indexes of various anthracycline antibiotics are very different in spite of the apparent slight differences in their chemical structures.1,2 It is therefore plausible that modification of the structure of anthracycline antibiotics caused by their immobilization within erythrocytes via glutaraldehyde could also modify the properties of the original drugs, toxicity included. In fact, in experiments with mice, an increase in the therapeutic index of doxobubicin immobilized in erythrocytes was observed along with an increase in its toxicity.65,66 In patients with various lymphoproliferative disorders, interesting results were obtained with carrier erythrocytes prepared by incubation with anthracycline antibiotics in an isotonic medium without the subsequent treatment with glutaraldehyde.6-8 We were not going to assess the antitumor activity of such carrier cells. The objective was only to study their pharmacokinetics and tolerability. The loaded cells were prepared aseptically by incubating heterologous or autologous erythrocytes (200 ml) with the antibiotic at 37°C for an hour. Its concentration was chosen to ensure a dose of 25-50 mg/m2. In three out of eight reported cases, doxorubicinloaded erythrocytes were used.6,7 In the other five patients, daunorubicin-loaded erythrocytes were studied 8. Despite the fact that antibiotics that had not been immobilized readily left the erythrocytes, their pharmacokinetics turned out significantly altered compared with the pharmacokinetics of their free forms. As a rule, the peak concentration of the antibiotic decreased, and it circulated in the blood longer. Two phases, fast and slow, could be distinguished in the

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kinetics of disappearance of anthracycline antibiotics from the blood, whether they were infused in the free or erythrocyte-loaded form. With both forms, the first (fast) phase was 30-60 min long. During this phase, a 10-30-fold decrease in the antibiotic concentration was observed. The slow phase was much longer if anthracycline antibiotics bound to erythrocytes were infused. With daunorubicin-loaded erythrocytes, the duration of the slow phase was about three days. In the same patients, the circulation half-life was estimated at 14.3 h for daunorubicin infused in the erythrocyte-bound form and at 5.4 h for the antibiotic infused in the free form. It is of interest that, for erythrocyte-bound daunorubicin, the half-life in the slow phase was close to that for DaunoXome.8 With doxorubicin-loaded erythrocytes, we observed a decrease in the antibiotic concentration to 5-10% of its peak level by the end of the first phase. Its concentration sluggishly decreased over the subsequent three days of observation in two patients and even slightly increased in one patient. As the slow phase was prolonged when anthracycline antibiotics were infused loaded in erythrocytes, the area under the pharmacokinetic curve was several times larger compared with that observed after administration of their free forms. Anthracycline antibiotics bound to erythrocytes without immobilization with glutaraldehyde were well tolerated in all cases. No immediate reactions of the patients to infusion of such carrier erythrocytes were observed. The toxic effects typical of cytostatic chemotherapy (nausea, vomiting, mucositis, alopecia) were significantly less pronounced in response to infusion of anthracycline antibiotics bound to erythrocytes. No prolonged or severe myelosuppression was observed. In none of the cases, echocardiographic signs of cardiotoxicity were detected.

Conclusions Anthracycline antibiotics loaded in erythrocytes without glutaraldehyde treatment are a promising therapeutic modality. Their reduced toxicity affords an opportunity of administering them at higher doses. The prolonged circulation time may enhance the therapeutic efficacy of these antibiotics. Of great importance is the problem of preferential delivery of the antibiotics to the foci of tumor growth by erythrocytes. In this context, it is tempting to study a possibility of anthracycline antibiotic transport by erythrocytes carrying antibodies or other ligands on their surface that are capable of specifically binding to tumor cells. The pharmacokinetics of carrier erythrocytes prepared using high antibiotic concentrations (on the order of 1 mg/ml) also awaits future studies. The potential problem of low deformability of such erythrocytes may be put to advantage by using them to route the antibiotics to particular organs or tissues. It is expedient that future studies also address ways to immobilize anthracycline antibiotics in erythrocytes not related to glutaraldehyde treatment.

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37. Magnani M, Rossi L, Brandi G et al. Targeting antiretroviral nucleoside analogues in phosphorilated form to macrophages: in vitro and in vivo studies. Proc Natl Acad Sci USA 1992; 89:6477-6481. 38. Magnani M, Casabianca A, Fraternale A et al. Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection. Proc Natl Acad Sci USA 1996; 93:4403-4408. 39. Chiarantini L, Droleskey R, Magnani M et al. In vitro targeting of erythrocytes to cytotoxic Tcells by coupling of Thy-1.2 monoclonal antibody. Biotechnol Appl Biochem 1992; 15:171-184. 40. Bax BE, Bain MD, Fairbanks LD et al. In vitro and in vivo studies with human carrier erythrocytes loaded with polyethylene glycol-conjugated and native adenosine deaminase. Br J Haematol 2000; 109:549-554. 41. Pei L, Petrikovics I, Way JL. Antagonism of the lethal effects of paraoxon by carrier erythrocytes containing phosphotriesterase. Fundam Appl Toxicol 1995; 28:209-214. 42. Ataullakhanov FI, Vitvitsky VM, Zhabotinsky AM et al. Permeability of human erythrocytes to asparagine. Biokhimiia 1985; 50:1733-1737. (in Russian) 43. Kravtzoff R, Colombat PH, Desbois I et al. Tolerance evaluation of L-asparaginase loaded red blood cells. Eur J Clin Pharmacol 1996; 51:221-225. 44. Fraternale A, Rossi L, Magnani M. Encapsulation, metabolism and release of 2-fluoro-ara-AMP from human erythrocytes. Biochim Biophys Acta 1996; 1291:149-154. 45. Benatti U, Giovine M, Damonte G et al. Azidothymidine homodinucleotide-loaded erythrocytes and bioreactors for slow delivery of the antiretroviral drug azidothymidine. Biochem Biophys Res Commun 1996; 220:20-25. 46. Pitt E, Johnson CM, Lewis DA. Encapsulation of drugs in intact erythrocytes: An intravenous delivery system. Biochem Pharmacol 1983; 32:3359-3368. 47. DeLoach JR, Tangner CH, Barton C. Hepatic pharmackinetics of glutaraldehyde-treated methotrexate-loaded carrier erythrocytes in dogs. Res Exp Med 1983; 183:167-175. 48. Tonetti M, Gasparini A, Giovine M et al. Interaction of carboplatin with carrier human eythrocytes. Biotechnol Appl Biochem 1992; 15:267-277. 49. Kitao T, Hattori K Takeshita M. Agglutination of leukemic cells and daunomycin entrapped erythrocytes with lectin in vitro and in vivo. Experientia 1978; 34:94-95. 50. Tyrrell DA, Ryman BE. The entrapment of therapeutic agents in resealed erythrocyte ‘ghosts’ and their fate in vivo. Biochem Soc Trans 1976; 4:677-680. 51. De Flora A, Benatti U, Guida L et al. Encapsulation of adriamycin in human erythrocytes. Proc Natl Acad Sci USA 1986; 83:7029-7033. 52. Tonetti M, Astroff B, Satterfield W et al. Construction and characterization of adriamycin-loaded canine red blood cells as a potential slow delivery system. Biotechnol Appl Biochem 1990; 12:621-629. 53. Kitao T, Hattori K. Erythrocyte entrapment of daunomycin by amphotericin B without hemolysis. Cancer Res 1980; 40:1351-1353. 54. Gaudreault RC, Bellemare B, Lacroix J. Erythrocyte membrane-bound daunorubicin as a delivery system in anticancer treatment. Anticancer Res 1989; 9:1201-1205. 55. DeLoach JR, Droleskey R. Interaction of anthracycline drugs with canine and bovine carrier erythrocytes. J Appl Biochem 1985; 7:332-340. 56. Ataullakhanov FI, Vitvitsky VM, Kovaleva VL et al. Rubomycin loaded erythrocytes in the treatment of mouse tumor P388. Adv Exp Med Biol 1992; 326:209-213. 57. Ataullakhanov FI, Batasheva TV, Vitvitsky VM et al. Effect of glutaraldehyde treatment on rubomycin and hemoglobin leakage from murine erythrocytes loaded with rubomycin. Biotekhnologiia 1993; (2):40-43. (in Russian) 58. Ataullakhanov FI, Batasheva TV, Vitvitsky VM. Effect of temperature, daunorubicin concentration and suspension hematocrit on daunorubicin binding by human erythrocytes. Antibiotiki i Khimioterapiia 1994; 39(9-10):26-29. (in Russian) 59. Tonetti M, Zocchi E, Guida Z et al. Use of glutaraldehyde treated autologous human erythrocytes for hepatic targeting of doxorubicin. Adv Exp Med Biol 1992; 326:307-317. 60. Ataullakhanov FI, Kulikova EV, Vitvitsky VM. Doxorubicin binding by human erythrocytes. Adv Biosci 1994; 92:163-168. 61. Ataullakhanov FI, Batasheva TV, Bukhman VM et al. Treatment of Rausher virus induced murine erythroblastic leukemia with rubomycin loaded erythrocytes. Adv Biosci 1994; 92:177-183. 62. Ataullakhanov FI, Kulikova EV, Vitvitsky VM. Reversible binding of anthracycline antibiotics to erythrocytes treated with glutaraldehyde. Biotechnol Appl Biochem 1996; 24:241-244. 63. Ataullakhanov FI, Kulikova EV, Vitvitsky VM. Binding of daunorubicin and doxorubicin to erythrocytes treated with glutaraldehyde. In: Sprandel U, Way JL, eds. Erythrocytes as Drug Carriers in Medicine. New York: Plenum Press, 1996:143-148.

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64. Avrameas S, Ternynck T. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunosorbents. Immunochemistry 1969; 6:53-66. 65. Zocchi E, Tonetti M, Polvani C et al. Encapsulation of doxorubicin in liver-targeted erythrocytes increases the therapeutic index of the drug in a murine metastatic model. Proc Natl Acad Sci USA 1989; 86:2040-2044. 66. Zocchi E, Tonetti M, Polvani C et al. Enhanced antineoplastic efficiency of erythrocyte encapsulated doxorubicin in a murine liver metastatic model. Adv Biosci 1991; 81:231-237. 67. Gasparini A, Chiarantini L, Kirch H et al. In vitro targeting of doxorubicin loaded canine erythrocytes to cytotoxic T-lymphocytes (CTLL). Adv Exp Med Biol 1992; 326:291-297. 68. Henderson CA, Metz EN, Balcerzak SP et al. Adriamycin and daunomycin generate reactive oxygen compounds in erythrocytes. Blood 1978; 52:878-885. 69. Shinohara K, Tanaka KR. The effect of adriamycin (doxorubicin HCL) on human red blood cells. Hemoglobin 1980; 4:735-745. 70. Arancia G, Bordi F, Calcabrini A et al. Ultrastructural and spectroscopic methods in the study of anthracycline-membrane interaction. Pharmacol Res 1995; 32:255-272. 71. Skorokhod AA, Vitvitsky VM, Kulmann RA et al. Detrimental effects of daunorubicin and doxorubicin on human erythrocytes in vitro. Voprosy Onkologii 1999; 45(4):374-379. (in Russian) 72. Sprandel U, Zollner N. Osmotic fragility of drug carrier erythrocytes. Res Exp Med 1985; 185:77-85. 73. Lisovskaya IL, Shurkhina ES, Yakovenko EE et al. Distribution of rheological parameters in populations of human erythrocytes. Biorheology 1999; 36:299-309. 74. Satterfield WC, Clarke MS, Hill MJ et al. Clinical evaluation of doxorubicin in canine carrier erythrocytes as therapy for canine lymphosarcoma. Adv Biosci 1991; 81:189-193. 75. Matherne CM, Satterfield WC, Gasparini A et al. Clinical efficacy and toxicity of doxorubicin encapsulated in glutaraldehyde-treated erythrocytes administered to dogs with limphosarcoma. Am J Vet Res 1994; 55:847-853. 76. Tonetti M, Astroff AB, Satterfield W et al. Pharmacokinetic properties of doxorubicin encapsulated in glutaraldehyde-treated canine erythrocytes. Am J Vet Res 1991; 52:1630-1635. 77. Gasparini A, Tonetti M, Astroff B et al. Pharmacokinetics of doxorubicin loaded and glutaraldehyde treated erythrocytes in healthy and lymphoma bearing dogs. Adv Exp Med Biol 1992; 326:299-304. 78. Tonetti M, Polvani C, Zocchi E et al. Liver targeting of autologus erythrocytes loaded with doxorubicin. Eur J Cancer 1991; 27:947-948. 79. Tonetti M, Bartolini A, Sobrero A et al. Organ distribution of glutaraldehyde treated erythrocytes in patients with hepatic metastases. Adv Biosci 1994; 92:169-176.

CHAPTER 9

Drug-Loaded Red Blood Cells for the Control of the Inflammatory Response: Selective Targeting of Nuclear Factor-!B (NF-!B) R. Crinelli, A. Antonelli, M. Bianchi, L. Gentilini and M. Magnani

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hile the causes of inflammatory diseases remain unknown, transcription factors are increasingly being found to play a pivotal role in the development and maintenance of these pathologies. In particular, the broad involvement of nuclear factor-!B (NF!B) in various aspects of the pathology of chronic inflammation makes it an extremely attractive target for therapeutic intervention. Despite the progress already made in understanding the molecular events governing NF-!B activation, several outstanding questions, including the development of specific inhibitors and the safety of totally blocking a factor involved in basic cellular functions, are still a matter of debate. In this regard, we investigated the route of drug delivery by means of red blood cells as an approach to selectively target NF-!B inhibitors to macrophages, one of the most important cellular compartments involved in inflammation.

Introduction Cells of the immune system encountering a microbial agent are able to develop a defense reaction, leading to the establishment of an inflammatory process with anti-microbial effects. This process requires de novo synthesis of specific proteins which activate residential macrophages and recruit blood leukocytes to the site of inflammation. New synthesized proteins include chemokines, which attract macrophages; enzymes, which generate pro-inflammatory mediators; cell adhesion molecules that recruit and allow transmigration of leukocytes across the blood barrier and cytokines, which amplify and spread the primary pathogenic signal. Since multiple gene products have been identified at sites of inflammation, there has been a surge of interest in identifying the intracellular signaling cascades and targets, including transcription factors, which control inflammatory gene expression. Understanding the function and regulation of transcription factors involved in immune and inflammatory responses is of particular interest in developing new therapeutic interventions directed to treat diseases in which the initiation of chronic inflammation is associated with development of autoimmune responses which progress to sustained, self-perpetuated inflammation.1 Indeed, most human autoimmune and inflammatory pathologies can be ascribed to the aberrant activation and expression of genes whose products are involved in the initiation and progression of pathogenesis.2, 3 Due to its pleiotropic function in immunity, nuclear transcription factor -!B (NF–!B) is considered to be one of the most promising targets in chronic inflammation.4 In response to pathogens and/or pro-inflammatory cytokines, transient activation of NF-!B occurs as part of normal physiological regulation in most every cell type involved in inflammation (i.e., endothelial cells, lymphocytes and macrophages). NF-!B regulates the expression of a wide array of genes including those encoding for cytokines, chemokines, cell adhesion molecules, inducible nitric oxide synthase and cyclooxygenase 2 (COX-2), all of which are central to the inflammatory Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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response.5,6 Furthermore, cytokines that are induced by NF-!B, such as interleukin 1∋ (IL-1∋) and tumor necrosis factor % (TNF-%), can also directly activate the NF-!B pathway, thus establishing a positive regulatory loop. On the other hand, abnormal and constitutive activation of NF-!B has been found to be associated with a number of auto-immune and inflammatory disorders, such as rheumatoid arthritis, inflammatory bowel diseases, multiple sclerosis, psoriasis and asthma.4,7,8 In this context, NF-!B shows aberrant, constitutive nuclear localization and enhanced transcriptional activity, presumably resulting from defects in the regulatory mechanism controlling its activation.9 NF-!B is a collective name for the inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence termed the -!B motif. In unstimulated cells NF-!B is held in the cytoplasm in a latent state by association with a member of the -!B family of inhibitors (IkB) so that the NF-!B nuclear localization signal (NLS) is blocked. A wide spectrum of agents, including bacterial and viral pathogens, lipopolysaccharide (LPS) and cytokines activate NF-!B. Activation involves liberation of NF-!B from cytoplasmic complexes containing I!B proteins (i.e., I!B%, I!B∋, I!B&). This is achieved through signal-induced degradation of I!B that is phosphorylated by a cytokineinducible kinase complex (IKK) and subsequently targeted through ubiquitination to the 26S proteasome.10 The signaling pathway in which IkB is phosphorylated, ubiquitinated, and subsequently degraded by the proteasome provides numerous potential target points for interference. However, aside from a number of nonspecific NF-!B blockers, including antioxidants, proteasome inhibitors, corticosteroids and other immunosuppressants, very few specific inhibitors have been described so far.7,11 One of the main problems emerging from this area of research is that the design of any new drug should not only address the specificity issue but also the risk of compromising other functions of NF-!B (such as protection against apoptosis) or affecting other signaling cascades that involve similar kinases or ubiquitin ligases. Studies on knockout mice have clearly revealed that disruption of the p65 (RelA) component of NF-!B results in embryonic lethality due to liver apoptosis;12 whereas, inhibition of the E3∋-TrCP ligase which catalyzes IkB ubiquitination is thought to result in stabilization of other substrates such as ∋catenin whose accumulation is known to induce tumorigenicity.13 Therefore, as it is most likely unwise to totally abrogate NF-!B activation and considering the lack of specific inhibitors, the development of drug delivery systems able to selectively target inhibitors to a specific cell population would be extremely advantageous in improving efficiency and decreasing toxicity of the drug. Studies performed in our laboratory have focused on the use of red blood cells as a delivery system to target some selected NF-kB inhibitors to macrophages, one of the most important cellular compartments involved in inflammation. This system takes advantage of the phagocytic capacity of macrophages, a function shared with only a few other cell types in the body. Because of this capability, drug delivery through phagocytosis should induce both selective targeting of drugs to organs and cells of the mononuclear phagocyte system and a minimization of drug-mediated cytotoxic effects on non-phagocytic cells.

Targeting Glucocorticoids to Macrophages: Inhibition of NF-kB Activation and Cytokine Release Glucocorticoids suppress many functions in activated monocytes/macrophages, including the release of TNF-%, which plays an important role in the pathogenesis of several autoimmune diseases.14 However, their therapeutic use is limited because of their well-known side effects on endocrine function and metabolism.15 This evidence prompted us to investigate the use of red blood cell as a delivery system with which to target glucocorticoids to macrophages. With this goal, non-diffusible glucocorticoid analogues such as dexamethasone-21-P and prednisolone-21-P were selected and efficiently entrapped in human erythocytes by a procedure of hypotonic hemolysis and isotonic resealing, reaching intracellular concentrations of up to 10

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Fig. 1. Mechanisms of and approaches to interference with I!B% degradation. Activation of NF-!B is controlled by inducible degradation of I!B. This process involves the covalent attachment of a multiubiquitin chain to I!B, which is consequently recognized and degraded by the 26S proteasome, allowing NF-!B nuclear translocation. Two different approaches to interference with I!B degradation are shown: (A) induction of I!B% mRNA synthesis by glucocorticoids; (B) inhibition of I!B% degradation by ubiquitin analogues. (A1) RT-PCR and immunoblot analysis of I!B% mRNA and protein levels in macrophages receiving dexamethasone-loaded erythrocytes (L) and free dexamethasone (Dex). For use as controls, macrophages were either left untreated (C) or incubated with unloaded erythrocytes (UL). (A2) Immunoblot analysis of residual I!B% levels upon LPS stimulation and electrophoretic mobility shift assay (EMSA) of NF-!B nuclear content using a probe corresponding to the -!B site from the Igk gene.22 (B1) Immunoblot analysis of ubiquitin conjugates and free ubiquitin in macrophages receiving ubiquitinLys48Arg-loaded erythrocytes (L) and unloaded erythrocytes (UL). (B2) Immunoblot analysis of I!B% protein levels in unstimulated and LPS-stimulated macrophages upon delivery of Ub-Lys48Arg-loaded RBC (L) or unloaded RBC (UL). Untreated macrophages (C) were used as controls.

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and 6 mM, respectively.16 The procedure can be easily adapted to internalize different concentrations of the drug simply by varying the amount added to the red blood cell (RBC) suspension during the encapsulation step. Significantly, glucocorticoid analogues do not interfere with erythrocyte metabolism even at the highest concentrations tested. In fact, both the rate of glycolysis (as measured in terms of lactate production) and ATP content were in the normal range. Furthermore, inside the erythrocytes these drugs display a high degree of stability, as they are only slowly dephosphorylated to the corresponding glucocorticoid by resident enzymes.16 Targeting of the drug-loaded RBC to macrophages can be obtained by inducing an artificial clustering of band 3 via the addition of ZnCl2 as clustering agent and bis(sulfosuccinimidyl) suberate (BS3), which makes the clusters irreversible. Once formed, these clusters are opsonized by autologous antibodies and C3b and then recognized by Fc- and C3b-receptors.17 This procedure has been demonstrated to increase the recognition and phagocytosis of modified RBC by macrophages, resulting in the delivery of the drug directly into the phagocytic cells where high concentrations can be attained with minimal systemic toxicity.16 Glucocorticoids have been used for decades as pharmacological inhibitors of immune and inflammatory processes despite the almost total lack of clues regarding their mechanisms of action. Recent advances in the field have led to the finding that most of their anti-inflammatory potential relies on their ability to interfere with the activity of pro-inflammatory transcription factors such as NF-!B. The inhibitory effect of glucocorticoids on NF-!B activation seems to be exerted through several mechanisms in different cell types which are referred to in the literature as the inhibitor kappaB-alpha up-regulatory model,18,19 the protein-protein interaction model,20 and the competition model.21 The first mechanism is consistent with a role for glucocorticoids in inducing the expression of I!B%, which compensates for the rapid degradation of the inhibitor upon cell stimulation, resulting in cytoplasmic retention of NF-!B. This mechanism is restricted to certain cell types such as monocytes. Indeed, delivery of dexamethasone to monocyte-derived macrophages by simple diffusion from the culture medium or by phagocytosis of dexamethasone-21-P-loaded RBC results in an increased intracellular concentration of I!B% mRNA and protein (Fig. 1, panel A1).22 In both cases, increased levels of the inhibitor correlate with inhibition of NF-kB nuclear translocation (Fig. 1, panel A2) and TNF% mRNA synthesis upon LPS-stimulation.22 This indicates that when dexamethasone-21-P is delivered to macrophages by means of opsonized RBC it is able to efficiently interfere with NF-!B activation through the same molecular mechanisms and with the same inhibitory effect described for the free molecule. Furthermore, the effects produced by the targeted delivery of dexamethasone-21-P to macrophages are also made evident by a reduced release of pro-inflammatory mediators such as TNF-% and IL-1∋ in the culture medium (Fig. 2) (unpublished results). This is of particular interest since these cytokines are responsible for the over-expression of leukocyte-endothelial cell adhesion molecules which mediate extravasation and the migration of leukocytes across the blood barrier to the site of inflammation (Fig. 3).23 For this reason, a limited release of pro-inflammatory mediators upon targeted delivery of dexamethasone to macrophages is expected to interfere with activation of the endothelial compartment without exposing it to the adverse effects of glucocorticoids. Recent results obtained in our laboratory demonstrate that exposure of the human cell line ECV304 (which exhibits endothelial characteristics) to the conditioned medium derived from LPS-stimulated macrophages leads to a reduced expression of intercellular adhesion molecule 1 (ICAM-1) mRNA, when macrophages are pretreated with dexamenthasone-loaded RBC (Fig. 3) (unpublished results). Further studies are required to determine the efficacy of this glucocorticoid delivery system in repressing acute and chronic inflammatory reactions in vivo.

Targeting Ubiquitin Analogues to Macrophages: A New Approach !B Activation to Interfering with NF-! The development of approaches allowing modulation of NF-!B activation via targeting of IkB% ubiquitination and/or degradation is a fascinating alternative to glucocorticoid administration. As

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Figure 2. TNF-% and IL-1∋ release by macrophages targeted with dexamethasone-21-P-loaded erythrocytes. Macrophages were left unstimulated or stimulated with 1 ∝g/ml of LPS (an endotoxic lipopolysaccharide from E. coli 0111:B4). After 30 min of stimulation, cells were washed with fresh medium; cytokine release in the culture supernatants was determined 24h later using an enzyme-linked immunosorbent assay (ELISA). Macrophages left untreated or targeted with unloaded erythrocytes (UL-RBC) were used as controls.

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Figure 3. Modulation of macrophage activation and effects on endothelial adhesion molecule expression. A variety of stimuli activate NF-!B in macrophages (1), leading to the release of pro-inflammatory mediators including TNF-% and IL-1∋ (2). Through the same molecular mechanisms, these soluble molecules are in turn able to induce the expression of the endothelial adhesion molecules involved in the extravasation of leukocytes across the blood barrier (3). Selective targeting of NF-kB inhibitors to the monocyte/macrophage compartment allows the release of pro-inflammatory mediators to be modulated and, as a consequence, a reduction in the expression of adhesion molecules in the endothelium. The bottom panel shows an RTPCR analysis of ICAM-1 mRNA levels in ECV304 cells incubated with conditioned medium derived from untreated macrophages (C), macrophages targeted with unloaded RBC (UL) or dexamethasone-21-Ploaded erythrocytes (L), LPS-stimulated macrophages (C+) and LPS-stimulated macrophages targeted with unloaded (UL+) or drug-loaded erythrocytes (L+).

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stated above, IkB% degradation occurs through a highly selective degradation pathway that involves the covalent conjugation of multiple ubiquitin molecules to target proteins, leading to the formation of multiubiquitin chains (Fig. 1). These chains have a repeating structure in which the carboxyl terminus of each ubiquitin monomer is linked to Lys48 of the preceding moiety to form a motif that is selectively recognized by the 26S proteasome.24 It has been demonstrated that reductively methylated ubiquitin or a ubiquitin mutant in which lysine 48 has been replaced by arginine act as polyubiquitin chain terminators, as they are unable to function as acceptors of further ubiquitin molecules.25,26 Based on this evidence, we reasoned that UbLys48Arg might be efficient in stabilizing the levels of I!B% and, as a consequence, in preventing NF-!B activation. In this context, the use of the erythrocyte-based delivery system offers a significant advantage since it allows efficient internalization of ubiquitin analogues into macrophages. In our experience, recombinant ubiquitin-Lys48Arg can be easily encapsulated in red blood cells, as described for glucocorticoids, with an efficiency of 28.8 pmol/106 RBC and targeted to macrophages.27 Under these experimental conditions, most of the mutant remains as free molecule and can compete with the endogenous wild-type ubiquitin monomer for conjugation to cellular proteins (Fig. 1, panel B1). Targeting of UbLys48Arg-loaded RBC to macrophages produces a significant inhibition of I!B% degradation upon LPS stimulation (Fig. 1, panel B2).27 Furthermore, the ability of UbLys48Arg to interfere with IkB% turnover, and as a consequence to limit NF-!B activation, is also demonstrated by a strong reduction of TNF-% release.27 An interesting aspect of this approach is that, once inside the macrophages, UbLys48Arg seems to only partially affect the endogenous ubiquitin conjugate pool. Since cells contain isopeptidase enzymes with “editing” functions on polyubiquitin chains,28 it is possible that the mutant ubiquitin could be removed from the chain and substituted with wildtype ubiquitin. However, based on the results we have obtained (Fig. 1, panel B1),27 it could also be speculated that long-lived proteins with slow rates of degradation as a consequence of ubiquitination are more sensitive to the activity of these enzymes in reverting the incorporation of the mutant ubiquitin. On the other hand, short-lived proteins such as I!B% are probably more rapidly ubiquitinated than de-ubiquitinated by isospeptidases. For these reasons, ubiquitinLys48Arg, by affecting the turnover of only a limited population of intracellular proteins (the short-lived), can be expected to offer the advantage of strongly limiting the cytotoxic effects produced, for example, by proteasome inhibitors. These molecules are potent inhibitors of NF-!B activation, but the wisdom of using them as drugs is currently a matter of debate since they produce severe side effects, including accumulation of ubiquitin-conjugated proteins, depletion of free ubiquitin and stabilization of proteins which regulate cellular functions and cell cycling.29,30 Thus, modulation of ubiquitination could provide the means by which to achieve a greater selectivity unattainable with proteasome inhibitors.

Conclusions The selective delivery of anti-inflammatory drugs to macrophages is feasible and results in the control of activation of NF-!B, the main transcription factor responsible for the release of pro-inflammatory mediators by these cells. Regulation of NF-!B activation can be a powerful therapeutic strategy with which to block the inflammatory response in instances where this process becomes chronic or dysregulated. However, a complete and persistent blockage of NF!B will lead to immune deficiency and apoptosis of healthy cells. For this reason, the development of drug delivery systems able to target specific cellular compartments is of great interest. The results described herein indicate that red blood cells represent a valuable tool that can be used to achieve this goal. In vitro studies clearly indicate that targeting NF-!B inhibitors, such as glucocorticoid analogues or recombinant peptides able to compete with components of the NF-!B signaling pathway, results in suppression of cytokine release. Interestingly, this approach not only allows the regulation of macrophage activation, but in turn has an effect on the activation of other cells (e.g., endothelial cells) involved in inflammation. This aspect is extremely encouraging and should lead to further investigations, possibly using in vivo animal models of pathological conditions involving an exaggerated activation of macrophages.

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References 1. Papavassiliou AG. Transcription-factor-modulating agents: Precision and selectivity in drug design. Mol Med Today 1998; 4(8):358-366. 2. Brennan FM, Maini RN, Feldmann M. Cytokine expression in chronic inflammatory diseases. Br Med Bull 1995; 51:368-384. 3. Staudt LM. Gene expression physiology and pathophysiology of the immune system. Trends Immunol 2001; 22(1):35-40. 4. Makarov SS. NF-!B as a therapeutic target in chronic inflammation: Recent advances. Mol Med Today 2000; 6(11):441-448. 5. Baeuerle PA, Henkel T. Function and activation of NF-!B in the immune system. Annu Rev Immunol 1994; 12:141-179. 6. Baldwin AS. The NF-!B and IkB proteins: New discoveries and insights. Annu Rev Immunol 1996; 14:649-681. 7. Perkins ND. The Rel/NF-kappa B family: Friend and foe. Trends Biochem Sci 2000; 25(9):434-440. 8. Tak PP, Firestein GS. NF-!B: A key role in inflammatory diseases. J Clin Invest 2001; 107(1):7-11. 9. Barnes PJ, Karin M. Nuclear factor-kappaB: A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336(15):1066-1071. 10. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: The control of NF-!B activity. Annu Rev Immunol 2000; 18:621-663. 11. Yamamoto Y, Gaynor RB. Therapeutic potential of the NF-!B pathway in the treatment of inflammation and cancer. J Clin Invest 2001; 107(2):135-142. 12. Beg AA, Sha WC, Bronson RT et al. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 1995; 376(6536):167-170. 13. Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis: A look outside the nucleus. Science 2000; 287(5458):1606-1609. 14. Joyce DA, Steer JH, Abraham LJ. Glucocorticoid modulation of human monocyte/macrophage function: Control of TNF-% secretion. Inflamm Res 1997; 46(11):447-451. 15. Goulding NJ. Corticosteroids: A case of mistaken identity? Br J Rheumatol 1998; 37(5):477-480. 16. Magnani M, D’Ascenzo M, Chiarantini L et al. Targeting Dexamethasone to macrophages. Drug Delivery 1995; 2:151-155. 17. Magnani M, Rossi L, Brandi G et al. Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages: In vitro and in vivo studies. Proc Natl Acad Sci USA 1992; 89(14):6477-6481. 18. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS Jr. Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science. 1995; 270(5234):283-286. 19. Auphan N, DiDonato JA, Rosette C et al. Immunosuppression by glucocorticoids: Inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science. 1995; 270(5234):286-290. 20. Scheinman RI, Gualberto A, Jewell CM et al. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol. 1995; 15(2):943-953. 21. Jenkins BD, Pullen CB, Darimont BD. Novel glucocorticoid receptor coactivator effector mechanisms. Trends Endocrinol Metab. 2001; 12(3):122-126. 22. Crinelli R, Antonelli A, Bianchi M et al. Selective inhibition of NF-!B activation and TNF-alpha production in macrophages by red blood cell-mediated delivery of dexamethasone. Blood Cells Mol Dis 2000; 26(3):211-222. 23. Vaday GG, Franitza S, Schor H et al. Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix. J Leukoc Biol 2001; 69(6):885-892. 24. Dubiel W, Gordon C. Ubiquitin pathway: Another link in the polyubiquitin chain? Curr Biol 1999; 9(15):R554-557. 25. Chau V, Tobias JW, Bachmair A et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 1989; 243(4898):1576-1583. 26. Gregori L, Poosch MS, Cousins G et al. A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis. J Biol Chem 1990; 265(15):8354-8357. 27. Antonelli A, Crinelli R, Bianchi M et al. Efficient inhibition of macrophage TNF-alpha production upon targeted delivery of K48R ubiquitin. Br J Haematol 1999; 104(3):475-481. 28. Voges D, Zwickl P, Baumeister W. The 26S proteasome: A molecular machine designed for controlled proteolysis. Annu Rev Biochem 1999; 68:1015-1068.

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29. Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 2001; 292(5521):1552-1555. 30. Murray RZ, Norbury C. Proteasome inhibitors as anti-cancer agents. Anticancer Drugs 2000; 11(6):407-417.

CHAPTER 10

Design and Synthesis of New Pro-Drugs to be Used With Carrier Red Blood Cells S. Scarfì, G. Damonte and U. Benatti

Introduction

N

owadays, increasing attention is devoted to new systems of controlled in vivo delivery of biologically active molecules, the primary aim being to overcome some disadvan tages of conventional schedules of administration.1 Side effects are mostly critical for chemicals that display cytotoxic activities, as often observed with chemotherapic agents, especially antiviral and antitumoral drugs. A promising strategy towards improvement of therapies with highly toxic molecules is the exploitation of autologous cells as bioreactors competent for the time- and site-controlled production of pro-drugs. Within this frame, erythocytes have been proposed for two decades as potential carriers and bioreactors of medical interest.2 Internalization of many different molecules within red cells proved to be feasible by virtue of the properties of the erythrocyte membranes, whose elasticity and deformability allow the hypotonically induced opening of transient pores large enough to be crossed by externally placed macromolecules, before their closure in isotonic conditions.3. The erythrocytes so engineered have an in vivo life-span strictly comparable to that of unloaded ones when they are reinjected in the circulatory system of an autologous or compatible organism. Once loaded, red cells can behave as slow delivery systems for molecules of pharmacological interest, improving their pharmacokinetic patterns and therapeutic performances.3-10 There are two major ways of achieving erythrocyte-based release of drugs: direct encapsulation of membrane-diffusable drug molecules or internalization of impermeant and more encapsulable pro-drugs susceptible to be metabolically converted by endogenous erythrocyte enzymes to membrane-releasable active drugs. Obviously, the specificity and kinetic properties of intraerythrocytic enzymes involved in bioconversion reactions should dictate the choice of molecules that can be encapsulated in red blood cells to achieve the production of a specific drug. In all cases, the efficiency of the bioreactor, as measured by the output of the active drug, relies on several biochemical features including activity and substrate affinity of the bioconverting red cell enzymes and also patterns of exit of the drug across the membrane. Paradoxically, such efficiency may prove to be too high, posing challenges to the development of a pharmacokinetically useful system of drug delivery. In principle, these problems can be faced by the design and synthesis of metabolically more remote precursors of the final drug. In other words, the exploitation of specific bioconverting enzymes in the erythrocyte can suggest the final production of a membrane-diffusable active drug through a multi-step pathway that starts from an encapsulated pre-prodrug. An example of this strategy is represented by the slow delivery system developed in our laboratory for 5-F-2’-deoxyuridine (FdUR) an antineoplastic drug showing selective cytoxicity towards liver metastases from colorectal carcinomas.11 In this case human erythrocytes can Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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behave in vitro as bioreactors competent to release FdUR in a two-step pathway involving first a phosphodiesterase-catalysed hydrolysis of an ad hoc synthesized dimeric fluoropyrimidine of which we here report the design, chemical synthesis and characterization, followed by conversion of 5-F-2’-deoxyuridine monophosphate (FdUMP) to FdUR.12 Ideally, the design of such encapsulable precursors should follow extensive biochemical characterization of the relevant bioconverting enzymes, suggesting best fit-based structures to be tailored accordingly. In both cases of engineered erythrocytes, either passive carriers or bioreactors, the release of drugs can be obtained in the circulatory system or at specific sites.13 The natural property of erythrophagocytosis of monocyte/macrophages makes these cells ideal compartments for erythrocyte-based delivery of drugs. Furthermore, monocyte-derived macrophages (MDMs) are important in vivo reservoirs for different kinds of viruses and microorganisms. Some typical examples are: 1. Human immunodeficiency virus (HIV), which selectively kills CD4 lymphocytes, but in macrophages induces an infection characterized by a long–term production of virus particles.14 2. Various herpesviruses such as HSV-1/2.15 for which macrophages can become natural reservoirs of latent HSV in the lungs, central nervous system and other tissues. Moreover, HSV-1/2 infection is common among individuals infected with HIV. 3. Microorganisms of the Mycobacterium avium-M. intracellulare complex (MAC) which represent the most common cause of systemic bacterial infections in patients with AIDS.16-19 In fact, while human infection with MAC is rare in immunocompetent individuals,17 it is widespread in HIV-infected patients, its incidence in the late stages of AIDS being from 25 to 40%.20,21 In addition MAC infection, as well as HSV, is able to activate and increase HIV replication,22 thereby accelerating the progression of the disease. Hence, therapeutic strategies able to inhibit replication of HIV alone or in the presence of HSV or MAC co-infections in macrophages are needed. The nucleoside analogues zidovudine (AZT) and acyclovir (ACV) are among the drugs of choice against HIV-1 and HSV-1 infections, while the antimicrobial agent ethambutol (EMB) is frequently used to control MAC infections. We report here the design, chemical synthesis and characterization of a series of new AZT analogues: an AZT homodinucleotide (AZTp2AZT) designed for HIV infection, and two heterodinucleotides AZTp2ACV designed for HIV and HSV co-infections and AZTp2EMB for HIV and MAC co-infections, respectively consisting of an antiretroviral (AZT) and an antiherpetic drug (ACV) or an antiretroviral (AZT) and an antimicrobial agent (EMB). All the dinucleotides were bound by a pyrophosphate bridge opportunely designed to be hydrolized once inside macrophages after phagocytosis of encapsulated erythrocytes used as delivery system specifically targeted to the macrophagic compartment. Finally, the affinity of the different dinucleotides-loaded erythrocytes to opportunely infect human macrophages provided effective and almost complete in vitro protection from HIV-1, HSV-1 or MAC replication as described elsewhere.23-25

Dimeric Fluoropyrimidine Synthesis and Intraerythrocytic Biochemical Pathway De Flora et al have previously described the conversion of the encapsulated pro-drug FdUMP to its pharmacologically active form FdUR in human and mouse erythrocytes.26,27 Although such pro-drug to drug conversion was unequivocally demonstrated to occur in the FdUMPloaded erythrocytes, its rate was found to be too high to be compatible to an efficient anticancer therapy. Accordingly, an alternative approach was developed in order to down-regulate FdUR release: the reconstruction within the erythrocytes of a longer sequence of reactions still terminating with the FdUMP to FdUR conversion. This study.12 involved the chemical synthesis of a properly tailored pre-prodrug represented by a new fluoropyrimidine dimer that was

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Figure 1. molecular chemical structure of 5-fluoro-2’-deoxyuridyl-(5’(3’)-5-fluoro-2’-deoxy-5’-uridylic acid (Compound 1) followed by MS characterization performed in negative ion mode. Spectrum of Compound 1: [M – H]- = 632.5, [M + Li+- 2H]- = 638.5, [M + 2Li+—3H]- = 644.2. Fragments of the molecule were identified as A, B and C where 503.3 = [M—A – H]-, 404.5 = [B – H]-and 324.7 = [C – H]-.

demonstrated to generate FdUMP within erythrocytes. The kinetic and metabolic features of this model of drug production and release seem to fit the requirements of chemotherapy with FdUR. Synthesis of the dimeric fluoropyrimidine compound: This compound: 5-fluoro-2’deoxyuridyl-(5’(3’)-5-fluoro-2’-deoxy-5’-uridylic acid, (Compound 1 reported in Fig. 1) was

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chemically synthesized from two separately deblocked 5-fluoro-2’-deoxyuridine mononucleotide moieties according to the general method described by Mazzei et al28 starting from the completely protected monomer ∋-cyanoethyl-p-clorophenyl-5’-(5-fluoro-2’-deoxyuridine phosphate)-3’-levulinate (C 0). Details on synthesis of the protected monomer (C 0) and on its treatment in order to obtain separate removal of the levulinic group from the 3’ position (C 02) and of the ∋-cyanoethyl group form the 5’ position (C 01), respectively, have been reported elsewhere.29 The mixture of C 01 and C 02 (0.025 mmol each) was heated at 40°C for 30 min in the presence of 2,4,6-triisopropylbenzenesulphonyl chloride (TIPS) (0.1 mmol) and 1methylimidazole (MI) (60 ∝l), using 1 ml of anhydrous pyridine as solvent. After cooling, the solution was evaporated with toluene and the residue dissolved in chloroform and purified by silica gel chromatography as described by Gasparini et al.12 The collected fractions were evaporated under reduced pressure and precipitated in petroleum ether. Deprotection of Compound 1 and purification by HPLC was obtained as described by Gasparini et al.12 Finally, the eluates were evaporated under reduced pressure and the residue suspended in 0.3 ml of water and precipitated as lithium salt by adding 3 ml of 2% LiClO4 in acetone. Results and discussion: The protected dimer was obtained at a 70% yield. The final compound (C 1) was obtained at a 50% yield. The molecular weight of C 1 was confirmed using an electrospray mass spectrometer. The instrument was set in the negative ion mode for the presence of phosphate groups in the structure. The analysis showed an m/z of 632.5 consistent with the expected mass as reported in Figure 1. The metabolic pattern of conversion of C 1 was investigated in intact erythrocytes following encapsulation of this compound as described by Gasparini et al.12 Both intra-erythrocytic metabolites and those released into the supernatant plasma during a 30 hours sterile incubation were analyzed. The results of these experiments showed that approximately 30% of the original amount of C 1 was still present in the red cells after 30 hours, furthermore three major by-products were detectable in the same extracts. Of these, one was immediately identified as FdUMP while the other two proved to be the corresponding diphosphate and triphosphate forms, FdUDP and FdUTP respectively. At the same time very low amounts of FdUR and fluorouracile (FU) were also detected. In fact the two were produced intracellularly and then released from the red cells, FdUR very rapidly over the first 3 hours and much more slowly from 3 to 30 hours of incubation while FU showed a delay formation as compared to FdUR and afterwards increased in a parallel fashion to FdUR until reaching relatively high extra-erythrocytic levels at 30 hours. In conclusion the introduction of an additional, phosphodiesterase-catalysed step upstream to the final bioconversion reaction resulting in the slow production and release of FdUR has two major and distinctive consequences as compared to simple FdUMP metabolism26: a) the formation of FU in addition to FdUR and b) the partial challenging of FdUMP to FdUDP and FdUTP derivatives that represent transient fluoropyrimidine reservoirs for further formation of FdUR (and FU) through sequential dephosphorylation steps. A biochemical approach to therapy like the one pursued in this study seems to be promising. In fact the present findings represent the basis for the development of animal models of pre-prodrug-loaded erythrocytes behaving as active slow delivery systems of FdUR in the treatment of sensible hepatic and colorectal carcinomas.

AZTp2AZT Homodinucleotide Synthesis and Characterization

Most antiviral nucleoside analogues (AZT, dideoxycitidine etc.) to be pharmacologically active, must first be phosphorylated by cellular kinases.30,31 The activity level of these enzymes depends on the cell type and the cell activation state.32,33 Usually, quiescent cells have low levels of the enzymes responsible for nucleoside analogue phosphorylation, while activation results in increased activity levels. Macrophages phosphorylate AZT and dideoxycytidine (ddC) at a much lower rate than peripheral blood mononuclear cells.34,35 To overcome the low ability of resting macrophages in phosphorylating the antiviral dideoxynucleosides, Magnani et al36 have previously developed a drug-delivery system, based

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on the use of autologous erythrocytes, for the direct administration of phosphorylated nucleoside analogues. In the past few years it has been shown that these analogues are responsible in the cell for antiviral activity and cell toxicity.37. Based on these considerations, we thought that an antiviral drug designed to protect macrophages should ideally be administered in a phosphorylated form, yet not as the nucleoside 5’-triphosphate, to avoid cell toxicity, but as a close precursor of it. We report here the chemical synthesis, characterization, biochemical activation and anti-retroviral activity in macrophages of a new AZT analogue, an AZT homodinucleotide di(thymidine-3’-azido-2’,3’-dideoxy-D-riboside)-5’,5’-p1,p2-pyrophosphate (AZTp2AZT) reported in Figure 2, which pyrophosphate bond biochemical hydrolysis generates two azidothymidine monophosphate (AZT-MP) moieties, that fits the above mentioned requirements. Synthesis and characterization of AZTp2AZT: AZT-MP was synthesized and characterized as described Magnani et al.38 Synthesis of P1-AZT-5’-P2-diphenyl pyrophosphate was obtained according to Michelson.39 AZT-MP tri-n-octylammonium salt was prepared by dissolving 0.25 mmol of AZT-MP free acid (100 mg) in 3 ml of methanol and 0.45 ml of tri-noctylammine. The mixture was stirred for 30 min at 25°C and vacuum dried. The residue was suspended in 2 ml of N,N-dimethylformamide and dried under reduced pressure. P1-AZT-5’-P2-diphenyl pyrophosphate (0.25 mmol) was then dissolved in anhydrous pyridine and added to 0.25 mmol of the AZT-MP tri-n-octylammonium salt, prepared as above and treated to obtain the final compound as described by Magnani et al.23 The dimer was then purified by HPLC.23 and then submitted to chemical analyses. The purification resulted in the salt-free compound with a 99.1% purity and with a final yield of the process of 60%. The mass spectrum of the purified product, acquired with an electrospray mass spectrometer set in the negative ion mode, showed a molecular ion at m/z 675.9, consistent with the expected mass of the AZTp2AZT molecule as shown in Figure 11.2. The UV absorption spectrum of the same purified product, acquired in phosphate buffer (pH 4.9) and 40% methanol in water, exhibited an absorption maximum at 265 nm. Finally, the 1H nuclear magnetic resonance (NMR) spectrum acquired at 200 MHz in the temperature range of 20-30°C on a Varian Gemini spectrometer in 2H2O showed the most significant chemical shifts at 1.639 (ppm (-CH3), 2.225 (ppm (O-CH2), 3.943 (ppm (broad 1’-CH and 2’-CH2), 4.275 (ppm (3’-CH), 5.955 (ppm (4’-CH) and 7.439 (ppm (aromatic proton 6-CH). Taken together, these properties are consistent with the structure expected from the above-described procedure of synthesis. Results and discussion: Murine, feline and human RBC were used to encapsulate AZT homodinucleotide yielding final intra-erythrocytic concentrations up to 4 mM in the specific case of human RBC, however never below 1 mM in the other species tested. These results confirm the high encapsulability of the dinucleotide, as expected from the molecular chemical structure, this being one of the principal features required in the design of this drug. Stability experiments indicated that as much as 90% of AZTp2AZT was still present after incubation in isotonic, sterile conditions of the loaded human RBC for 24 hours at 37°C. This remarkable stability within the carriers allowed to introduce the membrane alterations that induce opsonization of the loaded RBC and their consequent recognition and phagocytosis by macrophages.36 In macrophages the dimer showed a progressive decay, due to a specific pyrophosphatase enzymatic activity, until becoming undetectable within three days after erythro-phagocytosis with a paralell increase of its metabolite AZT-MP that is subsequently converted to the pharmacologically active triphosphate form. These data are consistent for metabolism of AZTp2AZT taking place largely in macrophages rather than in the carrier RBC, thus fulfilling a major requirement of the newly synthesized molecule. Accordingly, AZTp2AZT-loaded erythrocytes administered to macrophages inhibited HIV-1 pro-viral formation by 93-97% and p24 production by 80%.23 Cell-targeted synthesis of pharmacologically active derivatives of AZT seems to be optimal for display of anti-retroviral activity, even starting from quite low intracellular concentrations of the AZTp2AZT pro-drug in macrophages and thereby preventing cell toxicity. Therefore, The RBC-based system of targeted delivery of precursors of triphosphorylated nucleoside

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Figure 2. molecular chemical structure of AZT homodinucleotide di(thymidine-3’-azido-2’,3’-dideoxy-Driboside)-5’,5’-p1,p2-pyrophosphate (AZTp2AZT) and its MS analysis after purification. The acquisition performed in negative ion mode revealed a unique peak at 613.4 corresponding to [M – H]-.

analogues in virus-infected macrophages proves to be a promising way to inhibit HIV replication in these cells.

AZTp2ACV Heterodinucleotide Synthesis and Characterization

Human herpesviruses (HSVs) are distributed worldwide and are among the most frequent causes of viral infections in HIV-1 immuno-compromised patients. Hence, therapeutic strategies able to inhibit HSV-1 and HIV-1 replication are sorely needed. Until now, the most common therapies against HSV-1 and HIV-1 infectivity have been based on the administration of nucleoside analogues; however, to be active, these antiviral drugs must be converted to their triphosphorylated derivatives by viral and/or cellular kinases. At the cellular level, the main problems concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations, we designed a new compound consisting of azidothymidine (AZT) and acyclovir (ACV) bound by a pyrophosphate bridge, P1thymidine-3’-azido-2’-3’-dideoxy-D-riboside-5’-P2guanine-9(2-hydroxyethoxymethyl) pyrophosphate (AZTp2ACV) reported in Figure 3, which could act as a pro-drug for the production of partially phosphorylated antiviral drugs. The impermeant and highly encapsulable AZTp2ACV was encapsulated into autologous erythrocytes to undergo phagocytosis by human macrophages, where, once inside, metabolic activation of the drug occurred. We report here the chemical synthesis and characterization, metabolism and antiviral activities of the above-mentioned heterodimer. The results obtained show that the administration of AZTp2ACV-loaded RBCs to macrophage is possible and that it protects macrophages from both HIV-1 and HSV-1 infections.24 Synthesis and characterization of AZTp2ACV: The AZTp2ACV heterodimer (P1thymidine3’-azido-2’-3’-dideoxy-D-riboside-5’-P2 guanine-9-(2-hydroxyethoxymethyl)pyrophosphate was obtained using the coupling method of the phosphoromorpholidate by Hostetler et al40 with several modifications.

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Figure 3. molecular chemical structure of P1thymidine-3’-azido-2’-3’-dideoxy-D-riboside-5’-P2guanine-9(2-hydroxyethoxymethyl) pyrophosphate (AZTp2ACV) and its MS analysis after purification. The acquisition performed in negative ion mode revealed a unique peak at 634.0 corresponding to [M – H]-.

ACV-MP was obtained as described by Yoshikawa et al,41 except that after the reaction the mixture was poured into diethyl ether, and the resulting precipitate was separated by centrifugation. The powder was then dissolved in water and the acqueous solution adjusted to pH 7.5 with a 1 M NaOH solution, then the solvent was removed under reduced pressure and dried in vacuo. The product (ACV-MP), obtained with a 100% yield, was identified using a mass spectrometer equipped with an electrospray ionization source (API-ESPI), and set in the negative ion mode, that confirmed the expected molecular mass of 305.5. The azidothymidine-phosphoromorpholidate compound was synthesized following the method of Hostetler et al.40 The product was analyzed by mass spectrometry as described above and showed a mass value of 416.8, consistent with the expected molecular weight. The coupling of azidothymidine-phosphoromorfolidate to acycloguanosine-monophosphate was obtained as follows: 1 mmol of ACV-MP tributylammonium salt, obtained as described by van Wijk et al,42 was dissolved in 5 ml of anhydrous pyridine and added to a 5 ml solution of the same solvent containing 1 mmol of AZT-MP. The mixture was then treated as described by Rossi et al24 and purified accordingly. The final product, raising a 31% yield, was analyzed by mass spectrometry with an expected m/z ratio of 634.0 as shown in Figure 3. All measurements were performed in the negative ion mode, scanning in the 300-800 mass range. Results and discussion: AZTp2ACV (Fig. 3) was encapsulated into human erythrocytes yielding different intracellular concentrations (from 1.0 up to 10 mM) by adding different amounts of AZTp2ACV during the dialysis step. Also in this case, as already occurred for AZTp2AZT homodimer, the compound showed high encapsulability features confirming the winning strategy of the design of these kinds of molecules. Once loaded with 4.0 ∝mol/ml of AZTp2ACV, RBCs were targeted to human macrophages following the usual procedure,36 showing the presence of 0.4 nmol of AZTp2ACV/106 macrophages at time zero (that is, after 18 hours of incubation of loaded RBCs with macrophages) and a progressive decay until reaching 30% at 48 hours after the onset of erythro-phagocytosis. Once delivered to macrophages,

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AZTp2ACV degradation occurred to yield its pharmacologically active metabolites, as demonstrated by the following reported antiviral activities. The suggested mechanism is as follows: AZTp2ACV is first cleaved into two nucleoside 5’-phosphate moieties (AZT-MP and ACVMP) by a specific pyrophosphatase, after which, the partially phosphorylated drugs are converted to the corresponding 5’-triphosphate derivatives that are ultimately responsible for inhibition of HIV-1 and HSV-1 infectivity. In fact, once encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis, the heterodimer is able to protect macrophages from de novo infection by HIV-1 with a maximum inhibition of 80%, and HSV1 with an inhibition of more than 90%.24 Concluding, we have shown that it is possible to protect macrophages against de novo HIV and HSV infections by the administration of a single pro-drug consisting of two nucleoside analogues (AZT and ACV) bound by a pyrophosphate bridge. These promising in vitro data must be further evaluated in an animal model of immunodeficiency.

AZTp2EMB Heterodinucleotide Synthesis and Characterization

Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS.16-19 The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in immuno-compromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology.20,21 Hence, a therapeutic strategy able to inhibit HIV and Mycobacterium replication could be very useful. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance.43 Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Prompted by these considerations, we designed and synthesized a new heterodimer consisting of AZT and ethambutol (EMB), a commonly used bactericidal drug, interconnected by a pyrophosphate bridge (AZTp2EMB), reported in Figure 4, susceptible to be cleaved by a specific pyrophosphatase enzyme present in both human erythrocytes and macrophages as already described in this article. Once encapsulated into RBCs, the compound could act as a pro-drug for the release of AZT and EMB in the circulatory system or alternatively be directly targeted to infected macrophages. Here we report the chemical synthesis, characterization, biochemical activation and antibacterial effect of the above mentioned heterodimer. Synthesis and characterization of AZTp2EMB: The heterodimer was obtained using a modified version of the phosphoromorpholidate coupling method. The azidothymidine phosphoromorpholidate compound was synthesized following the method of Hostetler et al.40 The product was analyzed by mass spectrometry using an electrospray source that revealed a mass value of 416.8 m/z, consistent with the expected molecular structure. The synthesis of the compound ethambutol monophosphate was based on a significantly modified version of the method described by Cherbuliez et al.44 Equimolar ratios (2 mmol each) of ethambutol and pyrophosphoric acid were heated at 150°C for 6 hours in an oil bath. Water (30 ml) was added to the cooled reaction mixture, and the pH was adjusted to 9.0 by adding a 1 N Ba(OH)2 solution to precipitate inorganic phosphates, which were then removed by filtration. The mixture was acidified to pH 3.0 with a 5 N H2SO4 solution to obtain the precipitation of BaSO4, which was then removed by centrifugation. The acidic solution was then concentrated in vacuo and loaded onto a DEAE Sephadex A-50 cation-exchange resin to remove excess ethambutol. The product, obtained with a 90% yield, was analyzed by mass spectrometry as described above, showing the correct value of 283.5. With regard to the synthesis of the AZTp2EMB heterodimer P1-thymidine-3’-azido-2’,3’-dideoxy-D-riboside-5’P2(+)2,2’-(ethylenediimmino)-di-1-butanol-pyrophosphate, the final coupling of AZT phosphoromorpholidate to ethambutol monophosphate was obtained and purified as described by Rossi et al.25 The final product, obtained with a 21% yield, was analyzed by electrospray mass spectrometry, which gave the expected m/z ratio of 613.4 as shown in Figure 4. The

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Figure 4. molecular chemical structure of P1-thymidine-3’-azido-2’,3’-dideoxy-D-riboside-5’-P2(+)2,2’(ethylenediimmino)-di-1-butanol-pyrophosphate (AZTp2EMB) and its MS analysis after purification. The acquisition performed in negative ion mode revealed a unique peak at 675.9 corresponding to [M – H]-.

measurements were performed both in the negative and positive ion mode due to the contemporary presence of phosphate and aminic groups in the molecule, scanning in the 300-800 mass range. Results and discussion: The heterodimer AZTp2EMB (Fig. 4) obtained as described above was encapsulated in human erythrocytes loaded with different concentrations (from 1.0 mM to more than 10 mM) by adding different amounts of compound during the dialysis step. A slow linear decrease in intracellular AZTp2EMB was observed, reducing by one-half its concentration about every 48 hours. The decrease in the amount of heterodimer in the RBCs was paralleled by stoichiometric production of EMB, AZT and AZT-MP in the incubation medium. The erythrocytes showed a high efficiency of EMB and AZT formation and export. So, MAC-infected human macrophages were cultured in the presence of AZTp2EMB-loaded nonopsonized RBCs or by simply adding the media pre-incubated with loaded erythrocytes for different time intervals. Both loaded RBCs or the pre-incubated media were able to inhibit the growth of bacilli in infected macrophages in a time-dependent manner. In particular, the administration of loaded RBCs was able to inhibit the intracellular replication of M. avium of about 22% in one day and more of 90% in six days.25 With this strategy we suggest the possibility of protecting individuals against HIV and MAC infections by the administration of a single pro-drug consisting of two compounds (azidothymidine and ethambutol) bound by a pyrophosphate bridge, which has two major advantages: it improves the pharmacokinetics and decreases toxic side effects of these drugs. The data here reported prove that RBCs loaded with the new heterodimer AZTp2EMB act as bioreactors for slow delivery of the antiviral drug AZT and the antimycobacterial ethambutol with promising perspective for the development of macrophage-targeted delivery of AZTp2EMB-loaded erythrocytes.

Conclusions The design and build-up of pharmacologically active new molecules requires a careful and multi-faceted study of the specific cell targets and of the enzymatic pathways involved in drug

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metabolism. In particular, the procedure of chemical synthesis of Compound 1 allowed us to investigate properly its putative role as precursor of pharmacologically active fluoropyrimidine molecules in biological systems. The metabolic fate of Compound 1 within human erythrocytes seems to involve an immediate phosphodiesterase-catalyzed conversion of the dimeric structure to FdUMP. As soon as this moiety is produced, it is partially rephosphorylated to both FdUDP and FdUTP. These two molecules are then slowly reconverted back to FdUMP and finally to FdUR in 3-30 hours. The pool of phosphorylated precursors created by such a metabolic pathway is able to ensure an almost linear production in, and release of, FdUR by the erythrocytes. These findings indicate that human RBCs behave as bioreactors competent to transform a non-membrane diffusable pro-drug molecule into a diffusable drug according to patterns that are compatible with pharmacokinetic requirements of human cancer therapy.12 In the case of the development of new therapeutic systems against AIDS, based on nucleotide analogues and designed to achieve anti-retroviral activity in specific cell types like macrophages, a combination of different features is required. In fact, pharmacologically active triphosphorylated nucleoside analogues can display unacceptable cell toxicity. For this reason, such molecules should be formed directly in the desired cell type at low concentrations such as to inhibit viral reverse transcriptase selectively; therefore metabolically suitable pro-drugs, structurally tailored to be converted by endogenous enzyme systems, are necessary. All the above requirements seem to be fulfilled by the homo- and heterodinucleotides (AZTp2AZT, AZTp2ACV, AZTp2EMB) azidothymidine-derived that have been designed, synthesized and characterized in our laboratory and here reported. Central to the design of these new molecules is their high encapsulability and susceptibility to dinucleotide pyrophosphatase activity. In fact, the enzyme promotes the activation of the drugs directly in macrophages with the release of AZT-MP alone, or the same plus ACV-MP or EMB now able to display their pharmacological action on HIV-1, HSV-1 or MAC infections or co-infections as clearly demonstrated in our studies.23-25 The versatility of the chemical procedure for synthesizing such molecules should allow extension to other homodinucleotides (e.g., of ddC, ddA and ddI) or heterodinucleotides carrying nucleoside analogues of comparable anti-retroviral activity.

References 1. Langer R. New method of drug delivery. Science 1990; 249:1527-1533. 2. Ihler GM, Glew RH, Schnure FW. Enzyme loading of erythrocytes. Proc Natl Acad Sci USA 1973; 70:2663-2666. 3. Deloach JR. Encapsulation of exogenous agents in erythrocytes and the circulating survival of carrier erythrocytes. J Appl Biochem 1983; 5:149-157. 4. Deloach JR, Sprandel U. Red Blood Cells as Carriers for Drugs. Basel: Karger 1985. 5. De Flora A, Benatti U, Guida L et al. Encapsulation of adriamycin in human erythrocytes. Proc Natl Acad Sci USA 1986; 83:7029-7033. 6. Ropars C, Chassaigne M, Nicolau C. Red Blood Cells as Carriers For Drugs. Potential therapeutic applications. Oxford: Pergamon Press, 1987. 7. Zocchi E, Tonetti M, Guida L et al. Encapsulation of doxorubicin in liver targeted erythrocytes increases the therapeutic index of the drug in a murine metastatic model. Proc Natl Acad Sci USA 1989; 86:2040-2044. 8. Green R, Deloach JR. Resealed Erythrocytes as Carriers and Bioreactors. Oxford: Pergamon Press, 1991. 9. Magnani M, Deloach JR. The Use of Resealed Erythrocytes as Carriers and Bioreactors. New York: Plenum Press, 1991 10. De Flora A, Tonetti M, Zocchi E et al. Engineerd erythrocytes as carriers and bioreactors. In: Terhorst C, Malavasi F, Albertini A, eds. The Year of Immunology. Basel: Karger, 1993:168-174. 11. Chabner BA. Pharmacologic Principles of Cancer Treatment. Philadelphia: Saunders, 1982. 12. Gasparini A, Giovine M, Damonte G et al. A novel dimeric fluoropyrimidine molecule behaves as a remote precursor of 5-Fluoro-2’-deoxyuridine in human erythrocytes. Biochem Pharmacol 1994, 48(6):1121-1128. 13. Tonetti M, Bartolini A, Sobrero A et al. Organ distribution of glutaraldheide treated erythrocytes in patients with hepatic metastases. In: Deloach JR, Way JL, eds. Advances in the Biosciences.

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Carrier and Bioreactor Red Blood Cells for Drug Delivery and Targeting. 1994, 92: 169-176.AUTHOR, IS THIS A BOOK OR A JOURNAL? 14. Meltzer MS, Skillman DR, Gomatos PJ et al. Role of mononuclear phagocytes in the pathogenesis of human immunodeficiency virus infection. Annu Rev Immunol 1990; 8:169-194. 15. Wu L, Morahan PS, Hendrzak JA et al. Herpes simplex virus type 1 replication and IL-1 beta gene expression in mouse peritoneal macrophages activated in vivo by an attenuated Salmonella typhimurium vaccine or Corynebacterium parvum. Microbiol Pathog 1994; 16:387-399. 16. Benson DA, Ellner JJ. Mycobacterium avium complex infection and AIDS: Advances in theory and practice. Clin Infect Dis 1993; 17:7-20. 17. Inderlied CB, Kemper CA, Bermudez LEM The Mycobaterium avium complex. Clin Microbiol Rev 1993; 6:266-310. 18. Low N, Pfluger D, Egger M. Disseminated Mycobacterium avium complex disease in the Swiss HIV Cohort Study: Increasing incidence, unchanged prognosis. AIDS 1997; 11:1165-1171. 19. Young LS. Mycobacterium avium complex infection. J Infect Dis 1998; 157:863-867. 20. Nightingale SD, Byrd LT, Southern PM et al. Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J Infect Dis 1992; 165:1082-1085. 21. Ellner JJ, Goldberger MJ, Parenti DM. Mycobacterium avium infection and AIDS: A therapeutic dilemma in rapid evolution. J Infect Dis 1991; 163:1326-1335. 22. Havlir DV, Haubrich R, Hwang J et al. Human immunodeficiency virus replication in AIDS patients with Mycobaterium avium complex bacteremia: a case control study. J Infect Dis 1998; 177:595-599. 23. Magnani M, Casabianca A, Fraternale A et al. Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection. Proc Natl Acad Sci USA 1996; 93:4403-4408. 24. Rossi L, Brandi G, Schiavano GF et al. Macrophage protection against Human Immunodeficiency Virus or Herpes Simplex by red blood cells-mediated delivery of a heterodinucleotide of azidothymidine and acyclovir. AIDS Res Hum Retrov 1998; 14 (5):435-444. 25. Rossi L, Brandi G, Schiavano G et al. Heterodimer-loaded erthrocytes as bioreactor for slow delivery of the antiviral drug azidothymidine and the antimycobacterial drug ethambutol. AIDS Res Hum Retrov.1999; 15(4):345-353. 26. De Flora A, Zocchi E, Guida L et al. Conversion of encapsulated 5-fluoro-2’-deoxyuridine-5’monophosphate to the antineoplastic drug 5-fluoro-2’-deoxyuridine in human erythrocyte. Proc Natl Acad Sci USA 1988; 85:3145-3149. 27. Zocchi E, Guida L, Polvani C et al. Human and murine erythrocyte as bioreactor releasing the antineoplastic drug 5-fluoro-2’-deoxyuridine. In: Green R, Deloach JR, eds. Resealed Erythrocyte as Carrier and Bioreactor. Oxford: Pergamon Press 1991:51-57. 28. Mazzei M, Balbi A, Grandi T et al. Synthesis in solution of oligodeoxynucleotides and some of their 5’- and 3’- linked derivatives. Il Farmaco 1993; 48:1649-1661. 29. Mazzei M, Grandi T, Balbi A et al. Protected 5-fluoro-2’-deoxyuridine monophosphate for solution-phase synthesis of oligonucleotides. Il Farmaco 1994; 49:793-797. 30. Mitzuja H, Yarchoan R, Brother S. Molecular targets for AIDS therapy. Science 1990; 249:1533-1544. 31. De Clercq E. Basic approaches to anti-retroviral treatment. J AIDS 1991; 4:207-218. 32. Gao W, Cara A, Gallo R et al. Low levels of deoxynucleotides in peripheral blood lymphocytes: A strategy to inhibit human immunodeficiency virus type 1 replication. Proc Natl Acad Sci USA 1993; 90:8925-8928. 33. Gao W, Agbaria R, Driscoll JS et al. Divergent anti-human immunodeficiency virus activity and anabolic phosphorylation of 2',3'-dideoxynucleoside analogs in resting and activated human cells. J Biol Chem 1994; 269:12633-12638. 34. Perno CF, Yarchoan R, Cooney DA et al. Inhibition of human immunodeficiency virus (HIV-1/ HTLV-IIIBa-L) replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides. J Exp Med 1988; 168:1111-1125. 35. Richman DD, Kornbluth RS, Carson DA. Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages. J Exp Med 1987; 166:1144-1149. 36. Magnani M, Rossi L, Brandi G et al. Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages: In vitro and in vivo studies. Proc Natl Acad Sci USA 1992; 89:6477-6481. 37. Lewis W, Dalakas MC. Mitochondrial toxicity of antiviral drugs. Nat Med 1995; 1:417-422. 38. Magnani M, Giovine M, Fraternale A et al. Red blood cells as a delivery system for AZT. Drug Delivery 1995; 2:57-61.

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39. Michelson AM. Synthesis of nucleotide anhydrides by anion exchange. Biochim Biophys Acta 1964; 91:1-13. 40. Hostetler KY, Stuhmiller LM, Lenting BM et al. Synthesis and antiretroviral activity of phospholipid analogues of azidothymidine and other antiviral nucleosides. J Biol Chem 1990; 265:6112-6117. 41. Yoshikawa M, Kato T, Takenishi T. A novel method for phosphorylation of nucleosides to 5’nucleotides. Tetrahedron Lett 1967; 50:5065-5068. 42. van Wijk GMT, Hostetler KY, van den Bosch H. Antiviral nucleoside diphosphate diglycerides: Improved synthesis and facilitated purification. J Lipid Res 1992; 33:1211-1219. 43. Bermudez LE, Inderlied CB, Young L.S. Mycobacterium avium complex in AIDS. Curr Clin Top Infect Dis 1992; 12:257-281. 44. Cherbuliez E, Rabinovitz J. Recherches sur la formation et la transformation des esters. Helv Chim Acta 1956; 39:1455-1461.

CHAPTER 11

Engineered Red Blood Cells as Circulating Bioreactors P. Ninfali and E. Biagiotti

O

ver the last twenty years, enzymes encapsulated in red blood cells (RBC) have proven to be efficient bioreactors in the clearance of undesired molecules from the blood stream and have shown an ability to correct congenital metabolic disorders. Highly efficient methods of entrapment based on hypotonic dialysis, resealing and annealing were set up and the loaded RBC were used in clinical trials. One of the most important applications was the use of asparaginase-loaded-RBC as a therapeutic tool in the treatment of lymphosarcomas and acute lymphoblastic anemia. In our laboratory, two main applications of enzyme-loaded RBC were tested for the clearance of toxic products of alcohol metabolism and the control of the glycemic state. Erythrocytes loaded with aldehyde dehydrogenase were shown to favour the depletion of acetaldehyde and ethanol in normal or alcoholic mice, and alcohol oxidase-loadedRBC were efficiently used to deplete methanol and formaldehyde during methanol poisoning. The coentrapment of glucose oxidase and hexokinase was shown to be useful in the control of glycemia in a strain of diabetic mice. Although liposomes are usually used nowadays to carry proteins, RBC loaded with proteins still remain an important tool in clinical biochemistry, particularly when a long in vivo circulation is required.

Introduction The carbohydrate metabolism in mature mammalian RBC is constituted by the EmbdenMeyerhoff pathway (EMP) and the connected pentose phosphate pathway (PPP). The EMP generates ATP, 2,3DPG and NADH, which are important for cell survival and function. The final products of EPM are pyruvate and lactate, whose intracellular levels are in equilibrium with their plasma levels. The PPP starts from glucose-6-phosphate (G6P) and bypasses some steps of the EMP; final products of G6P metabolism then enter EMP at the level of F6P and triose-P after having released NADPH and pentose-P. In RBC, the percentage of glucose which passes through the PPP is normally 10%, but it can increase under oxidative stress to produce higher amounts of reducing equivalents. The human RBC also have the capacity to metabolize galactose as well as purine nucleotides and nucleosides in order to regulate the adenine nucleotide turnover; moreover the ATPase enzyme systems, which are tightly bound to the membrane, maintain a high level of intracellular K+ and a low level of Na+ by pumping these cations against a concentration gradient.1 Few enzymes of the Krebs-cycle, carbonic anhydrase, carboxylic esterase, acid phosphatase, glyoxalase I and II, SOD, transaminases, glutamate dehydrogenase and other minor enzymes have also been found in RBC.1;2 The metabolic system of RBC is relatively simple and quite also stable when exogenous enzymes are included in the cytoplasm by loading procedures based, for example, on hypotonic dialysis followed by resealing and reannealing.3 This possibility has led to methods able to Erythrocyte Engineering for Drug Delivery and Targeting, edited by Mauro Magnani. ©2002 Eurekah.com.

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Figure 1. Example of an enzyme encapsulated in the erythrocyte—The enzyme may be a circulating bioreactor able to transform undesired molecules into harmless or easily metabolized products. Alternatively, it may be used to study the cytotoxicity of some products generated by the enzymatic reaction.

yield long surviving erythrocytes encapsulated with enzymes to correct or solve congenital metabolic disorders or to clear undesired molecules from the blood stream.3;4 This approach is shown in Figure 1 Comparative studies, using erythrocytes of different species, demonstrated that erythrocytes of humans, mice and dogs offer the best conditions for the entrapment of enzymes.3 The percentage of entrapment is dependent on the molecular size of the protein, while the loss of enzyme is caused by hemolysis or destruction of RBC in the reticuloendotelial system.4

The Entrapped Enzymes Most of the enzymes whose entrapment has been set up over the last twenty years are listed in Table1.

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Table 1. Enzymes entrapped in erythrocytes and putative applications Entrapped enzyme

Source and conditions of use

Modified property

Clinical usefulness

Ref

Urate oxidase Uricase %-glucosidase Glucose oxidase

Commercial from different souces, native Commercial, native Native from A. niger

Uric acid concentration

Uricemia

4

Humans and A. niger, native

Amyloidosis Study of H2O2 cytotoxicity Diabetes

4 27

Hexokinase + Glucose oxidase (-aminolevulinate dehydratase Arginase

Liver glycogen ATP and GSH depletion, H2O2 accumulation Glucose consumption with normal ATP (-aminolevulinate concentration To lower arginine concentration

Toxic porphyria

28

Hyperargininemia

29

To lower cyanide

Poisoning by CN-

30,31

Asparagine anemia/lymphosarcomas Glucose consumption rate Bilirubin level in plasma Levels of ATP

Acute lymphoblast.

7,11

Study of aspects of glucose metabolism Jaundice

13

Severe combined immunodeficiency Gaucher’s desease

32 18,2

Methanol poisoning

26

Human blood, native

Hexokinase

Commercial + S2O3=, native concentration Humans, native plasmatic level Humans, native

Bilirubin oxidase

Humans, native

Adenosine deaminase

Humans, PEG-conjugated enzyme

Alglucerase

Humans, native glucocerebrosides

Lysosomal

Alcohol oxidase

Yeast (Pichea Pastores), native

Methanol concentration in blood

Rhodanase Asparaginase

13

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Erythrocyte Engineering for Drug Delivery and Targeting

Rat or bovine liver, native

14

Alcohol dehydrogenase and/or Aldehyde dehydrogenase

Bovine liver / Alcaligenes euthropus

Alcohol and acetaldehyde level in blood

Chronic ethanol consumption

33,13, 15,25, 34,6

Glutammate dehydrogenase Glucose-6-phosphate dehydrogenase

Bovine liver, native

Ammonia level in blood

Renal failure

33

Human erythrocytes or granulocytes, native

Oxidative stress in red cells

Study of the biochemistry of favism

35,12

Superoxide dismutase

Human erythrocytes

Oxidative stress in red cells

Study of the biochemistry of the superoxide anion

6

Engineered Red Blood Cells as Circulating Bioreactors

Table 1. Cont.

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Considering the whole period of studies on the loading techniques and applications, we can identify three phases of research: 1) The first (1973-1984) addressed the problem of loading methodology and the possibility of encapsulating as much enzyme as possible, with little focus on therapeutical applications and greater focus on the modifications of the loaded RBC at a structural and functional level. In 1977, Beutler conducted the first of the few studies on clinical applications carried out during this period. He reported clinical trials limited to a single patient with Gaucher’s disease who was given the enzyme glucocerebrosidase.5 2) The second period (1985-1991) focused mainly on therapeutic applications to correct metabolic deficiencies with a long term efficacy in the blood stream. RBC were shown to be as effective as liposomes in the delivery of the enzymes and experiments were also performed with RBC modified in the surface lipids to permit targeting of specific cells. Some interesting findings in this period made it possible to further define the role of antioxidant enzymes by elevating their concentrations within the cell to see if antioxidant defence was improved or imbalanced.6 3) In the third period (1992-2000) basic studies focused on the use of carrier blood cells in commercial applications. Clinical trials were initiated to test the dose-response curves employing recombinant or bacterial enzymes in human patients since this carrier system had been shown to provide greater applications by minimizing immunologic reactions and enhancing stability of the encapsulated enzyme.

Among all the enzymes tested in this period, L-asparaginase proved to be one of the most important therapeutic tools.7 In fact, leukemic lymphoblastic cells are unable to synthesize the essential amino acid asparagine, therefore these cells are killed by the substrate depleting action of this enzyme.8 A comparison between the metabolic state of cancerous and normal cells in the presence or in the absence of the asparaginase is shown in Figure 2. This tool had been used previously by giving the free bacterial asparaginase to patients with lymphosarcomas and acute lymphoblastic anemia but strong antigenicity in most patients and anaphylactic shock in about 20% of the patients were reported.9;10 Comparative analysis of intolerance reactions in patients receiving asparaginase encapsulated in autologous RBC and patients receiving free enzyme showed that if asparaginase is entrapped in RBC the immediate hypersensitivity reactions is prevented.11

Coentrapment of Two Enzymes In our laboratory many enzymes were successfully encapsulated and used to solve physiological problems. In some cases, two enzymes were coentrapped to correct or compensate the additional effects derived by the entrapment of a single enzyme. This was the case of glucose oxidase (GOD) and hexokinase (HK) entrapment. When entrapped alone, GOD produced marked oxidant damage due to H2O2 produced in the oxidation of glucose; therefore, it was suitable for studying the H2O2-mediated cytotoxicity. H2O2 induces very low levels of ATP and GSH, which injure RBC rapidly captured by the splenic bed.12 On the other hand, when hexokinase was entrapped alone, the RBC consumed twice as much glucose as unloaded cells and this was shown to be a useful model to identify the metabolic steps which become critical in the maintenance of ATP and 2,3DPG during red cell storage or circulation.13 On the basis of the results obtained with a single enzyme, Rossi et al.14 decided to focus on the coentrapment of hexokinase and glucose oxidase to obtain RBC with quite normal ATP and GSH levels, a high capacity to metabolize glucose and acceptable biological properties. Erythrocytes coentrapped with HK and GOD were shown to be able to regulate blood glucose around physiological concentrations in hyperglycemic states.14 Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) were coentrapped in human RBC,15 using electroporation, a method which has proven to be a convenient alternative to the hypotonic dialysis method.16 However no application of RBC incorporating both enzymes was shown by these authors.

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Figure 2. A comparison between the metabolic situation of cancerous and normal cells in the absence (A) or in the presence (B) of the asparaginase—L-Asparaginase is used in th etherapy of acute lymphoblastic leukemia and some lymphosarcomas. The cancerous cell is unable to synthesize Asparqagine (Asn) so that it must support the protein synthesis with the exogenous Asn. On the contrary, the normal cell is able to synthesize Asn and it may survive with the endogenous Asn. Asparaginase may be administered as a free enzyme or encapsulated in RBC to transform Asn into aspartate (Asp) and stop protein synthesis of the cancerous cell. Asparaginase encapsulated into autologous RBC markedly reduces the intolerance reactions.

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Enzymes Bound to RBC Membrane In some cases it appeared more advantageous to attach the enzyme outside the RBC instead of entrapping it in the erythrocyte. An example was uricase, which was coupled to the RBC membrane by biotin-avidin-biotin enzyme bridge.14 This strategy was adopted to overcome the limiting step, which was the permeability of uric acid through the RBC membrane. In this case, more than 1 IU/ml packed RBC was coupled to the external side of the erythrocyte membrane and the uric acid degradation capacity of these RBC was compared with RBC loaded with a similar amount of uricase activity. The uric acid degradation capacity of uricase bound to RBC membranes was shown to be more efficient than that of the uricase-loadedRBC.17 The experiment was repeated in in vivo conditions and the results showed that animals receiving uricase bound to RBC were able to maintain lower concentrations of uric acid than animals receiving uricase loaded RBC.17 Other authors utilized enzymes bound to polyethyleneglycol (PEG) and then loaded in RBC18 to obtain higher half life of the enzyme, but in this case the percentage of entrapment was low and this approach appeared less effective than loading with native enzymes.

Enzymes Encapsulated in RBC to Lower Alcohol Toxicity In our laboratory, particular attention was devoted to alcohol intoxication problems which are mainly due to the chemical reactivity of alcohol-derived aldehydes.19 For example acetaldehyde (Ach) causes damage by inactivation of several enzymes or interference in many mitochondrial shuttles, with inhibition of oxidative phosphorylation and reduced availability of reducing equivalents.19,20 Since 1984, we have been interested in the role of Ach and AlDH in human erythrocytes.21 These studies have demonstrated that acetaldehyde at mM concentration increases glucose consumption, PPP flux, and NADH/NAD+ ratio21, but it maintains constant the ATP/ADP ratio. Furthermore, kinetic studies on the AlDH, from adult or newborn RBC, have shown that the human enzyme has a Km value in the range 25-35 mM.22 A second aspect we have been interested in is the concentration of acetaldehyde in the blood after heavy alcohol intake. Using gas chromatography, we determined an average value of 5 mM.23 These results led us to search for an AlDH able to work at 1-10 mM Ach concentrations for the encapsulation in RBC. Aldehyde dehydrogenase from Alkaligenes eutrophus grown on ethanol showed these characteristics.24 This enzyme has a Km = 4 mM and a molecular mass of 195 ± 6 kDa that makes the entrapment in RBC possible. The enzyme was thus purified to homogeneity and entrapped in human or mice RBC with 21% entrapment and 78% cell recovery.25 Erythrocytes were incubated in the presence of Ach at concentrations ranging from 1 to 100 mM and their lactate/pyruvate ratio or ATP/ADP ratio did not change significantly, showing the metabolic integrity of RBC.25 The Ach consumption of the RBC loaded with AlDH was 9 times higher than that of the unloaded RBC (Table 2) and AlDH-loaded RBC were tested in mice. We first used normal mice and showed that the clearance of both ethanol and acetaldehyde were favoured by the presence of circulating loaded RBC. In a second experiment, we used alcohol-treated mice to simulate a condition of intoxication typical of chronic alcohol abuse. Mice received ethanol in their drinking water for 6 months and a marked increase in the level of circulating Ach was observed. An 88% increase in serum GOT compared to the basal level showed the presence of alcoholic liver disease. After ethanol intake, normal mice were able to reduce the plasma Ach to normal levels in 7 hours, while alcoholic mice were unable to deplete the Ach level significantly during the same period (Fig. 3). On the contrary, when the alcoholic mice received AlDH-loaded RBC they were able to reduce Ach to normal levels in approximately the same time as normal mice (Fig. 3). This means that the loaded RBC were able to support the metabolism of Ach by compensating for the lost metabolic efficiency of the liver due to chronic alcohol intake. The loaded RBC were also shown to be very efficient in the presence of methanol. The poisoning effect of methanol in humans is caused by the metabolic formation of formaldehyde

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Table 2. Encapsulation of AlDH and Acetaldehyde consumption of human RBC

AlDH (I.U/mlRBC) Ach cons. (mmol/h/ml RBC)

Native

Unloaded

Loaded

0.10 ± 0.03 0.60 ± 0.15

0.12 ± 0.04 0.57 ± 0.15

1.55 ± 0.25 4.05 ± 0.88

Aldehyde dehydrogenase (AlDH) from Alkaligenes Eutrophus was encapsulated in human RBC by hypotonic dialysis technique then incubated at 37°C in PBS in the presence of Acetaldehyde (Ach) for the measurement of Ach consumption. The hemolysis degree over four hours was less than 10%.

by alcohol dehydrogenase in the liver and formic acid by aldehyde dehydrogenase.26 Since these transformations are very slow in humans, it would be possible to intervene with RBC loaded with a specific alcohol oxidase (AlOx) after diagnosis of methanol intoxication. An automatic mechanism was set up to prepare RBC loaded with AlOx. We chose to entrap AlOx from Pichea pastoris with Km of 0.31 and 14.92 mM for methanol and ethanol, respectively. After loading procedures, human erythrocytes had an AlOx activity of 1.45 ± 0.2 U/ml of RBC with a 12% entrapment, while the native or unloaded RBC had undetectable AlOx activity. The metabolic properties of AlOx loaded RBC showed that, in the presence of 2 mM methanol, these RBC were able to metabolize methanol very quickly and the rates of methanol catabolism increased with the percentage of AlOx loaded into the RBC. Other metabolic properties showed that lactate consumption was decreased and the PPP activity was stimulated about 6 times more than with unloaded RBC.26 The increase of PPP was expected due to the production of H2O2 in AlOx reaction and to methemoglobin formation. These modifications were well tolerated by the erythrocytes, which did not change their essential metabolic properties while the methanol consumption was accelerated. Mouse erythrocytes loaded with AlOx (1.09 U/ml) were given to a group of mice intraperitoneally and these mice later received 0.7 g of methanol per Kg body weight. The rate of methanol consumption was measured in the blood taken from the caudal vein and compared with that of another group of mice receiving unloaded RBC and the same dose of methanol. Figure 4 shows the methanol consumption in the two mice groups. Mice receiving AlOx loaded RBC mantained the peak of methanol at one half the concentration of the mice receiving unloaded RBC. Moreover, when a second dose of methanol was given to mice after 24-36 h the efficiency of the RBC in depleting methanol was maintained.26

Conclusion Erythrocytes have been used over the last twenty years as a carrier of enzymes in order to deplete toxic molecules in the blood stream of animals or humans. This system has been shown to be a useful therapeutic tool in the treatment of certain pathologic conditions and for the prevention of poisoning by toxic substances. Some important therapeutic systems have been set up including the asparaginase-loaded-RBC, the HK/GOD coentrapment and the AlDH or AlOx system. The availability of commercial recombinant human enzymes may enlarge the therapeutic applications. Coupling enzymes on the outer RBC membrane is an interesting strategy to be used when metabolites enter the red cell too slowly. Unfortunately, this system induces the production of antibodies that soon inactivate the coupled enzyme. Notwithstanding their potential, the enzyme-loaded RBC are scarcely used in therapy and liposomes seem to be preferred in the delivery of proteins. However, the use of RBC appeared to be just as effective as liposomes except for fusion capacity. Indeed, liposomes are thought to be potentially capable of fusing with cell

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Figure 3. Blood acetaldehyde level in alcohol treated mice receiving unloaded or loaded RBC compared with normal control mice. Alcohol treated mice received 10% ethanol in their drinking water for 6 months. Twenty-four hours before alcohol assumption they were injected intraperitoneally with 0.6 ml of a suspension (40%Ht) of AlDH loaded or unloaded RBC, then 2g/Kg ethanol in 0.9% NaCl was given intraperitoneally and acetaldehyde concentration was assayed during the time course. Normal mice received the ethanol dose only. (a) and (b) : values are in each case significantly different from unloaded alcoholic mice, but (b) is never significantly different from (a).

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Figure 4. Methanol concentration in blood of mice receiving unloaded or Alcohol oxidase (AlOx) loaded erythrocytes. Mice received introgastrically 0.7 g of methanol/Kg in a final volume of 300 ml of PBS. At each time blood was drawn from the caudal vein of 5 animals from each group and methanol concentration was determined. (a) significantly different and (b) not significantly different from the correspondent unloaded value.

membranes in vivo, while the potential for erythrocyte fusion with target cells is not great. On the other hand, erythrocytes have some advantages over liposomes. For example, it is much easier to build up large stocks of RBC for use as enzyme carriers than to prepare similar quantities of liposomes. In addition, RBC are non-immunogenic and non-toxic and their circulating time can be readily controlled and may be varied from minutes to days or weeks. Because of the long half-life of the sealed RBC, it is also possible to have a circulating pool of enzymes slowly released from the RBC over its life span. In any case, the use of enzyme-loaded RBC should be preferred to the administration of free enzymes, which is the main therapeutic system in the treatment of some diseases. For instance, free asparaginase is still administered to patients with lymphosarcomas and free alglucerase is used in treating Gaucher’s disease. The

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possibility of setting up automatic and sterile equipment for the loading procedure should help the clinicians in the preparation of the loaded RBC which may reduce the number of administrations of the enzyme and its immunogenicity. Further developments can be expected in all cases where recombinant human enzymes are commercially available for the treatment of diseases or intoxications where the bacterial enzyme has limited the application. For all these reasons, we think that the use of resealed RBC as bioreactors is a useful tool in clinical biochemistry.

Acknowledgement Authors wish to thank Sara Ninfali for her help in the preparation of the figures.

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