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Factors influencing the biodistribution of liposomal systems Sommerman, Eric Frank 1986

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FACTORS INFLUENCING THE BIODISTRIBUTION OF LIPOSOMAL SYSTEMS by ERIC F. SOMMERMAN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in FACULTY OF GRADUATE STUDIES BIOCHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1986 ® ERIC F. SOMMERMAN, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of B i o c h e m i s t r y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date N o v e m b e r 2 1 , 1 9 8 6 DE-6(3/81) ABSTRACT Liposomes have important potential as drug delivery vehicles. However, in order to realize this potential, much basic research is required to elucidate the interactions experienced by liposomes in vivo. In this thesis two aspects of these interactions are investigated: the influence of vesicle size and lipid composition on the biodistribution observed in viva, and the interaction of liposomes with plasma proteins. In order to determine the in vivo behavior of liposomal systems, a new vesicle marker is synthesized (n5I-tyraminyl-inulin, U5ITI) and tested in vivo. It is shown that this probe satisfies the necessary criteria for an accurate marker of liposome behavior, and is superior to probes used by other workers in terms of accuracy, convenience, high specific activity, low tissue quenching and cost. The use of 125ITI as a vesicle marker allows accurate measurements to be made with lower doses of liposomes than previously employed. The influence of vesicle size, composition, and dose on the blood residency times, leakage and tissue distributions of vesicles was therefore investigated at these lower doses, employing a cannulation procedure to monitor vesicles. It is demonstrated that the clearance of vesicles from the circulation exhibits biphasic kinetics. The relative number of vesicles cleared during the early phase (halflife < 2 0 min) is decreased by increasing the vesicle dose or decreasing the size. The behavior of small vesicles produced by extrusion is also investigated, and the in vivo behavior of these systems is shown to be equivalent to conventional sonicated systems. The second part of this thesis investigates the binding of plasma proteins to vesicles in vitro. It is shown that vesicles bind a large number of plasma components and that the binding is strongly dependent on the surface charge of the vesicle. Some of the proteins have been tentatively identified with 2 - D electrophoresis and several were positively identified via immuno- autoradiography. A hypothesis is advanced regarding the role of plasma proteins in the fate of liposomes in vivo. iii Table of Contents ABSTRACT iii LIST OF TABLES ix LIST OF FIGURES x ABBREVIATIONS USED xiii ACKNOWLEDGEMENTS xvi 1. INTRODUCTION 1 1.1 The Concept of Directed Drug Delivery 1 1.1.1 The Properties and Preparation of Liposomes 1 1.1.1.1 MLVS 3 1.1.1.2 FATMLVS 3 1.1.1.3 SUVs 3 1.1.1.4 LUVS 4 1.1.2 Targeting of Liposomes In Vivo: A Historical Overview 4 1.2 Rationale of Work „ 7 1.3 Liposomal Interactions with Plasma Proteins 8 1.3.1 Interactions with HDL 8 1.3.2 Interactions with Other Lipoproteins 10 1.3.3 Interactions with Clotting Factors 11 1.3.4 Fibronectin 11 1.3.5 Interactions with Albumin 13 1.3.6 Interactions with a and fi Globulins 13 1.3.7 Interactions with the Immune System 13 1.3.7.1 Antigenicity of Liposomes 14 1.3.7.2 Nonspecific Interactions with Immunoglobulins 14 1.3.8 The Interaction with the Complement System 14 1.4 The Mononuclear Phagocytic System (MPS) 17 1.4.1 Function of the MPS In Vivo 17 iv 1.4.2 Histological Origin and Development of the MPS 17 1.4.3 Phagocytosis of Foreign Particles 19 1.5 Other Cell Types Involved in Liposomal Clearance 21 1.5.1 Hepatocytes 21 1.5.2 Leukocytes 22 1.6 The In Vivo System: A Summary 22 1.7 Thesis Outline 24 1.7.1 Problems in the Literature 24 1.7.1.1 In Vivo Studies 24 1.7.1.2 In Vitro Protein Work 25 1.7.1.3 Cell Studies 25 1.7.2 Experimental Approach 27 2. 1 "I-Tyraminyl-Inulin: a Convenient Marker for Deposition of Liposomes In Vivo 29 2.1 Introduction 29 2.2 Materials and Methods 35 2.2.1 Chemicals 35 2.2.2 Assay of Periodic Acid 35 2.2.3 Assay of Carbohydrate 36 2.2.4 Assay of Phosphate 36 2.2.5 Synthesis of Tyraminyl-Inulin 36 2.2.6 Iodination of the Tyraminyl-Inulin Adduct 38 2.2.7 Preparation of Large Unilamellar Vesicles 39 2.2.8 In Vivo Experiments 39 2.3 Results and Discussion 40 3. Influence of Size and Lipid Composition on Liposome Clearance, Leakage and Tissue Distribution In Vivo 47 3.1 Introduction 47 v 3.2 Materials and Methods 51 3.2.1 Materials 51 3.2.2 Synthesis of Phosphatidylserine 51 3.2.3 Liposome Preparation 53 3.2.3.1 Preparation of MLVs 53 3.2.3.2 Preparation of VETs 54 3.2.3.3 Preparation of SUVs 54 3.2.3.4 Vesicle Work Up 54 3.2.4 In Vivo Blood Clearance and Tissue Distribution Studies 55 3.2.5 In Vitro Studies 57 3.2.5.1 Control Studies With Ultrogel Ac 34 Chromatography 57 3.2.5.2 Plasma Stability Studies 57 3.2.5.3 Studies with Whole Rat Blood 59 '3.3 Results 59 3.3.1 In Vivo Studies 59 3.3.1.1 Clearance of Free U 5ITI 59 3.3.1.2 Dose Dependency Studies 60 3.3.1.3 Size and Lipid Composition Studies 60 Blood Clearance Determinations 60 Tissue Distributions 65 3.3.2 Effect of Surface Charge on the In Vivo Fate of FATMLVs 68 3.3.2.1 Phosphatidylserine Containing Vesicles 68 3.3.2.2 Stearylamine Containing Vesicles 70 3.3.3 In Vitro Studies 74 3.3.3.1 Plasma Stability Studies 74 3.3.3.2 Studies with Whole Blood 74 3.4 Discussion 76 vi 4. Liposomal Plasma Protein Binding: Surface Charge Effects 81 4.1 Introduction 81 4.2 Materials and Methods 82 4.2.1 Materials 82 4.2.2 Preparation of Vesicles 83 4.2.3 Preparation of Human Plasma or Serums 83 4.2.4 MLV Incubations in Plasma 84 4.2.5 Determination of Liposomal Protein Binding 84 4.2.6 Gel Electrophoresis 85 4.2.6.1 Buffers and Solutions 85 Stock Solutions 85 Gel Solutions 86 4.2.6.2 Sample Preparation for Gel Electrophoresis 87 4.2.6.3 SDS-PAGE 87 4.2.6.4 Two-Dimensional Gel Electrophoresis 89 4.2.6.5 Staining 90 4.2.6.6 Gel Densitometry 90 4.2.6.7 Immuno-Autoradiography of Gels ("Western Blot") 91 Antibody Purification 91 Iodination of Rabbit Anti-Goat IgG 91 Protein Transfer to Nitrocellulose 92 Incubation with IgG 92 4.2.7 Assays for Clotting Factors 93 4.2.7.1 Factor II 93 4.2.7.2 Factor V 93 4.2.7.3 Factor IX 94 4.2.7.4 Factor X 94 vii 4.3 Results 95 4.3.1 Preliminary Studies 95 4.3.2 Partial Characterization of Proteins 101 4.3.2.1 Symbolic Designation of Proteins 101 4.3.2.2 2-D Electrophoresis 101 4.3.2.3 Tentative Protein Identification 108 4.3.2.4 Identification of Proteins Using "Western" Blotting 108 4.3.3 Effect of Surface Charge on Protein Binding 113 4.3.3.1 Total Protein Binding 113 4.3.3.2 Relative Protein Binding 113 4.3.3.3 Densitometric Results 117 4.3.4 Studies With Different Plasma Preparations 120 4.3.5 Clotting Assays 128 4.4 Discussion 128 5. CONCLUSIONS 135 BIBLIOGRAPHY 140 viii LIST OF TABLES 1.1 Major plasma proteins which bind to liposomes 9 2.1 Tissue distribution of free 125ITI 42 3.1 A comparison of vesicle doses, compositions, and labels employed in the literature 48 3.2 Trapped volumes and injection doses of vesicle preparations .. 56 3.3 Tissue distributions of 125ITI encapsulated in various liposome preparations 67 3.4 Effect of SA on the tissue distribution of 125ITI entrapped in FATMLVs 73 3.5 Stability of VET 1 0 0s in plasma 75 3.6 Fate of 125ITI entrapped in VETi00s after incubation in blood 77 4.1 Composition of solutions used for SDS-PAGE 88 4.2 Plasma protein binding to MLVs 98 4.3 Molecular weights and tentative identities of MLV bound plasma proteins 110 4.4 Quantitation of protein binding to 1:1:2 EPS:EPC:Cholesterol MLVs after incubation with plasma or serum 125 4.5 Quantitation of protein binding to EPS: EPC:Cholesterol MLVs after incubation with dialyzed plasma or serum 125 4.6 Quantitation of clotting factor activity in plasma and on MLVs. 129 ix LIST OF FIGURES 1.1 An illustration of the three types of capillaries 6 1.2 The clotting cascade 12 1.3 The complement system 13 1.4 Mononuclear phagocyte development 18 1.5 Interactions of liposomes with the in vivo system 23 2.1 The synthesis of tyraminyl-inulin 37 2.2 Clearance and excretion of free 125ITI from the rat circulation 41 2.3 Blood clearance and excretion of 125ITI encapsulated in VET 1 0 0 systems 44 2.4 Tissue distribution of U 5ITI entrapped in VET 1 0 0 systems 45 3.1 Chromatographic separation of 3H-inulin from VET 1 0 0s 58 3.2 Clearance of free U 5ITI from the rat circulation 61 3.3 Effect of dose on the blood clearance of 125ITI entrapped in VET 1 0 0s 62 3.4 Effect of size on the blood clearance of 125ITI entrapped in liposomes composed of EPC-Cholesterol (1:1) 64 3.5 Effect of Size on the blood clearance of 125ITI entrapped in liposomes composed of DSPC-Cholesterol (1:1) 66 3.6 Dose effect on the tissue distribution of 125ITI entrapped in FATMLVs composed of EPC:EPS:Cholesterol (4:1:5) 69 3.7 Tissues distribution of 125ITI entrapped in FATMLVs composed of EPC:EPS:Cholesterol (4:1:5) 71 x 3.8 Effect of SA on the tissue distribution of 125ITI entrapped in FATMLVs .". 72 4.1 SDS-PAGE analysis of MLV associated protein 99 4.2 SDS-PAGE analyis of MLV associated protein using an acrylamide gradient 100 4.3 Designation of major liposome bound proteins 102 4.4 2-D gel electrophoresis map of human plasma 103 4.5 2-D gel electrophoresis of human plasma 105 4.6 2-D gel electrophoresis of plasma proteins bound to MLVs composed of 1:1 EPC-Cholesterol 106 4.7 2-D gel electrophoresis of plasma proteins bound to MLVs composed of 1:1:2 EPS:EPC:Cholesterol 107 4.8 2-D gel electrophoresis of plasma proteins bound to MLVs composed of 1:1 EPC-Cholesterol containing 5% SA 109 4.9 Immunoautoradiography of albumin and IgG bound to 1:1 EPC-Cholesterol MLVs 112 4.10 Surface charge effect on plasma protein binding to MLVs .. 114 4.11 SDS-PAGE analysis of MLV protein binding as a function of surface charge 115 4.12 SDS-PAGE analysis of MLV protein binding as a function of surface charge 116 4.13 Densitometric scans of MLV bound plasma proteins after SDS-PAGE 118 4.14 Densitometric scans of MLV bound plasma proteins after SDS-PAGE 119 xi 4.15 Effect of surface charge on the binding of Band E and Band F to MLVs 121 4.16 Effect of surface charge on the binding of Band C and Band D to MLVs 122 4.17 Effect of surface charge on the binding of Band I to MLVs 123 4.18 SDS-PAGE analysis of proteins bound to 1:1:2 EPC:EPS:Cholesterol MLVs 127 xii ABBREVIATIONS USED Apo Apolipoprotein APT Activated partial thromboplastin Ba(OH)2 Barium hydroxide BSA Bovine serum albumin CL Cardiolipin C3B Complement factor 3B CRP C-reactive protein DSPC Distearylphosphatidylcholine DF Deferoxamine EDTA Ethylenediaminetetraacetic acid EPC Egg phosphatidylcholine EPS Egg phosphatidylserine FATMLV Frozen and thawed multilamellar vesicles FC Folin and Ciocalteu HBS HEPES buffered saline HDL High density lipoprotein HEPES [4~(2-Hydroxyethyl)]-piperazine ethane sulfonic acid IEF Isoelectric focusing IgG Immunoglobulin G 1 2 5ITI 125I-tyraminyl-inulin KI Potassium iodide LDL Low density lipoprotein MgCl2 magnesium chloride MeOH Methanol MLV Multilamellar vesicles MPS Mononuclear phagocytic system xiii NaBH4 Sodium borohydride NaBHjCN Sodium cyanobOrohydride NaCl Sodium chloride NaH 2P0 4 Sodium dihydrogen phosphate NaHS04 Sodium bisulfite NaCl Sodium chloride Na2S04 Sodium sulfite Na 2S 20 5 Sodium thiosulfate NH 4OH Ammonium hydroxide (NH4)2S04 Ammonium sulfate NP40 Nonidet P40 PA Phosphatidic acid PAC Perturbed angular correlation PAGE Polyacrylamide gel electrophoresis PC Phosphatidylcholine PE Phosphatidylethanolamine PG Phosphatidylglycerol PMN Polymorphonuclear leukocyte PS Phosphatidylserine psi pounds per square inch RE Recticuloendothelial system SDS Sodium dodecylsulfate SM Sphingomyelin SnCl2 Stannous chloride SUV Small unilamellar vesicles (generated via sonication) TEMED N,N,N',N'-tetramethylethylenediamine xiv Thin layer chromatography Vesicles produced by extrusion technology Very low density lipoprotein XV ACKNOWLEDGEMENTS First and foremost, I'd like to thank the amazing P. R. Cullis, Professor at large, for buying me a beer during a D.O.A. gig at the PIT, and then talking me into doing a Ph.D. Since that .time, I have been thoroughly impressed with his rare ability to put together a lab which is very productive and a really fun place to work at the same time. In addition, I also want to thank Mick Hope, Colin Tilcock, Tom Madden, Laurence Mayer, Marcel Bally, Kim Wong, Tom Redelmeier, Helen Loughrey, Larry Reinish, Richard Harrigan, Linda Tai, Simon Eastman, and Diane Tanguay for being the most helpful and friendly people I've ever worked with, and probably ever will. Special mention must be made of Marcel Bally who, in addition to being sympathetic to alternative trips, is also the world's most useful person. Working with you guys has been a real treat I'd also like to acknowledge H. P. Pritchard and company, for helping me to plummet the depths of vivisection, and simultaneously shorten my life span with large quantities of 1251. Thanks also goes to R. Samborski, Dr. Gamma Ray himself, for doing the liposome-monocyte experiments, even though the data was boring. And now, in the true spirit of Wholism, I'd also like to thank: the Department of Biochemistry, for making me move off my boat and keep regular hours; the City of Vancouver, for keeping up the E. Coli. population at Wreck Beach; the country of Canada, for paying for my education; the planet Earth, for being a nice place to grow up; the Solar System, for keeping us all in orbit; the Milky Way, for making such a great candy bar; and the Universe, for obeying the laws of physics long enough for me to Finish my degree. xvi To My Dear Mother and Father xvii 1. INTRODUCTION 1.1 THR CONCEPT OF DIRECTED DRUG DELIVERY The ability to specifically "target" drugs to intracellular sites in vivo has long been one of the highest aspirations of medical research. The use of drug carriers with an affinity for a specific organ was first suggested 80 years ago (Ehrlich 1906). In recent years, this approach has generated enormous interest as the limitations of conventional chemotherapy, particularly with regard to the toxic side effects of virtually all drugs, have become clear. In particular, in many cases it is the toxicity of drugs which limit the allowable dose and thus many diseases such as cancer, and some fungal and parasitic infections, are currently incurable by chemotherapy. Thus any advance in the ability to target drugs, thereby diminishing systematic effects as well as decreasing the required dose, would constitute a major clinical advance. However, before such targeting protocols can be used in clinical situations, a basic knowledge of the interactions of liposomes with the in vivo system is required. The work presented in this thesis has been done in order to gain basic information about these interactions. 1.1.1 THE PROPERTIES AND PREPARATION OF LIPOSOMES Throughout the literature, many different types of drug carriers have been employed in targeting protocols, including macromolecules (Gregoriadis, 1979), cells or cell envelopes (Gregoriadis, 1979), nanocapsules (Couvreur et al., 1980) or nanospheres (Couvreur et al., 1979), microprotein aggregates (Couvreur et al., 1980) and synthetic lipidic vesicles or liposomes (Fidler, 1980; Gregoriadis, 1979; Juliano and Stamp, 1975). Among all the systems used, liposomes possess many important advantages. As they are made of endogenous lipid, liposomes are basically nontoxic at a reasonable dose. They are simple and inexpensive to make, and are very adaptable in that their size, composition, and retention properties can be readily modified to suit various purposes. 1 2 Although the use of lipid emulsions as drug carriers dates back at least to 1932 (Johnson, 1932), systematic elucidation of the properties of various lipid dispersions began in the early 60's. At that time, Bangham (Bangham, 1963; Bangham and Home, 1964) presented evidence that phospholipids in water adopt closed lamellar structures similar to biological membranes, and trap solvent within the inner compartment. They also reported that hand shaken dispersions developed large, multilamellar vesicles (MLVs) while ultrasonication of these preparations resulted in small, unilamellar vesicles (SUVs) (Bangham, 1963; Bangham and Home, 1964). Subsequently, these vesicles have been used in an enormous number of membrane modeling experiments, and their physicochemical properties are well established. The application bf these systems as drug delivery vehicles soon followed (Weissmann, et al., 1975; Rahman, et al., 1973; Magee and Miller, 1972; Allison and Gregoriadis, 1974; Juliano and Stamp, 1975). As the interest in liposomes increased, various techniques for their production were developed, all producing preparations with different structural properties, and all having particular advantages and disadvantages. Only the systems used in the experimental part of this work will be described here, as a complete discussion of all the different systems reported in the literature is beyond the scope of this thesis. However, the reader is referred to an excellent review on the subject (Hope et al., 1986). There are three main parameters used to define the structural properties of a given liposomal preparation: the size of the vesicle; the number of lamellae; and the trapped volume. The size is commonly measured by freeze fracture (van Venetie, 1980) or light scattering (Mayer et al., 1986). The lamellarity is indicated by the proportion of lipid exposed to the outer environment, as it will approach 50% only for unilamellar systems. The trapped volume can be determined by dispersion in a nonpermeable radiolabeled solute, with subsequent removal of the external label, and is 3 usually expressed as trapped volume per mole lipid. The various liposomal preparations used will be discussed with regard to these properties. 1.1.1.1 MLVS MLVs are the simplest liposomes to make, forming spontaneously from the mechanical dispersion of dry lipid in an aqueous solution. MLVs have an "onion" configuration, with many concentric lamellae. With neutral lipids, the lamellae are packed closely together, leading to a relatively low trapped volume. Additionally, these systems exhibit a nonequilibrium solute distribution across the various lamellae which reflects the differential permeabilities of the solvent and solutes (Gruner et al., 1985). These preparations are very heterogeneous, with a wide distribution in size, lamellarity, and trapped volume. Unilamellar structures are also present in "MLV" preparations. 1.1.1.2 FATMLVS A different type of MLV preparation (Mayer et al., 1985) results from freezing and thawing MLVs (FATMLVs). These vesicles have a much larger trapped volume, and exhibit equilibrium solute distributions. They are also very heterogeneous in size and contain unique intravesicular structures, such as vesicles in vesicles and vesicles between lamellae. Closely packed lamellae are rare, in comparison to normal MLVs. 1.1.1.3 SUVs Small unilamellar vesicles (SUVs) can be produced from MLVs by sonication (Huang, 1969). As the name implies, they are small (about 25 nm in diameter), and only have one bilayer. Their trapped volume is relatively low, and they are relatively unstable as they can spontaneously fuse to form larger systems (Parente and Lentz, 1984). 4 1.1.1.4 LUVS Large unilamellar vesicles (LUVs) are very similar to SUVs except that they are larger and are not formed by sonication. There have been various methods employed for the production of these vesicles, such as reverse phase evaporation (Szoka and Papahadjopoulos, 1978), detergent dialysis (Enoch and Strittmatter, 1979), and extrusion technology (Hope et al., 1985). The production of vesicles by extrusion is a comparatively new method of LUV production and has many advantages over other protocols (see Hope et al., 1986, for a review). In this procedure, MLVs are extruded through polycarbonate filters of various sizes generating vesicles by extrusion technology (VETs). Depending on the filter pore size (30-600nm), uniform and reproducible vesicles can be produced. The size and trapped volume vary according to the pore size employed. When using pore sizes up to lOOnm, these vesicles are unilamellar. When using larger pore sizes, the use of FATMLVs as a precursor decreases the lamellarity, and increases the trapped volume of the respective VET systems (Mayer et al., 1986). 1.1.2 TARGETING OF LIPOSOMES IN VIVO: A HISTORICAL OVERVIEW Early studies done with lipid emulsions administered i.v. showed that the liver and spleen are the primary deposition sites (Saxl and Donath, 1925; Jaffe and Berman, 1928). The cells primarily responsible for clearing lipid from circulation are the mononuclear phagocyte system (MPS), to be discussed below. All subsequent work has shown that, regardless of the size, dose, or composition of a vesicle preparation, the ultimate deposition site is in the MPS. This can be exploited therapeutically in diseases affecting the MPS. This type of approach is called "passive targeting", as vesicles passively accumulate in these tissues in the absence of any targeting protocol. In contrast to passive targeting, "active targeting" is the term used to denote the targeting of vesicles to cells other than the MPS. Active targeting is a challenging 5 goal. In order to be successful the vesicle must meet stringent requirements. In the first place, it must have access to the targeted tissue site in vivo. Thus if the site is extravascular, the vesicle must be capable of extravasation and movement in the interstitial space. Secondly, the vesicle must be capable of a selective interaction with the site of interest, by having an appropriate antibody or other targeting molecule on the outer surface to facilitate binding. Thirdly, the vesicle must be able to avoid uptake by the MPS or breakdown in the blood long enough for the specific binding to take place. Most of the work done in the recent literature has attempted to achieve active targeting. In regard to the first criterion above, the work of Poste (Poste, 1983; Poste et al., 1984) has been very important in clarifying the problems. As he has pointed out several times, the anatomy of the microcirculation in the respective tissue is of critical importance in assessing the ability of the vesicle to extravasate. As shown in Figure 1.1, there are three types of capillaries. Only in the sinusoidal capillaries can vesicles be expected to extravasate. Sinusoidal capillaries are found only in the liver, spleen, and bone marrow, the tissues with major MPS activity. Thus, it appears unlikely that active targeting to extravascular sites other than the liver, spleen, and bone marrow will be feasible in the foreseeable future. However, even if active targeting is restricted to these tissues many important clinical applications can still be developed. Most of the work in the literature has been concerned with the second and third points indicated above. There are now many methods available for conjugating antibodies to liposomes (Leserman et al., 1980; Martin and Papahadjopoulos, 1982), and these have been shown to result in specific cell binding with subsequent endocytosis in vitro (Machy et al., 1982; Heath et al., 1983). Also liposome preparations have been developed which exhibit relatively long half lives in the circulation in vivo (Gregoriadis and Davis, 1979; Kirby et al., 1980). Thus there have been significant advances made in the development of systems for active targeting within the vasculature. 6 Figure 1.1. Schematic diagram illustrating the structural differences between the various classes of blood capillaries (redrawn from Poste, 1984). (e) denotes the endothelial cells, (b) denotes the subendothelial basement membrane, and (p) denotes the parenchymal cells, (con); an illustration of the continuous capillary, with a continuous endothelial layer with tight junctions between adjacent cells, as well as a continuous basement membrane, (fen); an illustration of a fenestrated capillary, in which the endothelium is periodically interrupted with fenestrae, which are spanned by a membranous diaphragm. The basement membrane is continuous, (sin); a sinusoidal capillary, as found in liver, spleen and bone marrow. The gaps may be as large as several 100 nm in diameter. The liver lacks a basement membrane whereas the spleen and bone marrow have a discontinuous basement membrane. 7 1.2 RATIONALE OF WORK In light of the above considerations, active targeting within the vasculature is seen as a reasonable goal. There are many clinical situations in which this approach could be useful, for example: in leukemia, targeting to stem cells could be advantagous; in liver cancer, targeting to hepatoma cells may be feasible; it may be advantageous to target to specific capillary beds or to atherosclerotic plaques; or perhaps vesicles could be attached "piggyback" to erythrocytes as a controlled drug release system. Thus, any advances in this area can be viewed as potentially important advances. The work presented in this thesis has been developed with this goal in mind. Additionally, work done in this area sheds light on basic issues in immunology, opsonization processes, foreign particle recognition, and RBC senesence, among others. The interactions experienced by liposomal systems in vivo, subsequent to i.v. administration, are exceedingly complex. As a first step, it is important to define the parameters that determine the behavior of liposomes in vivo. When liposomes are injected i.v., there are two general fates for entrapped materials: either release in the blood due to liposomal disruption, or uptake along with the liposomes into various tissues. Thus, in defining the fate of liposomes and liposomal contents in vivo, it is important to quantitate the percentage of intact liposomes remaining in the blood, the percentage broken open in the blood, and the percentage of intact liposomes taken up by each tissue. At any time post injection, these values should add up to the total amount of liposomes injected. The importance of correctly measuring these parameters will be especially stressed in the experimental part of this work. In order to modify the in vivo behavior of liposomes to suit a particular therapeutic goal, it is important to be able to modify the above parameters in the appropriate manner. This requires an understanding of the complex mechanisms that determine the fate of liposomes in vivo. Some of these mechanisms have been investigated in this thesis. There are two general classes of interactions which together 8 are thought to be the major determinants of liposome behavior: the interaction with plasma proteins and the interaction with the MPS system. These two groups of interactions will receive detailed attention in this thesis. Other types of possible interactions with capillary walls, red blood cells, etc., are not treated in this work. 1.3 LIPOSOMAL INTERACTIONS WITH PLASMA PROTEINS The interaction of liposomes with the plasma proteins can be expected to be a major factor in determining their ultimate fate in vivo. The interaction with plasma proteins affect the stability of intact liposomes in blood (Arakawa et al., 1975; Krupp et al., 1976) as well as modulate their subsequent interaction with cells (Finkelstein, et al., 1981; Tyrrell et al., 1977). However, the literature in this area is fragmentary. The subject has been reviewed several times (Morrisett et al., 1977; Scherphof et al., 1981; Bonte and Juliano, 1986). Some of the major plasma proteins which are known to interact with liposomes are given in Table 1.1. It can be assumed that this roster will grow as more is learned. In principle, there are several modes in which liposomes can interact with plasma proteins (Bonte and Juliano, 1986). Proteins can bind electrostatically, as in the case of calcium mediated vitamin K dependent clotting factors; proteins can bind electrostatically with subsequent protein insertion into the bilayer, as in the case of apo lipoprotein insertion; proteins can bind in a hydrophobic fashion, resulting in protein insertion into the bilayer; lipid transfer and/or lipid exchange can also occur with the lipoproteins. The importance of these interactions varies for each plasma protein. 1.3.1 INTERACTIONS WITH HDL As mentioned above, when liposomes are added to plasma, a certain percentage of their contents are found to leak out (Scherphof et al., 1975) indicating that a fraction of the vesicles have ruptured. Investigations into the mechanism of plasma 9 Table 1.1. The major plasma proteins systems, with a literature reference. PROTEIN HDL LDL.VLDL Fibronectin Albumin a-2 Macroglobulin IgG C-Reactive Protein The Clotting System The Complement System h are known to interact with liposomal REFERENCE Krupp et al., 1976 Zierenberg et al., 1979 Rossi and Wallace, 1983 Van Rooijen and Van Nieuwmegen, 1980 Black and Gregoriadis, 1976 Weissmann et al., 1974 Gewurz et al., 1982 Jackson, 1980 Cunningham et al., 1979 10 induced liposome breakage have shown that HDL is the plasma component primarily responsible for this (Krupp et al., 1976; Zierenberg et al., 1979; Tall, 1980), and that lipid transfer from the vesicle to HDL accompanies leakage. There is evidence that other plasma proteins stimulate the HDL interaction (Damen et al., 1980). Further studies have demonstrated that the lipid transfer as well as the leakage of contents can be modified by the size, composition, and concentration of liposomes in plasma (Scherphof et al., 1984). Thus, it has been shown that while 100% PC vesicles are very susceptible to HDL disruption, addition of equimolar cholesterol effectively prevents vesicle disruption. Smaller size vesicles appear to be more sensitive to HDL but again disruption can be prevented by addition of cholesterol. Modification of the PC headgroup (Gupta et al., 1981) or the use of sphingomyelin (SM) (Damen et al., 1981) also inhibits the interaction. The interaction increases extensively above the phase transition temperature of pure PCs (Scherphof et al., 1984). Recent studies (Klausner et al., 1985) indicate that a complex is formed with Apo AI and the vesicle, and that this interaction is primarily responsible for the disrupting effect. There is considerable controversy as to the further details of this interaction. 1.3.2 INTERACTIONS WITH OTHER LIPOPROTEINS Besides HDL, other lipoproteins are known to interact with liposomes in plasma. For instance, it has been established that LDL and VLDL exchange lipid with liposomes under a variety of conditions (Zierenberg et al., 1979). This exchange is dependent on the presence of HDL. Whether this has any effect on the fate of liposomal contents in vivo is not clear at this time. There have also been some recent studies done with apo H and liposomes (Wurm, 1984). It was found that Apo H interacts very strongly with negatively charged lipids such as phosphatidylserine (PS) or phosphatidylinositol (PI), but scarcely interacts with neutral lipid species. Again the significance of this interaction is not clear at this 11 time. Although there have been no studies done on the effect of chylomicrons on liposome behavior in plasma, it seems quite possible that they would interact in some way. If this is the case, then it could be expected that the nutritional state of the experimental subject would effect the liposomal fate in vivo. In regard to this, the experiments presented later in this thesis were done under uniform feeding conditions. 1.3.3 INTERACTIONS WITH CLOTTING FACTORS The effect of liposomes containing negatively charged phospholipids on the clotting system has been extensively studied. As outlined in Figure 1.2, anionic phospholipid is a cofactor in several steps in the clotting cascade (see Jackson, 1980, for a review). The phospholipid is bridged to the vitamin K dependent (carboxy- glutamic acid containing) serine proteases by calcium bridges. As the clotting reactions procede, the profactors are cleaved to forming an active enzyme which in most cases is still bound to the carboxy-glutamic acid containing fragment by disulphide bridges. Such fragments may continue to bind to PS containing liposomes. Inspite of the enormous amount of literature on the involvement of lipid in the clotting cascade, there has been nothing done on the effect of clotting factors on the fate of liposomes in vivo. It may be expected that clotting factors may exert an important influence on the fate of liposomes containing acidic phospholipid. 1.3.4 FIBRONECTIN Although fibronectin is widely recognized as an important factor in many surface phenomena including phagocytosis, cell-cell adhesion, and hemostasis, very little work has been done on its effect on the fate of liposomes in vivo. One report (Rossi and Wallace, 1983) has demonstrated that fibronectin binds to phospholipid vesicles composed of PC and phosphatide acid (PA), phosphatidylethanolamine (PE), or 12 Figure 1.2. An illustration of the clotting cascade, including the intrinsic and extrinsic pathways. Most of the clotting factors are serine proteases in their active form. The Roman numerals denote the specific clotting factors, the "a" subscripts designate the activated species of each factor. "Ca-PL" denotes a phospholipid bilayer containing acidic phospholipids in the presence of calcium, whereas "TF" denotes tissue factor, a lipoprotein type species obtained from damaged tissues. The intrinsic pathway is contact activated with a negatively charged surface, resulting in the activation of Factor XII to Xlla. Factor Xlla activates Factor XI to XIa, and then XIa activates Factor IX to IXa. Factors IXa and Villa bind to acidic phospholipid through calcium bridges, and the combined complex activates Factor X to Xa. In the extrinsic pathway, Factor VII is activated to Vila by a number of different factors. Factor Vila, in the presence of tissue factor, activates Factor X to Xa. The intrinsic and extrinsic pathways converge at the activation of Factor Xa. Factor Xa and Factor Va bind to acidic phospholipid in the presence of calcium and convert Factor II to Factor Ha (Thrombin), which converts Factor I to Factor la (Fibrin). Factor Xllla cross links the fibrin into a clot. INTRINSIC EXTRINSIC surface, kinogen, prekal likrein i XI l a -« • XII Xi ! • XIa i i IX ! — - ixa Vlllg Ca-PL i Ca-PL i i cross linked fibrin VI I; kal I ikrein, Xlla X l a I i VII TF I I 13 phosphatidylglycerol (PG). Binding was independent of composition and lipid state. They also showed that the binding of fibronectin to liposomes resulted in aggregation and fusion even in the absence of calcium. Fibronectin binding appears to be irreversible, and results in a change in the conformation of the fibronectin molecule. There has been one report (Hsu and Juliano, 1982) that demonstrated that fibronectin enhances liposomal uptake by macrophages in vitro. 1.3.5 INTERACTIONS WITH ALBUMIN It is well established that albumin interacts with liposomes. Although it has been shown that albumin adsorbs to liposomes (Van Rooijen and Van Nieuwmegen, 1980), it appears that it has no significant effect on their structural integrity. The binding of albumin to liposomes does diminish the ability of hepatocytes (Hoekstra and Scherphof, 1979) to exchange lipid with the vesicles in vitro. 1.3.6 INTERACTIONS WITH a AND 6 GLOBULINS There is an extensive and confusing literature on the interactions of globulins with liposomes. In general, various globulin fractions are known to influence the exchange of lipid between vesicles and cells. However, due to the heterogeneity of the fractions employed, it is too early to conclude anything pertaining to the mechanism or importance of these phenomena. Black and Gregoriadis (1976) have provided evidence that o-2 macroglobulin binds liposomes in plasma and affects their mobility. 1.3.7 INTERACTIONS WITH THE IMMUNE SYSTEM The interactions of liposomes with the immune system are complex. In the following, only the interactions with the plasma proteins of the immune system are reviewed. The interactions with lymphocytes and other circulating cells are not treated here. 14 1.3.7.1 Antigenicity of Liposomes It is widely established that liposomes act as powerful immune adjuvants when used with lipidic antigens. It is well established that liposomes are highly efficient in this regard (Uemura et al., 1974; Yasuda et al., 1977). It has also been shown that repeated injection of liposomes containing naturally occurring glycolipids (Alving and Richards, 1977), as well as the acidic phospholipids PS,PA,PG, and cardiolipin (CL) (Alving, 1977), can generate an immune response. In an experiment using bacterial lipopolysaccharide as an antigen, it was also shown (Shuster et al., 1979) that antibodies could even be raised against PC when used as an adjuvant However, vesicles containing PC alone failed to generate an immune response. It is highly unlikely that antibody-antigen reactions are significant in first time injections with phospholipids as used in this study. 1.3.7.2 Nonspecific Interactions with Immunoglobulins Nonspecific absorption of immunoglobulins IgG onto liposomes is known to promote their uptake by macrophages. It was found that heat-aggregated immunoglobulins stimulate uptake more than native IgG (Weissmann et al., 1974). It was also shown that the various subclasses of IgG have differential effects on liposome stability. Fab fragments were not found to be active at all in this regard. The significance of these findings is controversial, and further evidence will be required to establish the in vivo significance of nonspecific IgG binding to vesicles. 1.3.8 THE INTERACTION WITH THE COMPLEMENT SYSTEM The complement system is the name for the set of serum proteins involved in foreign cell lysis, phagocyte chemotaxis, and facilitation of cell uptake and destruction by phagocytes. As shown in Figure 1.3, the activation of the complement cascade can occur by two pathways termed the classical and alternate pathway (See Reid and 15 Figure 1.3. A schematic illustration of the complement system. The complement system is a series of proteins whose principal function is to lyse foreign cells. The individual complement factors are designated CI-9, and when a line is drawn across the top it denotes the activated form, which in some cases has serine protease activity. The complement system can be activated by two pathways termed the classical and the alternate pathways. The classical system is activated by antigen-antibody complexes (denoted Ag=Ab in the figure), which activates CI. The alternative pathway is activated by the production of C3b, which can be produced by a variety of factors in vivo. The pathways converge at the activation of C5. The C56789 complex, the end product of complement activation, is situated in the target membrane and causes lysis. CLASSICAL ALTERNATIVE C1 A 9 - -Ab C4 + C2 C3b + B C1 D C4b2a C3bBb C3b C5 C4b2a3b C5b + C6+C7+ C8 + C9 C56789 ( ly t ic complex) 16 Porter, 1981, for a review). In cases where liposomes are functioning as an antigen in vivo, it is very clear from the literature that the vesicles will sustain damage through activation of the classical pathway of the complement system. However, even in cases employing first time injection of phospholipid mixtures, the classical pathway can be activated by C-reactive protein (CRP). CRP is a serum protein which has been shown to gready increase in concentration during acute inflammation (Gewurz et al., 1982). CRP initiates the complement cascade in a manner similar to an antigen-antibody complex (Kaplan and Volanakis, 1974). CRP is known to bind to vesicles containing the phosphocholine headgroup (Volanakis and Kaplan, 1971), and subsequendy activates the complement system (Richards et al., 1977). The activation is drastically increased by the inclusion of lyso-PC into the vesicles (Volanakis and Wirtz, 1979). CRP also increases macrophage activity (Bama et al., 1984). Because plasma concentrations of CRP are dramatically increased during inflammation responses, it might be expected that liposomes would be degraded more rapidly during an acute inflammation. Besides activation of complement via the classical pathway, liposomes can also activate it via the alternate pathway, under some conditions (Lachmann et al., 1973). The activation of the alternate pathway is initiated by the conversion of Factor C3 to C3B. The effect of liposome composition on complement activation has been investigated (Cunningham et al., 1979). It was shown that, while positively charged liposomes or liposomes containing hexosyl ceramides or glycerides caused extensive activation of the complement system, negatively charged or neutral liposomes were inactive in this respect. Thus although it is clear that the complement system interacts with liposomes in vivo, the significance of the interaction depends on many factors and any conclusions in this area await further experiments. 17 1.4 THE MONONUCLEAR PHAGOCYTIC SYSTEM (MPS^ The MPS has been mentioned several times in the previous discussion. As this system is of key importance in the removal of liposomes from the circulation, it is worthwhile to point out some of the key features of the MPS and review the existing literature on interactions of the MPS with liposomes. 1.4.1 FUNCTION OF THE MPS IN VIVO The MPS has at least five main functions in vivo, (a) It is the principal system involved in the removal of senescent or damaged cells from the body, and also sequesters other debri, including liposomes, (b) Activated macrophages are cytotoxic to tumor cells and thus are important in controlling neoplasias, (c) The macrophages participate with the lymphocytes in various immune responses, (d) They produce various bioactive molecules such as hormones, complement components, prostaglandins, etc. (e) They participate in the host defense mechanisms against microorganisms and are important in sequestering obligate microorganisms. In view of the important role of the MPS in the host defence mechanism, it is clear that the toxicity of a given liposomal therapy on the MPS itself must be clearly assessed before any assurance of the safety of the protocol can be made. 1.4.2 HISTOLOGICAL ORIGIN AND DEVELOPMENT OF THE MPS The cells of the MPS are derived from pluripotent stem cells in the bone marrow, in common with all circulating cellular elements. As outlined in Figure 1.4 the differentiation of the stem cell through to the promonocyte stage consists of several stages (See Gallin and Fauci, 1982, for a general review). The promonocyte is the earliest stage at which MPS characteristics can be determined. This promonocyte has poorly developed phagocytic activity, a few Fc receptors, and has many morphological similarities to succeeding stages. Promonocytes undergo active division in the bone 18 Figure 1.4. The development of mononuclear phagocytes from bone marrow cells (modified from Ganong, 1979). (USC): uncommitted stem cell; (CSC): cornrnitted stem cell; (PM): promonocyte; (MC): monocyte; (TF); tissue macrophage. See text for details. BONE MAR ROW B L O O D TISSUE USC C S C PM MC TM 19 marrow. Monocytes leave the bone marrow shortly after the last round of division. The monocyte is the circulating element of the MPS, and has a certain amount of phagocytic capability. There is no significant reserve pool of monocytes in the bone marrow. When a monocyte extravasates, it becomes a tissue macrophage if it remains viable. Quantitatively, tissue macrophages form the bulk of the MPS, with the circulating monocytes contributing only a small fraction. Although tissue macrophages are ubiquitous, the number and density of macrophages varies greatly between tissues, and the density of cells varies within a tissue. The MPS is concentrated in the organs of the recticuloendothelial (RE) system, including the liver, spleen and bone marrow. Although macrophages are anatomically associated with the fibroblasts and endothelial cells of the RE system, they are developmentally distinct and hence the use of the term RE system to denote the tissue macrophages has been replaced by the more precise term MPS as used in this work. In accordance with the tissue in which they are found, tissue macrophages have different names. Hence in the liver they are called von Kupfer cells, in the lung they are alveolar macrophages, in the skin the Langerhans cell, in the bone the osteoclast, etc. The Kupfer cells of the liver are quantitatively most important, although when expressed per gram of tissue the spleen has the highest density of MPS cells. 1.4.3 PHAGOCYTOSIS OF FOREIGN PARTICLES As mentioned above, phagocytosis of liposomes by cells of the MPS is the most important pathway for their clearance from the circulation. The process of phagocytosis has been divided into three steps: binding of the particle to the cell membrane, generation of a phagocytic signal, and endocytosis with subsequent fusion of the endocytotic vesicle with a lysosome. Although none of these phases are currently understood in detail, it will be useful to present a brief outline of the existing 20 knowledge. The binding of foreign particles to macrophages is an intriguing problem, and the literature available in this area is complex. In many cases, foreign particles have been previously coated with a variety of plasma proteins (Gallin and Fauci, 1982), which facilitate their binding and uptake. Such proteins are called opsins, and include various classes of IgG and C3B. The binding of IgG coated particles to macrophages has been widely studied (Uhr and Phillips, 1966; Quie et al., 1968; Phillips-Quagliata et al., 1971). There is a receptor for the Fc region of the IgG on the surface of macrophages. Upon binding of an IgG-coated particle to the Fc receptors, phagocytosis is stimulated. There is also a receptor for C3B, but apparendy binding of C3B coated particles does not stimulate phagocytosis (Ehlenberger and Nussenzweig, 1977; Mantovani, 1975). Thus it appears that the C3B receptor is auxilary to the IgG receptor and only increases the binding of the particle. Although the binding of opsinized particles appears straightforward, macrophages also actively engulf foreign particles by less specific binding mechanisms. Thus carbon particles, latex beads, and quartz particles have all been shown (Rabinovitch, 1968) to be actively phagocytosed by macrophages. How the macrophage recognizes these particles and distinguishes them from lipoproteins, etc., is unknown. It has been suggested (Fenn, 1921; Mudd, 1935) that the hydrophobic!ty and surface charge of the particle are the determining factors, but this does not explain all the data (Kozel et al., 1980; Rabinovitch, 1968). Further experiments will need to be done to elucidate this process. However, the study of phagocytotic regulation mechanisms in vitro can be complicated by the fact that various types of macrophages are artifically activated during their isolation, whereby they engulf cell debri, endogenous lipoproteins, etc. (Haydn Pritchard, personal communication). Thus the phagocytosis process studied in vitro is often a poor representation of the finely tuned in vivo process. 21 The uptake step of phagocytosis is complex and poorly defined. There are several good reviews on the subject (van Furth, 1980; Gallin and Fauci, 1982; Aggler and Werb, 1982). It is energy dependent and is known to involve actin filaments, actin binding protein, myosin, clathrin, several other poorly characterized proteins, and is calcium dependent In the phagocytosis process, uptake appears to be rate limiting under normal conditions (Poste, 1983), and thus MPS cells may bind more particles than they are able to take up. Because these bound particles may subsequently be released back into the circulation, or broken open while on the cell surface, there are complications in the measurement of MPS uptake activity, particularly when particle dose exceeds the uptake capacity of the MPS. This problem will be discussed further in the experimental part of this work. 1.5 OTHER CELL TYPES INVOLVED IN LIPOSOMAL CLEARANCE Besides the MPS, many other cell types are undoubtedly involved in the clearance and metabolism of liposomes under various conditions. Of these, only the hepatocytes and leukocytes will be discussed as only these types have been studied in any detail. 1.5.1 HEPATOCYTES As was discussed in a previous section, small liposomes may exit the circulation in tissues having a sinusoidal capillary bed (ie; the RE tissues). In the liver, this allows the vesicles to interact with the hepatocytes. This interaction has been studied in several laboratories (Rahman et al., 1980; Roerink et al., 1981). It has been shown that SUVs are taken up by hepatocytes in vivo, whereas MLVs are not (Scherphof, 1982; Poste et al., 1982). It has been possible to target specifically to the hepatocytes using an appropriate liposomal surface glycoside (Ghosh et al., 1982). It has also been shown that liposomes exchange lipid with hepatocytes in vivo (Poste et al., 1982), and there is evidence that this exchange is mediated by the Kupfer cells (Roerdink et al., 22 1981). This fact has to be considered when using a lipid label to measure MPS uptake. 1.5.2 LEUKOCYTES The uptake of liposomes by various types of human leukocytes has been demonstrated in several reports (Finkelstein et al., 1980; Kuhn et cd., 1983). They demonstrated that the monocyte fraction was more active than the neutrophil fraction in this regard. They also showed that precoating vesicles with heat aggregated IgG increased the rate of uptake several fold. However, due to the relatively low number of monocytes and neutrophils their uptake activity is extremely low (on the order of several nmol lipid per ml blood per hour as calculated from Kuhn et al., 1983) and not expected to be of significance in most experiments. One report (Mauk et al., 1980) indicated that the incorporation of 6-amino-mannosyl-cholesterol into vesicles caused PMN binding and retention of intact vesicles within aggregates of PMNs in the axilary space when injected subcutaneously. 1.6 THE IN VIVO SYSTEM: A SUMMARY At this point, it should be clear that the interaction of liposomes with the in vivo system is enormously complex and depends on many parameters. Figure 1.5 summarizes the above discussion and delineates the major interactions. Liposomes, upon injection iv, interact with the plasma proteins. Through protein insertion, lipid transfer, and lipid exchange mechanisms with the lipoproteins, liposomes can be broken open in the blood. Through interaction with C3B, CRP, or IgG, complement damage leading to leakage can also be sustained. With acidic phospholipids, the clotting cascade can be activated. Fibronectin can bind, aggregate, and fuse vesicles. The remaining vesicles, now aggregated and opsinized, are moved through the blood stream to the sinusoidal capillaries of the RE system, where they are bound and phagocytosed by cells of the 23 Figure 1.5. An illustration of the major interactions of liposomes with the in vivo system. See text for details. 24 MPS. The removal of vesicles from the blood is thus similar to the clearance of other foreign particles, microorganisms, etc., and depends on the interaction of a wide variety of defence mechanisms. 1.7 THESIS OUTLINE In light of the previous introductory sections, it is clear that in vivo liposome targeting, from the experimental point of view, is an enormous problem with many facets. Thus, before outlining the work done in this thesis, it is appropriate to give a brief survey of approaches taken in the literature, in order to put the work presented here in context A more in-depth discussion of the literature will be found in the experimental sections of this thesis. 1.7.1 PROBLEMS IN THE LITERATURE The literature in the field of liposome targeting is broad and diverse, reflecting the multidisciplinary backgrounds of the clinicians and scientists in the field. In order to simplify an analysis of this work, it has been broken down into three main categories: in vivo studies; in vitro blood and protein work; and in vitro cell studies. The auxilary categories of liposomology including production of liposomes, physical and chemical characterizations, etc., are not treated in this thesis. There are some excellent reviews available in these areas. (Hope et at., 1986; Mayer et al., 1986; Killian and de Kruijff, 1986) 1.7.1.1 In Vivo Studies The major part of the liposome targeting work has been the study of the fate of liposomes subsequent to their iv injection into an animal. Basically, the approach is to label liposomes with a radiolabel or flourescent marker, and assay the blood decay and tissue distribution of lipid and entrapped material at various times post injection. In doing this type of study, the influence of the 25 vesicle dose, size and composition on these parameters can be estimated. The literature is replete with such experiments. However, in spite of there being dozens of such studies published, very few of them have actually achieved what they set out to do, ie; measure the true fate of liposomes and their contents in vivo. This is due to several flaws in experimental design, such as improper labeling, inappropriate dosage and bogus time points. These problems are particularly prominent in the earlier literature, and can mainly be attributed to the researchers and clinicians overzealously trying to "cure cancer" in one experiment This "going for the home run" approach to research has generated an enormous amount of virtually unusable data. This problem will be treated in detail in Chapters Two and Three. 1.7.1.2 In Vitro Protein Work As indicated above, the interactions of liposomes with plasma proteins is probably an important factor in the behavior of liposomes in vivo. However, a great deal of work needs to be done in order to elucidate these interactions. Most of the previous work has investigated the interaction of purified proteins with liposomes, and has provided much useful data. The major problem with this approach is that it is difficult to relate these studies to the genuine in vivo interactions, because of the possible influence of cofactors, ions, and competition effects. The several papers which have looked at protein binding in total plasma preparations will be discussed in Chapter Four. 1.7.1.3 Cell Studies Another method of studying liposome targeting in vitro is the use of tissue culture techniques. There have been a great number of studies done with tissue culture cells and lymphocytes (see review by Pagano and Weinstein, 1978). These investigations have been important in determining the mechanisms of 26 vesicle-cell interactions (Shroit et al., 1986). Tissue culture systems have also been employed in an attempt to do active targeting in vitro, by conjugating antibodies onto the liposomal surface which bind to antigens on the cell surface. Antibody dependent uptake of liposomes has been achieved in many systems and much useful data has been obtained. (Leserman, 1980; Martin and Papahadjopoulas, 1982; Machy et al., 1982; Heath et al., 1983). However, the utility of these results in an in vivo system remains unclear, as the preconditions for active targeting (as outlined above) have still not been met. And further, even if control liposomes can be made to last long enough in the circulation for active targeting within the vasculature to occur, the influence of antibody conjugation on the liposomal binding of plasma proteins and recognition by the MPS still need to be determined. There have been comparatively few studies done on the uptake of liposomes by phagocytes in vitro. The work done with human leukocytes has been mentioned above. Hsu and Juliano (1982) investigated the uptake of liposomes by mouse peritoneal macrophages, and obtained some interesting results. Also, Scherphof and colleagues (Roerdink et al., 1981) have initiated studies using a Kupfer cell preparation. This work has been very important in discerning some of the features of phagocytosis, in vitro kinetic constants, and the influence of various plasma protein preparations on the uptake process. However, it is difficult to extrapolate these results to the in vivo process, because it is uncertain that the in vivo tissue macrophages truly behave like their in vitro derivatives. It is known that phagocytes are effectively "activated" during their isolation process, and thus effectively clean any cellular debri out of their media. This could be especially important in studying liposome uptake to the extent that it involves relatively nonspecific uptake processes described above. Moreover, there are 27 obvious differences between in vitro culture conditions and the complex in vivo system in terms of cellular accessibility, competition by other cells, and media composition. Therefore any results obtained in vitro must be scrupulously compared to corresponding in vivo experiments in order to be clearly applicable to the in vivo system. In light of these difficulties in vitro cell studies are not a major part of this thesis. 1.7.2 EXPERIMENTAL APPROACH The experiments presented in this thesis have been divided into three parts. In the Chapter Two basic studies have been done to ensure adequate measurement of the relevant in vivo parameters, under clinically relevant conditions. In order to achieve this, a new radiolabel was synthesized to measure the fate of liposomes in vivo. The superiority of this label over other labels in the literature is demonstrated. Chapter Three is an extension of Chapter Two. Here the effect of dose, size and composition on the blood clearance and tissue distribution of liposomes is determined. Although experiments similar to these have been reported previously, the conditions of the experiments described here allow several new conclusions to be made about the mechanisms behind the differential clearance times and fates of various types of vesicles. Moreover, the ability of the VET procedure to conveniently generate a SUV is demonstrated. Additionally, the stability of vesicles in plasma is measured in vitro, and the uptake by leukocytes is also determined. These results shed light on the behavior of liposomes in vivo. The last chapter describes a study of the binding of plasma proteins to liposomes in vitro. Tentative identification of many of these proteins is made, and several have been positively identified. Additionally, the effect of vesicle surface charge on protein binding is investigated in an attempt to account for the in vivo results. A model for plasma protein binding is presented and the implications for liposome design 28 are discussed. 2. 1 2 5 1 - TYRAMINYL- INULIN: A CONVENIENT MARKER FOR DEPOSITION OF LIPOSOMES IN VWQ 2.1 INTRODUCTION Liposomes have important potential as targeted vehicles for drug delivery in vivo. However, in order to evaluate the the efficacy of any targeting protocol, a convenient and reliable procedure for assaying delivery of encapsulated material to particular tissues is required. Considering the complexity of the in vivo system, a liposomal probe must meet several rather stringent criteria in order to faithfully measure the true tissue distribution of vesicle contents at any time point First, the free probe must be cleared rapidly from the blood and excreted, without significant tissue uptake. Otherwise, when blood is assayed for the probe, it will not be possible to distinguish intact liposomes from ruptured ones, without tedious and error prone separation procedures. Also the free probe must be excreted so that vesicle rupture may be quantitated by assaying the urine and there must be no tissue uptake of the free probe to confound the assay for liposomal uptake. Secondly, the probe must not be permeable to the liposome membrane so that only liposome lysis will allow leakage of the probe. Thirdly, the probe must exhibit prolonged tissue retention times, in order to allow measurement of genuine cellular uptake. In the course of liposome clearance, vesicles can bind to various sites in the vasculature without being taken up by cells. Over longer periods of time, these vesicles will either be taken up, ruptured or removed to other tissue sites. Equilibrium is reached when all vesicles have either ruptured or been endocytosed, with no extracellular vesicles remaining. Assay of label in tissues before equilibrium has been reached can thus give values which are quite different from the final tissue distribution values, especially when doses are given that excede the uptake capacity of the mononuclear phagocytic system (MPS). The time taken for equilibrium to be reached depends on the size, dose and composition of the 29 30 injected liposomes. Thus, vesicles with a long half life will need longer times for equilibrium to be reached. Poste (1983) recommends that a label should be retained in a tissue for at least 48 hr post equilibrium to obtain an accurate measurement of actual liposome uptake. Otherwise, it is impossible to distinguish between vesicle binding and genuine cellular uptake. Only when all three of these conditions are met simultaneously can the final tissue distributions be assayed in a straightforward manner. Although a variety of liposome labeling techniques are available in the literature, most of these procedures fail to meet one or more of the above criteria. It is useful to provide a short review which outiines the pitfalls of some of these methods. The use of these markers for other purposes such as assaying the metabolism of vesicular lipid, distinguishing between vesicle absorption and uptake, and so on, will not be reviewed here. The reader is referred to Schroit et al., (1986), for a detailed review of these topics. One common method is to employ a lipid soluble label. Thus radiolabeled phospholipids (Souhami et al., 1981), cholesterol (Steger and Dessnick, 1977), cholesterol-oleate (Kao and Juliano, 1981), as well as synthetic lipid derivatives (Abra et al., 1982), and metal-lipid adducts (Richardson et al., 1977) have been employed. The use of lipid labels has the advantage that the lipid component of the vesicle may be followed. However, as was discussed in the last chapter, vesicle lipids exchange with lipoproteins as well as with cells. The exchange rate is known (Scherphof et al., 1984) to vary with the lipid species, liposome size and composition, and probably the dose and other experimental parameters. Moreover, most lipid labels undergo metabolism subsequent to their transfer to lipoproteins and/or cellular exchange and uptake, which further complicates their use. Synthetic lipids, such as 125I-p-hydroxybenzamidine phosphatidylethanolamine (1J5I-BPE), also undergo exchange in vivo (Abra et al., 1982), albeit at a slower rate. Lipid labels have also been generated by the use of various metals which are complexed with the lipid. The most commonly employed isotope is 31 "Tc, which has been used (Souhami et al., 1981; Richardson et al., 1977) to follow the tissue distribution of liposomes. The "Tc is linked to the lipid using SnCl2 by the method of Osborne et al., (1979). This label, being a powerful gamma emitter, has the advantage of allowing whole body images to be taken in vivo (Richardson et al., 1977). However, it has the same pitfalls as the other lipid labels and consequently other measurements need to be taken in order to follow the vesicle contents. In summary, lipid labels are only useful to follow a particular lipid species, and not an intact vesicle. In addition to labels for the lipid component of a vesicle, there are a large number of. methods for labeling the contents of a liposome. Among these are metal isotopes (Poste et al., 1982), radiolabeled proteins (Ghosh et al., 1982; Gotfredsen et al., 1983), a variety of small molecules (Jonah et al., 1975; Steger and Dessnick 1977), and various carbohydrates (Bosworth and Hunt 1982; Allen and Everest, 1983). Only a few representatives of each type will be discussed. Methods employing metal isotopes to measure the distribution of vesicle contents all suffer from the same pitfall. The major problem is that the free metal is not quantitatively excreted from animal but is taken up by tissues to various degrees. Additionally, the metal may not remain in a tissue long enough to get equilibrium distributions when taken up in the liposomal form. On the whole, metal isotopes produce rather ambiguous data when used in tissue distribution studies and thus must be supplemented with other measurements. One isotope that circumvents some of these problems is 11'In, as used by Mauk and Gamble (1979). During the process of radioactive decay, l l l In decays to n i C d by electron capture which subsequently undergoes a gamma ray cascade. Using a perturbed angular correlation (PAC) spectrophotometer, it is possible to measure the rotational correlation time for a molecule to which the nucleus is bound. By chelating m I n to nitriloacetic acid and encapsulating it in vesicles, it is possible to determine 32 the degree of vesicle disruption in vivo because the 11'In will preferentially bind to proteins when leaving the vesicle in vivo, and the protein bound u l In has a much smaller rotational correlation time than the nitriloacetic acid bound m In. Thus the free l u In can be distinguished from the liposomal m I n by combining PAC spectroscopy with conventional gamma counting. The method offers the additional advantage that the breakdown of vesicles within the tissues can be observed. However, U 1 l n methods have some of the same problems as the other metals. 11'In is taken up by tissues in the free form (Mauk and Gamble, 1979), and is not retained by a tissue after cellular uptake in the encapsulated form for a long enough time to obtain equilibrium distribution values for the vesicle contents. Thus true blood decays and tissue distributions can not be determined with this label, under many circumstances. Lastiy, the technique requires the use of a PAC spectrometer, which is a not available in most laboratories. Another popular method for labeling vesicles is the use of proteins. For example, 1 2 5I-BSA (Gotfredsen et al, 1983) as well as '"I-IgG (Ghosh et al., 1982) have been used to assay tissue distributions of liposomes. There are many difficulties with these labels. First, the label is metabolized in the lysosome upon vesicle uptake, and consequentiy the 125I-tyrosine residues may leave the tissue where the vesicles were first taken up. It therefore becomes difficult to establish the total amount of vesicles which are taken up by various tissue. Second, if the vesicle ruptures in the blood stream, most of the label remains in circulation. Thus, in order to assay the blood for intact vesicles, it becomes necessary to separate the vesicles from the free marker in blood for each assay. Although some laboratories have used this method (Gotfredsen et al., 1983), it is tedious and inaccurate, especially for short time points. An exception to the general inadequacy of proteins for assaying vesicle distributions is of the glycopeptide 11'In-bleomycin. It has been demonstrated (Senior et al., 1985) to be an effective marker in that it is rapidly excreted in the free form 33 with no tissue uptake and exhibits prolonged tissue retention times when taken up in a liposomal form. However, the material is prohibitively expensive for low dose studies where high specific activities (1 mCi/ml) are required. There have been a large number of small molecules used for labels such as EDTA (Jonah et al., 1975), glucose (Steger and Dessnick, 1977), and 59Fe-deferoxamine mesylate ( i 9Fe-DF) (Patel et al., 1983). The use of glucose as an marker for deposition of liposome contents is obviously subject to severe problems. As glucose is being taken up and metabolized by virtually every tissue a correct tissue distribution is difficult to obtain under any circumstances. Moreover, only diabetics excrete glucose rapidly so the blood decay values would be impossible to interpret in most cases. 1 4 C-EDTA was employed by Jonah et al., (1975) in an effort to target EDTA as a chelator for heavy metal poisoning. As a marker of liposome contents, it is excreted fairly rapidly and has low tissue uptake. However, the encapsulated form is not retained by the tissues long enough to obtain equilibrium values. 5 9 Fe-DF was used to quantitate distributions in an effort to target DF as a chelator for iron. According to the results given (Guilmette et al., 1978) 5 9 Fe-DF seems to meet the criteria fairly well. Unfortunately, the longer time points (> 24 hr) were not examined in this report nor were the very short time points (< 0.5 hr) and so it is not clear if the tissue retention time is long enough or the blood decay time of the free form short enough. As the last category, certain carbohydrates offer many important advantages and are thus the most promising of the vesicle markers. 14C-sucrose has been used (Allen and Everest, 1983) quite effectively as a liposome marker under some conditions. The free form is rapidly excreted, and is not taken up by tissues. However, it does not remain in the tissues long enough (halftime approximately 6 hr) for equilibrium tissue distributions to be obtained. 34 The last molecule to be mentioned is the carbohydrate inulin. Inulin, currendy available with either 3 H or 1 4 C labeling, is by most criteria the best liposome marker (Poste, 1983). It is excreted extremely rapidly (Smith, 1956), and has very long tissue retention times when taken up in the encapsulated form (Poste, 1983). The only disadvantage of inulin is that it is only available with 3 H or 1 4 C, in common with many of the other labels described above. Because of the high tissue quenching of these isotopes, tedious sample oxidation protocols are required prior to scintilation counting. These protocols also limit the amount of tissue per sample to 100 mg, which means that 20-30 uCi must be injected into a 200 g rat in order to obtain significant amounts of radioactivity in samples. Because a clinically realistic dose of lipid is on the order of 3 mg lipid/kg body weight (see Results and Discussion below), and the trapped volume of the vesicles employed is on the order of 1.5 wl/mg lipid, this corresponds to a specific activity in the vesicle of approximately 20-30 mCi/ml. As the vesicle production techniques require a volume of at least least 0.5 ml, 10-15 mCi 3 H or U C - inulin would be required for each preparation of vesicles. This is clearly economically impractical. In this chapter the rapid synthesis of an iodinated version of inulin (125I-tyraminyl-inulin, U5ITI) is described, and the properties of this probe as a reporter of liposome deposition are characterized. It is shown that 125ITI satisfies all the requirements for monitoring blood lifetimes and tissue distributions with the advantages of low cost, minimal tissue workup and high specific activities. This latter property allows tissue distributions to be ascertained for lower liposome doses than previously described. 35 2.2 MATERIALS AND METHODS 2.2.1 CHEMICALS Inulin, periodic acid, sodium-m-arsenite, tyramine, G-25 Sephadex, sodium cyanoborohydride, sodium borohydride, and cholesterol were obtained from Sigma. Ultrogel Ac34 was obtained from Pharmacia, carrier free Na125I (100 mCi/ml) was supplied by Amersham and iodogen was obtained from Pierce. All other reagents were of analytical grade or better. Egg phosphatidylcholine (EPC) was purified from hen egg yolks as follows. Thirty egg yolks were added to 1.2 1 of acetone and the precipitate was obtained via filtration, washed 5 times with 1 1 acetone and extracted 3 times with 500 ml CHCl3:MeOH (1:1 v/v). The material was dried down under rotary evaporation and taken up in 100 ml of CHCl3:MeOH (1:1) and purified using silica acid preparative liquid chromatography (Prep LC/System 500, Waters Associates) with a PrepPak-500/silica column as the stationary phase and CHCl 3:MeOH:H 20 (60:30:4, v/v/v) as the mobile phase. The resulting EPC fractions were shown to be greater than 99% pure as indicated by 2-dimensional thin layer chromatography according to Broekhuipe (1969), using silica gel 60 TLC plates (250 u m thick; E. Merck, Germany) run in CHCl 3:MeOH:NH 4OH:H 20 (90:54:5.7:5.4, v/v/v/v) in the first dimension and CHCl 3 /MeOH/CH 3 C0 2 H:H 2 0 (25:15:4:2, v/v/v/v) in the second dimension, with subsequent staining with I2 vapor. 2.2.2 ASSAY OF PERIODIC ACID Periodic acid analysis was done using the arsenite titration method according to Dyer (1956). Sodium bicarbonate (1.5 gm) is dissolved in 10 ml of a sample having an approximate periodate concentration of 0-100 mM, and then 25 ml of standard 0.1 N sodium arsenite was added, followed by 1 ml of 20% (w/v) KI. The solution was 36 then titrated in the usual way with standard 0.1 N I2. The concentration of the periodic acid solution was calculated from the amount of arsenite consumed. 2.2.3 ASSAY OF CARBOHYDRATE Carbohydrate assay was performed according to Roe (1955) using the anthrone reagent The anthrone reagent was prepared by dissolving 0.5 g anthrone and 10 g thiourea in 1 1 of 66% H 2 S0 4 (v/v) at 80° C. The solution was then cooled and stored at 4°C until use (within 2 wk). Anthrone reagent (10 ml) was added to a sample containing 0-400 M g carbohydrate in 1 ml, and the sample was vortexed, cooled to 27° C, then left in a boiling water bath (95° C) for 15 min. After cooling to 27° C the absorbance was measured at 620 nm. The assay followed Beers law from 0 to 10 a mol for standard glucose. 2.2.4 ASSAY OF PHOSPHATE Phosphate assays were performed according to method of Fiske and Subbarrow (1925). Samples containing 0.05-0.2 u mol lipid phosphate were digested for 2 hr at 190° C in 70% (v/v) HC104. Upon cooling, 7 ml of 0.22% ammonium molybdate (w/v) in 2% H 2S0 4 (v/v) and 0.6 ml of Fiske-Subbarrow reagent (30 gm NaHS04, 1 gm Na2SO„ and 0.5 gm l-amino-2-napthol-4-sulphonic acid in 200 ml H 20) were added and the samples were incubated at 95° C for 15 min. The samples were cooled to 27° C and the absorbance at 815 nm was determined. 2.2.5 SYNTHESIS OF TYRAMTNYL-INULIN Tyraminyl- inulin was synthesized according to the three step reaction sequence outiined in Figure 2.1. Inulin (1.0 gm) was dissolved in 90 ml H 2 0 and cooled to 4°C. Subsequendy, 10 ml of freshly prepared 0.1 M periodic acid was added and the solution was stirred for 15 min at 4 ° C in the dark. Periodate consumption was 37 Figure 2.1. The synthesis of tyraminyl-inulin. (A) Complete structure of inulin. (1) Reaction 1: Random, partial oxidation of a typical diol group on the inulin molecule with periodate. (2) Reaction 2: Formation of the tyramine adduct by reductive amination. (3) Reaction 3: Complete reduction of any remaining aldehyde groups with sodium borohydride. See text for details. 2 3 TYRAMINYL-INULIN OH 38 assayed by the arsenite method described above indicating approximately two oxidations per inulin molecule. The reaction was terminated by neutralization with a saturated Ba(OH)2 solution and the resulting barium iodate and periodate salts were removed by centrifugation at 3000 g for 10 min. Then, 4.3 gm NaH2P04 and 0.55 gm tyramine were dissolved in the supernatant and the pH was adjusted to 7.5 with 1.0 M HCl. Subsequently, 0.25 gm NaBH3CN was added and the solution was stirred for 4 hr at 27° C. The remaining aldehydes were reduced by careful addition of 0.2 gm NaBH, and stirring for another hour at 27° C. Aliquots of 25 ml were degassed under reduced pressure and applied to a 1.5 x 80 cm Sephadex G-25 column previously equilibrated with H 2 0 at 4°C. The flow rate was adjusted to 10 ml/hr and 4 ml fractions were collected. The fractions were assayed for tyramine residues by monitoring absorbance at 279 nm and for sugar by employing the anthrone technique described above. The sugar containing fraction eluted in the void volume and had a constant tyramine:inulin molar ratio of 0.6, based on inulin. The adduct was completely separated from the free tyramine and other salts as determined by rechromatography on G-25. The peak fractions were lyophilized giving an 80% yield, based on inulin. 2.2.6 IODINATION OF THE TYRAMINYL- INULIN APDUCT Tyraminyl-inulin (2.5 mg) was dissolved in 0.2 ml 20 mM HEPES, 145 mM NaCl, pH 7.4 (HEPES buffered saline, HBS), and placed in a stoppered vial in which 40 M g iodogen had previously been deposited from 300u. 1 CHC13. Then 4 mCi NaU 5I (100 mCi/ml, carrier free) was added and the reaction was allowed to proceed for 45 min at 27° C. The solution was then taken up into a syringe containing 10 u 1 0.1 M Na2S205, 0.05 M KI and and applied to a 1 x 20 cm Sephadex G-25 column previously equilibrated with HBS. Fractions of 0.5 ml were collected and the 1 2 SI containing fractions eluting in the void volume were pooled. The resulting 1 2 5I- tyraminyl-inulin (125ITI) solution routinely contained 1 M Ci/ncl l 2 5I, where <0.01% 39 was in the free iodide form (<0.01% was CHC13 extractable when made to 1.2% H 2 0 2 (w/v) and 0.4% KI (w/v)), and over 99% of the material eluted as one peak in the void volume upon re-chromatography on Sephadex G-25. In all studies the material was used within 2 wk of production. For injection as free U 5ITI the stock solution was diluted to 5 u Ci/ml with HBS. 2.2.7 PREPARATION OF LARGF UNILAMELLAR VESICLES Vesicles (100 nm) were prepared by the extrusion technique (VET100s) according to the method of Hope et al., (1985). Thirty u mol EPC and 30 M mol cholesterol were dried down from CHC13 and placed under high vacuum for 2 hr. The resulting lipid film was dispersed in 1 ml HBS containing 1 mCi 1 2 5ITI by vortexing. The multilamellar systems thus produced were then extruded 10 times through two stacked polycarbonate Nucleopore filters (0.1 jum pore size) under N 2 pressure (200-400 psi). Then, 0.1 ml aliquots of the VET i 0 o S were applied to an Ultrogel Ac34 column (poured in a 1 ml tuberculin syringe tube) previously equilibrated with HBS. The lipid containing fractions were pooled and rechromatography indicated that > 97% of the 1 2 5iTI was "trapped" in the vesicles. The vesicles were assayed for 1 2 5I and lipid phosphate according to the method described above and the trapped volume was calculated to be 1.5 M 1/mg lipid (0.9 M14* mol lipid). The average radius of these vesicles was 700 nm (Hope et al., 1985). The VET 1 0 0s were diluted to 0.6 mg phospholipid in 200 ul of HBS, stored at 4°C, and used within 2 days of preparation. 2.2.8 FN VIVO EXPERIMENTS Female Wistar rats (150-175 gm) were obtained from the UBC animal care unit and fed ad libitium prior to and during experiments. They were lighdy anesthetized with ether, weighed, and 200 n 1 of HBS containing either 0.5 u Ci I25ITI 40 encapsulated in VETi00 (0.6 mg lipid) or 1 u Ci free 125ITI was injected via the tail vein. Control studies demonstrated that passage of vesicles through a 28 ga. cannula does not cause leakage of 3H-inulin from MLVs at normal injection flow rates (approximately 400 ul/ min). Thus injection of the smaller sized vesicles used throughout this work would not be expected to cause vesicle disruption. The rats were allowed to recover in metabolic cages where the urine and feces were collected. At various dmes post injection the rats were anesthetized with ether and sacrificed by exsanguination via the vena cava. Blood was collected in a syringe containing 200 u 1 200 mM EDTA and recovery was approximately 85%, assuming 4.9 ml blood per 100 g rat (Altmann and Dittmer, 1971). The heart, liver, lung, spleen and kidney were removed and the urine remaining in the bladder was collected. The carcass was then dissolved in 200 ml 9 M NaOH at 70° C overnight. Aliquots of the carcass digest and samples of tissues were then assayed for the presence of 1 2 5I. 2.3 RESULTS AND DISCUSSION Experiments were first conducted to determine the clearance of free 125ITI from the circulation and subsequent excretion. Thus rats were injected with 1 yCi free 125ITI and clearance from the blood, uptake into various tissues, and appearance in the urine assayed. As shown in Figure 2.2 clearance from the blood is extremely rapid, as only 4% remains after 10 min. The 125ITI is eventually found in the urine, with recovery of 80% or more of the label in 24 hr. This indicates that free 125ITI in the rat circulation is rapidly cleared from blood and excreted. As shown in Table 2.1, this is consistent with results obtained for other tissues. No significant 125ITI uptake was observed except for the kidney which transientiy accumulated approximately 3% of the injected label at 1 hr. This can be explained by the excretion of 125ITI into the urine. It is important to note that the liver and spleen take up less than 1% of the label at any time post injection. Thus it can be concluded that I25ITI in the free 41 Figure 2.2. Clearance of free 1J5ITI from the rat circulation (•) and subsequent excretion in the urine (•). Female Wistar rats were injected via the tail vein with 1 yCi 1 2 5 m in 200 M 1 HBS. Urine was collected in metabolic cages. The animals were sacrificed at the times indicated. Recoveries of blood averaged 80% assuming 4.9 ml blood per 100 gm rat Results are expressed as percentages of the total 1 2 5 rn injected ± standard error (n=3). 100* HOURS 42 Table 2.1. The tissue distribution of free 1 J 5 r n in vivo at various times post injection: tissues were harvested from rats employed to obtain the data of Figure 2.2. Results are expressed as total 1 2 5 r n injected ±the standard error. Tissue 1 Hr 3 Hr 5 Hr 7 Hr Kidney 2.7 +0.4 Liver 0.22 + 0.06 Spleen 0.14 + 0.09 Heart 0.15+0.03 Lung 0.32 + 0.08 1.4 +0.4 0.9 0.09 + 0.01 0.05 0.02 + 0.01 0.03 0.02 + 0.003 0.01 0.05 + 0.01 0.02 +0.4 1.5 +0.7 + 0.004 0.07 + 0.02 + 0.02 0.02 + 0.005 + 0.003 0.01 + 0.0005 + 0.003 0.03 + 0.01 43 f o r m is cleared and excreted rapidly and is not taken up into body tissues. The next set of experiments were designed to determine the in vivo tissue distribution of 12SITI encapsulated into VET^ o systems when injected i.v. An associated objective was to detect the fate of relatively low doses of VETi 0 0 systems consistent with the levels required for possible clinical applications. In previous work (Mayer et al., 1985) it has been demonstrated that drugs such as adriamycin can be trapped in VET systems at concentrations > 125 mM. Thus a "clinical" dose of adriamycin (2.5 Atmol/kg body weight) for a 200 gm rat would require a trapped volume of 4 ul The trap volume of the EPC:Cholesterol VETi 0 0s used in this work is 1.5 u 1/mg lipid, leading to a VET 1 0 0 dose of 2.7 mg lipid. Any ability to target these carriers should appreciably reduce the drug levels required, and therefore a low dose of VET 1 0 0s corresponding to 0.6 mg (lu mol) lipid has been chosen to demonstrate the utility of U 5ITI in following the fate of liposomal systems. An advantage of such low dose levels concerns the reduced saturation of uptake processes (Poste 1983). The clearance from the circulation of 125ITI encapsulated in EPC:Cholesterol (1:1) VET l 0 0 s and the subsequent appearance of 125ITI in the urine is illustrated in Figure 2.3. In contrast to free 125ITI, the encapsulated material in the circulation is cleared much slower. By plotting the data as a semi-log plot, biphasic kinetics are evident, with an initial halflife of 1.3 hr and a second phase halflife of 106 hr. Further, only 30% of the injected dose is eventually found in the urine even after 3 days. This latter result clearly indicates tissue uptake and retention of VET, 0 0 encapsulated 125ITI. The actual tissue distributions are shown in Figure 2.4, where approximately 50% of the in vivo 125ITI is accumulated by the liver, 10% by the spleen, and the rest in the carcass. Less than 3% of the U SITI was found in the heart, lung or kidney at any time post injection (data not shown). The tissue distributions for the liposomal contents are similar to previous observations (Abra and Hunt, 1981), and any differences may be attributed to differences in liposome dose 44 Figure 2.3. Clearance of 1 J 5iTI entrapped in EPC-Cholesterol (1:1) VET 1 0 0 systems from the rat circulation (•) and subsequent excretion in the urine (•). The VET10oS were injected into the tail vein of 150-175 gm female Wistar rats at a dose level of 0.6 mg lipid in 200 a 1 HBS. Urine was collected in metabolic cages. Blood was withdrawn and the animals were sacrificed at the indicated times, and the total amount of 1 2 5 m in the blood was calculated assuming 4.9 ml blood per 100 gm rat Results are expressed as the total 1 2 3 rn injected ± standard error (n=3). 10 0* HOURS 45 Figure 2.4. Tissue distribution of "SITI entrapped in EPC-Cholesterol (1:1) VET 1 0 0 systems after injection of doses corresponding to 1.0u mol lipid in 200 u\ HBS. The symbols correspond to liver (•); carcass (*) and spleen (•). The tissues were harvested as indicated in Methods. Results are expressed as percentages of total n s ITI in vivo (total l J 5 m injected minus amount excreted) ±the standard error (n=4). 100i 46 and size, lipid composition, as well as the animal model employed. The important point is that once the encapsulated 125ITI is associated and presumably endocytosed by cells of a particular tissue, long retention times are observed. Such characteristics clearly support the utility of 125ITI as a label for the deposition of liposomal contents. In summary, 1251-tyraminyl-inulin has been demonstrated to satisfy all of the criteria required of a marker for the in vivo fate of liposomal systems as it is cleared rapidly in the free form and exhibits long tissue retention times when accumulated into tissues. These characteristics are similar to "C-methoxy- inulin (Abra and Hunt, 1981), and indicate that the addition of approximately 0.6 tyramine residues per inulin molecule does not markedly alter the properties of inulin. The advantages of 125ITI include straightforward synthesis, high specific activities, low cost and excellent quenching properties. 3. INFLUENCE OF SIZE AND LIPID COMPOSITION ON LIPOSOME CLEARANCE, LEAKAGE AND TISSUE DISTRIBUTION IN VIVO 3.1 INTRODUCTION The potential of liposomal systems for active targeting within the vasculature has been pointed out in Chapter One. In Chapter Two the problems involved with vesicle labeling techniques have been dealt with, and the importance of employing low doses was noted. However, there are still significant problems to be overcome. In particular, straightforward methods of generating liposomal carrier systems which exhibit high trapping efficiencies are required (Poste, 1983), as are procedures for avoiding the mononuclear phagocyte system (MPS). Subsequent problems include the development of appropriate vesicle targeting and drug uptake mechanisms. Previous investigations suggest that liposomal uptake by the MPS, as well as the residence time in circulation, is determined to a large extent by the size of the delivery system. Thus large diameter vesicles are rapidly cleared from the circulation with half lives of 30 min or less (Allen and Everest, 1983) whereas smaller vesicles can exhibit extended half lives of 10 hr or longer. Moreover, the vesicle composition has been shown to be an important determinant in liposome behavior in vivo. The importance of high cholesterol concentrations in preventing serum induced leakage has been elucidiated (Gregoriadis and Davis, 1979), and it has also been shown (Senior and Gregoriadis, 1982) that vesicles composed of saturated phospholipids have a longer lifetime in the circulation than those composed of unsaturated lipids. Taken together, these findings indicate that SUVs containing saturated lipids and cholesterol exhibit the longest reported blood residence times in the literature. However, there are several major impediments to the employment of these systems, as an examination of the literature demonstrates (see Table 3.1). Firstly, the methods of producing small unilamellar vesicles (SUVs) currently used (sonication 47 48 Table 3.1. Comparison of the vesicle production techniques, labeling methods and dosages found in the the literature. The blood half life is also shown, where it was possible to estimate it from the data. As most of the indicated reports have employed multiple liposomal compositions, only one or two representitive compositions are illustrated. The abbreviations used are as follows: PE, phosphatidylethanolamine; SM, sphingomyelin; a-toe, a-tocopherol; REV, vesicles generated by reverse phase evaporation (Chapman, 1976); 1 2 5I-BPE, 1J5I-p-hydroxybenzamidine PE LIPID VESICLE VESICLE DOSE 3L00D REFERENCE COMPOSITION TYPE -MARKER (mg/kg) KALFLIFE EPC:Choi:PE (7:2:2) ?C:Chol:glyco-l i D i d (13:10:3) DSPC:Chol:glyco-lipid:A23187 (2:0.5:.5:.004) DSPC:Chol:glyco-lipid:A23187 (2:0.5:.5:.004) EPC:DPPS:Choi: a-toc (4:1:5:0.1) EPC:DPPS:C h o i : a-toc (4:1:5:0.1) EPC:DPPS:Choi: a-toc (4:1:5:0.1) EPC:DPPS:Choi: a-toc (4:1:5:0.1) PC: PS (7:3) PC:Choi (8:1) PC:Choi (8:2) MLV sonicated 30 sec. MLV SUV 12 -'I-IsG 23.5 •.' 5 min. SUV MLVs MLVs SUV,MLV, V E T 1 0 0 S * SUV,MLV SUV MLV,SUV 1!>C-EDTA i l l In U l In V E T 1 0 0 s * 1 2 5 I - B P E 1.9-1000 <2 hr I 2 5 I - B P E 31 l l f C - I n u l i n 15-1500 C-Inul in 0.5-50 3H-DPPC 51 Cr 99 Tc 80 100 " T c 40-400 3 H-Chol , l^C-PC, l"C-Inul in <2 hr <1 hr N.D. N.D. <30 min. Ghosh ec a i , 1982 Jonah et a l , 1975 Wu et a l , 1982 Mauk et a l , 1980 Abra et a l , 1984 Abra et a l , 1982 Bosworth and Hunt, 1982 Abra and Hunt, 1982 Poste et a l , 1982 Richardson et a l , 1977 Souhami et a l , 1981 (continued) 49 Table 3.1 (continued) LIPID COMPOSITION VESICLE TYPE VESICLE MARKER DOSE (mg/kg) BLOOD HALFLIFE REFERENCE EPC:Choi (1:1) REV SUV 1 V c h o l -oleace 0-300 40 min. 10 hr rCun and Jul iano, 1981 DSPC:Choi:SA (1.5:1:0.4) MLV 5 9FE-Deferomin Mesylate 30 7 Patei ec a i , 1983 PC: PS (4:1) SUV 3H-BSA 1 4 C-DSPC 50 <2 hr Gotfredsen et a l , 1983 DPPC:Chol:DPPG (8:8:2) SUV 1 2 5 I - B P E 108 <2 hr Szoka et a l , 1983 EPC:Choi (1:1) SUV 6-carboxy-f luorescein 120 180 min. Kirby et a l , 1980 DSPC:Chol (1:1) SUV,REV, MLV 6-carboxy-f luorescein 21 5 hr . , 2 hr. <1 hr Senior et a l , 1985 SM:Chol (1:1) PC:Choi • (1:1) SUV 6-carboxy-f luorescein 14-30 8 hr. 4 hr. Gregoriadis et a l , 1985 SM:Chol (2:1) SUV l l l I . n 35-75 12 hr. Beaumier and Hwang, 1983 (•) The VETiooS used in this report were generated by one pass through a single polycarbonate filter. However, work from our laboratory has shown (Hope et al. 1985) that this is insufficient to generate a homogenous population of VETiooS. (t) Unable to determine values from the data given. 50 (Huang, 1969) or French press (Baranholz et al., 1979)) are tedious to employ, difficult or expensive to scale up, and result in low trapping efficiencies (e.g., 1% or less). The second problem concerns the fact that the dosages used in most of the literature are enough to cause saturation of the MPS (Poste, 1983). Any differences in liposomal blood residence times which have been quantitated at dosages that partially or completely saturate the MPS are difficult to extrapolate to low dose conditions. Moreover, the clinical utility of high doses remains doubtful because large amounts of exogenous lipid are known to damage the MPS (Poste, 1983). Repetition of such doses damage the system further (Allen et al., 1984), inhibiting the important host defense functions of the MPS. The third problem is a reiteration of the measurement problems discussed in Chapter Two. Most of the studies in the literature investigating size effects utilized error prone labeling methods making it difficult to determine precisely what is being measured. The several studies which did employ valid labels were operating at a high lipid dose. Moreover, the time points at which assays were made are too long to observe finer details of liposome uptake (particularly in the initial phases) in virtually all the literature. Consequently, the work described in this Chapter has been done in order to surmount these problems. In particular, the utility of the extrusion technique (Hope et al., 1985) for producing small vesicles (VET30s) is demonstrated. The VET 3 0 systems are shown to be virtually indistinguishable from SUVs formed by sonication as judged by their in vivo behavior. The VET procedure can be used at lipid concentrations as high as 400 mg/ml, with trapping efficiencies as high as 50%, and is easy to scale up. Thus the VET systems effectively surmount the difficulties encountered with sonication protocols. Additionally, the experiments reported in this Chapter were done such that the ambiguities of previous work were removed, and thus they should serve as a prototype for further studies. Moreover, new conclusions about the vesicle behavior in vivo have been made. It is demonstrated that vesicle clearance exhibits biphasic 51 kinetics. In light of the data, a hypothesis is advanced to explain the mechanisms causing the size effects on vesicle uptake. 3.2 MATERIALS AND METHODS 3.2.1 MATERIALS Egg yolk phosphatidylcholine (EPC) was isolated from hen egg yolks as described in Chapter Two. Distearoylphosphatidylcholine (DSPC) was from Avanti. Cholesterol and Stearylamine (SA) were from Sigma. All lipids were chromatographically pure. n5I-tyraminyl-inulin (12iITI) was prepared as described in Chapter Two. All other chemicals were reagent grade or better. 3.2.2 SYNTHESIS OF PHOSPHATIDYLSERINE Egg yolk phosphatidylserine (EPS) was synthesized from EPC by employing the headgroup exchange capacity of phospholipase D (Comfurius and Zwaal, 1977). The phospholipase D was partially purified from the inner leaves of savoy cabbage according to published procedures (Kates and Sastry, 1969). Routinely, 5 kg of the yellowish-green leaves were homogenized in a Warring blender with 3 L of H 2 0 at 4°C. The homogenate was strained through cheesecloth and the filtrate was centrifuged at 2000 g for 30 min. The supernatant was titrated to pH 5.5 with 4 M HCl and aliquots were heated to 55° C in a boiling water bath and immediately removed to an ice bath. Subsequently, the aliquots were centrifuged for 30 min at 13,000 g and the supematants were combined. The phospholipase D was then precipitated by the adding ice cold acetone (1 supernatant: 1 acetone, v/v) and stirring overnight. The precipitate was then allowed to settle and the bulk of the supernatant was removed by aspiration. The residual supernatant was then removed via centrifugation for 10 min at 1000 g. The pellet was then lyophilized and stored at -70° C until use. For phospholipid 5 2 conversion, 200-500 mg of the partially purified phospholipase D preparation was taken up in 20 ml 40 mM CaCl2, 0.2 M sodium acetate buffer (pH 5.6) and stirred at 4 ° C for 30 min. Any insoluble material was then removed by centrifugation at 17,000 g for 10 min. The synthesis of EPS was carried out as follows. Five gm EPC was dissolved in 100 ml ether and added to 100 mis' of 46% L-serine (w/v) in a solution containing 0.1 M CaCl2 and 0.1M sodium acetate (pH 5.6). To this was added 20 ml of the phospholipase D solution (prepared as described above) and the two phase system was incubated at 40° C with vigorous shaking. The progress of the reaction was followed by running small ( 2 x 7 cm) TLC plates which had been cut from 20 x 20 cm silica gel 60 TLC plates (250 uM; E. Merck, Darmstadt, Germany) in a basic solvent system (composed of CHCl3/MeOH/glacial acetic acid/H20, 25:15:4:2: v/v). The plates were stained with molybdate spray (40.1 gm Mo0 3, 2.56 gm sodium molybdate in 4 1 25% H 2S0 4 (v/v)) and heated on a hot plate. The phosphorus containing compounds stain blue and subsequendy all organic compounds are charred black due to the presence of H 2S0 4. Normally, the reaction was stopped after about 30 min by cooling to 4 ° C and centrifugation for 10 min at 500 g. The ether phase was collected and the ether was removed by rotary evaporation. The crude EPS was washed by taking it up in 25 ml of CHCl 3/MeOH (2:1), and then adding 6 ml of H 20, generating a two phase system. The organic phase was removed and dried down. The reaction generally yielded about 30% EPS based on the EPC substrate. The EPS was purified by carboxymethylcellulose chromatography according to Comfurius and Zwaal (1977). Routinely, 3 gm of crude EPS was dissolved in 20 ml CHC13, and applied to a 5 x 72 cm CM-52 carboxymethylcellulose (Whatman, England) column equilibrated with CHC13. The CM-52 was pre-washed in MeOH. The lipid was eluted with a continuous CHCl 3-MeOH gradient (0-50%). EPS containing fractions were identified on TLC plates run in acid solvent (as in Chapter Two) by 53 spraying with a ninhydrin spray (0.2% ninhydrin, w/v, in H20 saturated butanol) followed by heating. Molybdate spray was employed subsequently to observe contaminating EPC. The pure EPS fractions were pooled and dried down. The EPS was converted to the sodium salt as described by Hope and Cullis (1980). Briefly, the lipid was dissolved in a Bligh and Dyer monophase (CHCl 3/MeOH/H 20 1:2.1:1, v/v) where the aqueous component contained 0.4 M HCl. The solution was titrated to pH 8 with a Bligh and Dyer monophase where the aqueous component was 0.5 M NaCl and 0.5 M NaOH. Subsequently, 0.4 volumes of H 2 0 and then 0.4 volumes CHC13 were added generating a two phase system. The organic phase containing the EPS was dried down and stored in CHC13 at -20°C. 3.2.3 LIPOSOME PREPARATION 3.2.3.1 Preparation of MLVs Multilamellar vesicles (MLVs) were prepared from 40-100 u mol of EPC-cholesterol (1:1, mol/mol), or DSPC-cholesterol (1:1 mol/mol). For the plasma stability experiments, 1 yCi 14C-cholesterol-oleate per 100 mg lipid was also included as a lipid label, at the activity indicated below. In other experiments EPS or SA were added. These mixtures were dissolved in CHC13 and then the CHC13 was dried off. After storage under high vacuum for 2 hr the resulting lipid film was dispersed by vortex mixing in 1 ml HBS (20 mM HEPES, 145 mM NaCl, pH 7.4) which also contained 1 mCi l 2 5ITI, generating MLVs. For the plasma stability experiments, the U 5ITI was replaced with 3H-inulin, at an activity of 10 ud per 1 ml HBS. In some cases the MLVs were subjected to 5 freeze-thaw cycles employing liquid nitrogen generating FATMLVs (Mayer et al., 1985). 54 3.2.3.2 Preparation of VETs Vesicles by extrusion technique (VETs) were prepared as previously described (Hope et al., 1985). Briefly, MLVs or FATMLVs were extruded through polycarbonate Filters with pore sizes of 30 nm, 100 nm, or 600 nm (generating VETs designated VET 3 0 , V E T I 0 0 and VET 6 0 0 , respectively). In most cases the MLVs were extruded 10 times through two stacked Filters, and the approximate pressures required were 100 psi, 300 psi, and 700 psi for the VET 6 0 0 , VET 1 0 0 or VET 3 0 systems, respectively. In the case of DSPC-cholesterol (1:1) preparations, however, extrusion through the 30 nm Filter was facilitated by using only one Filter and elevating the temperature to 60° C. 3.2.3.3 Preparation of SUVs Small unilamellar vesicles (SUVs) were prepared from MLVs by employing the standard sonication method (Huang, 1969). Briefly, MLVs prepared as described above were sonicated for 10 minutes employing a probe sonicator (Fischer Sonic Dismembrator model 150), followed by centrifugation for 5 min in a Fischer Microfuge to remove titanium particles. 3.2.3.4 Vesicle Work Up Vesicles were separated from free 125ITI (or 3H-inulin for the plasma stability experiment) by one of two techniques. For the MLV, FATMLV or VET 6 0 0 systems, vesicles were precipitated by centrifugation for 5 minutes in a Fischer Microfuge and taken up in 1 ml HBS. This centrifugation-wash cycle was repeated 5 times and then the vesicles were taken up in 1 ml HBS. For the VET 3 0 , VET 1 0 o or SUV systems, vesicles were passed down a 1 ml Ultrogel Ac 34 column equibrated with HBS as described in Chapter Two. Phosphate assays were performed as described in Chapter Two and the trapped volumes were calculated using the m l T I (or 3 H - inulin for the plasma stability 55 experiments) as a trapped marker. Subsequently, vesicles were diluted with HBS such that 250 ul contained one dose for injection. For most studies, the dose of lipid was adjusted to give 0.8 al trapped volume (see Table 3.2). For the dose dependency studies, the dose was 12.5 M mol, 1.25 M mol, or 0.125 M mol/ml. The dose for the surface charge experiments was generally 2 M mol phospholipid. 3.2.4 IN VIVO BLOOD CLEARANCE AND TTSSIJF. DISTRIBUTION STUDIES In order to monitor liposome clearance from the circulation, a convenient jugular cannulation technique was employed. Male Wistar rats were weighed ( 2 2 5 - 2 7 5 gm) and anesthetized with sodium pentobarbitol (50 mg/kg body weight). For studies lasting longer than 1 hr, maintenance injections of 1 0 - 2 0 mg/kg body weight were required. When anesthesia was complete, the throat muscles were blunt-dissected to expose the trachea and the right jugular vein. A trachyotomy was performed by partially transecting the trachea and inserting an 8 gauge plastic cannula into the trachea. The right jugular was then cannulated with an 18 gauge cannula. Liposomes, prepared as described above and containing at least 1 M Ci U 5ITI, were then injected in a 250 M 1 volume of HBS. In one experiment, 0.5 M O of free 125ITI was injected in 250 M 1 HBS. Any residual material was washed into circulation by a further injection of 250 M 1 HBS. At subsequent intervals, 250 M 1 of blood was removed and replaced with 250 M 1 HBS, keeping the blood volume constant throughout the experiment. The n 5ITI in the blood samples was determined by gamma counting and the 1 2 5ITI dose remaining in circulation was calculated assuming 4.9 ml blood per 100 gm body weight (Altaian and Dittmer, 1971). The tissue distribution studies were performed exactly as described in Chapter Two. Unless otherwise indicated, rats were sacrificed at 24 hr post injection. Table 3.2. Trapped volumes and injection doses of EPC: Cholesterol (1:1) VET 6 0 0 V E T i a o VETjo SUV Trapped volume 1.95 0.8 0.88 0.44 (u V u mol lipid) Dose 0.95 2.4 2.1 4.4 (mg/kg) 56 vesicle preparations. DSPC: Cholesterol (1:1) VET 6 0 0 VET 1 0 0 VET 3 0 SUV 1.28 0.8 1.1 1.1 1.5 2.4 1.8 1.8 57 3.2.5 IN VITRO STUDIES 3.2.5.1 Control Studies With Ultrogel Ac 34 Chromatography VET 1 0 0 systems were made as described above and were composed of 1:1 EPC:Cholesterol containing 1 u Ci 14C-cholesterol-oleate per 100 mg lipid, at a concentration of 100 mg lipid/ml HBS. The vesicles were added to 10 u Ci 3 H-inulin and vortexed. Thus, the 14C-cholesterol-oleate served as a lipid marker and the 3H-inulin labeled the external solventThe vesicles (10 n\) were added to 40 u 1 human plasma (obtained in heparinized tubes from the Acute Care Hospital, UBC) and immediately loaded on to a 1 ml Ultrogel Ac 34 column previously equilibrated with HBS. One drop fractions (about 50 y 1) were collected and assayed for 3 H and 1 4 C . The results are shown in Figure 3.1. It can be seen that a baseline separation was obtained, demonstrating the utility of the column for analytical determination of 3H-inulin leakage from VET 1 0 0s, in the presence of plasma. The minor 4 H peak under the l 4 C peak is due to the channels ratio correction program on the scintillation counter, as chromatography in the absence of 3 H - inulin yields an identical peak. Recoveries of 3 H and 1 4 C were reproducibly quantitative. Further experiments showed that all the vesicles could be recovered in the first 600 u 1, whereas all the free 3 H-inulin could be recovered in the following 1 ml, and thus only two fractions needed to be collected to quantitate vesicle leakage. Vesicles which were vortexed and rechromatographed did not leak detectable amounts of JH-inulin. 3.2.5.2 Plasma Stability Studies Human plasma was obtained as described above. VET 1 0 0 systems were made of 1:1 EPC-Cholesterol (containing 0.75 u Ci 14C-cholesterol-oleate and 8 M Ci 3H-inulin per 100 mg lipid) as described above. The vesicles (500 u\) were added to 500 ul of plasma or HBS and incubated at 40°C. At various times, 58 Figure 3.1. Chromatographic separation of 3H-inulin from VET10oS in the presence of plasma. VET10oS, labeled with 14C-cholesterol-oleate, were added to plasma containing 3 H-inulin and applied to a 1 ml Ultrogel Ac 34 column (see text for details). 59 50 M 1 aliquots were assayed for 3 H-inulin leakage as described above. 3.2.5.3 Studies with Whole Rat Blood Rat blood was obtained from male Wistar rats under ether anesthesia by vena cava puncture. Approximately 10 ml of blood was collected into a syringe containing 200 M1 of 2 0 0 mM EDTA, and used immediately. -VETioo systems composed of 1:1 EPC:Cholesterol containing 0.2 M Q 3H-inulin trapped /nmol lipid were made as described above and diluted to 5 M mol lipid/ml in HBS. Aliquots of 50 M 1 were added to 2.5 ml fresh whole rat blood and incubated at 4 0 ° C. A control tube was also run containing 0.5 M Ci 3H-inulin in 2.5 ml blood. At various times, the tubes were removed and centrifuged at 3000 g for 10 min. 250 M1 of plasma was assayed for 3 H-inulin. Another 50 M1 aliquot of plasma was loaded on to an Ultrogel Ac 3 4 column and assayed for trapped or free 3 H - inulin as described above. The red blood cell pellet, which also contained all the leukocytes ("buffy coat"), was washed three dmes with 2.5 ml HBS containing 5 mM glucose, 1 mM CaCl2 > and 1 mM MgCl2, and the supernatants and final pellet were assayed for 3 H-inulin. 3.3 RESULTS 3.3.1 IN VIVO STUDIES 3.3.1.1 Clearance of Free 125ITI Initial control experiments were aimed at characterizing the rate of clearance of free 125ITI from the circulation. As shown in Chapter Two, the clearance is rapid (residence half life < 5 min). However, the cannulation procedure allows accurate 125ITI blood levels to be obtained at time intervals as small as 1 min post injection. As shown in Figure 3.2, 125ITI clearance is 60 biphasic. An initial extremely rapid clearance is observed (half life < 25 sec) which is followed by somewhat slower clearance (halflife = 20 min), resulting in only 10% of the injected dose remaining in the circulation after 5 min. 3.3.1.2 Dose Dependency Studies As shown elsewhere (Abra and Hunt, 1981), intravenous administration of high concentrations of liposomes results in enhanced blood retention times, which are presumably due to blockade of the mononuclear phagocytocytic system. However, previous studies (Abra and Hunt, 1981; Bosworth and Hunt, 1981) have employed lipid dosages in the range of 30-100 mg/kg body weight. As discussed in the Introduction, it is of interest to investigate the effect of dose on vesicle clearance in a lower dose range. It may be noted that the use of l 2 5ITI allows the turnover of low liposomal doses (<1 mg/kg body weight) to be followed. The influence of lipid dose levels of 0.4, 4 and 40 mg/kg body weight on the clearance of VET 1 0 0 s composed of EPC-Cholesterol (1:1) is illustrated in Figure 3.3. For the 0.4 mg/kg doses a rapid initial clearance rate (half life = 16 min) is dominant, which is followed by a slower clearance rate (half life = 2.8 hr). With the 4 mg/kg dose, the first phase is slightly longer (halflife = 25 min), but the second phase is the same (2.8 hr). The highest dose (40 mg/kg) exhibits clearance behavior where the initial phase (halflife = 1.1 hr) is less predominant, but with a similar second phase halflife of 2.9 hr. Thus the data indicate that the second phase clearance phase is dose independent, whereas the first phase halflife is dose dependent and saturable. 3.3.1.3 Size and Lipid Composition Studies Blood Clearance Determinations In order to determine the influence of size and lipid saturation on clearance rates, it is clearly most appropriate to employ lipid doses such 61 Figure 3.2. Clearance of free 12SITI from the rat circulation. The 125ITI (0.5 M Ci) in 250 M 1 of HBS was injected into the jugular vein of 225-275 gm male Wistar rats. At subsequent intervals 250 M1 of blood was removed and assayed for 125ITI. The total amount of 1 2 5IT! remaining in the circulation was calculated assuming 4.9 ml blood per 100 gm rat Results are expressed as percent 125ITI injected. The error bars indicate the standard error (n=3). For further details see Methods. 100 w so •• 62 Figure 3.3. Dose dependence of the clearance of 1 J iITI entrapped in EPC-cholesterol (1:1) VETioo systems from the rat circulation. The VETmoS were prepared by extrusion through 100 nm filters and injected into the jugular vein employing the carmulation technique (see Methods). The lipid doses injected were: (•) 0.4 mg/kg body weight; (•) 4 mg/kg body weight; (*) 40 mg/kg body weight Blood was withdrawn at the indicated intervals and assayed for l " r n . Results are expressed as a percentage of the total 1 1 5 m injected. The error bars indicate the standard error (n=3). I I I I L 0 1 2 3 4 TimQ (hrs) 63 that the large systems are cleared relatively rapidly over a convenient experimental timecourse (e.g., 30 min). As determined from Figure 3.3, dose levels in the range of 4 mg/kg or less are thus most appropriate. Such doses also correspond to levels required for delivery of clinically relevant quantities of drug (Mayer et al., 1985). An arbitrary decision to employ liposomal systems containing 0.8 yl trapped volume was therefore made, which corresponds to approximately 3 mg lipid/kg body weight, for most of the types of liposomal carrier systems employed (Table 3.1). The clearance of various size VET systems composed of EPC-Cholesterol (1:1), as well as sonicated vesicles, is illustrated in Figure 3.4. It can be seen that size greatiy effects the clearance of vesicles. VET60o systems exhibit biphasic kinetics with a first phase halflife of 6 min and a second phase halflife of 21 min. The VET 1 0 0 systems give a first phase halflife 18 min. The second phase halflife can not be accurately accessed from the data. The V E T 3 0 and SUV systems have essentially equivalent behavior, although the halflives cannot be dicerned from the data. In any case, it is apparent that both the first and second phase halflives are size dependent, and that the first phase is "saturable" with respect to size. The preceding results indicate that the smaller sonicated and extruded systems do not experience the initial rapid clearance (half life <20 min) observed for the large systems at low dose levels (4 mg/kg body weight). In order to establish the generality of this observation, the clearance of corresponding DSPC-Cholesterol (1:1) systems was also investigated. It has been demonstrated (Gregoriadis and Senior, 1980) that DSPC-Cholesterol SUVs exhibit longer blood residence times than comparable EPC-Cholesterol systems, at doses on the order of 100 mg/kg. It was of interest to examine this effect in the low dose range. As shown 64 Figure 3.4. Size dependence of the clearance of 1 2 5 r n encapsulated in EPC-cholesterol (1:1) liposomes of varying size: (o) systems extruded through 600 nm filters (VET 6 0o); (•) systems extruded through 100 nm filters (VET 1 0 0); (•) systems extruded through 30 nm filters (VET3o); (•) sonicated vesicles (SUV). Vesicles were injected in the jugular vein via the cannulation technique and blood aliquots withdrawn at subsequent intervals and assayed for 1 2 5 r n . The amount of 125ITI determined in the blood is expressed as a percentage of the total 1 2 iITI injected. The error bars indicate the standard error (n=3). 10 15 20 25 Tlma (min) 65 in Figure 3.5, DSPC-Cholesterol (1:1) VET 3 0s exhibit similar clearance rates (half life >1.5 hr) as their sonicated SUV counterparts, again illustrating the similar clearance behavior of SUVs and VET3 0s. The V E T l 0 0 DSPC-Cholesterol systems are removed more rapidly than the corresponding EPC-Cholesterol systems, exhibiting a biphasic clearance behavior with an initial rapid (half life = 7 min) phase, and a second slower phase (halflife = 1.5 hr). Finally, the VET 6 0 0 systems experience equally rapid clearance ( half life = 4 min) followed by a second slower phase (halflife = 2.3 hr), as the similarly sized EPC-Cholesterol systems. Tissue Distributions The different clearance rates of the vesicle systems with different sizes implies size dependent interactions with blood and tissue components, and it may be expected that different final tissue distributions will result. As shown in Chapter Two for VET 1 0 0 systems composed of EPC-Cholesterol (1:1), equilibrium tissue distributions of entrapped U 5ITI are achieved at approximately 12 hr, and remain stable for at least 60 hr thereafter. The distributions of U 5ITI delivered via the EPC-Cholesterol (1:1) and DSPC-Cholesterol (1:1) systems were therefore determined at 24 hr post-injection and are given in Table 3.3. It should be noted that for the DSPC-Cholesterol (1:1) SUVs and VET 3 0s the distributions are not quite equilibrium values as 10-15% of the 12SITI remains in the circulation. In any event, three major trends are apparent. First, the amount of 125ITI accumulated in the liver increases somewhat for the smaller EPC-Cholesterol (1:1) systems, in contrast to the DSPC-Cholesterol systems, where only 15% is retained in the liver for the VET 3 0 systems. Second, the larger systems are somewhat more leaky than their smaller counterparts, as reflected by the 125ITI found in urine. This is particularly true of the 66 Figure 3.5. Size dependence of the clearance of l"ITI encapsulated in DSPC-cholesterol (1:1) liposomes systems of various sizes: (o) systems extruded through 600 nm filters (VETJOO); ( • ) systems extruded through 100 nm filters (VETioo); (•) systems extruded through 30 nm filters (VET30); (•) sonicated systems (SUV). Vesicles were injected in the jugular vein via the cannulation technique and blood aliquots withdrawn at subsequent intervals and assayed for 1 I 5 r n . The amount of l " m determined in the blood is expressed as a percentage of the total l 2 5ITI injected. The error bars indicate the standard error (n=3). 0 5 10 15 20 25 30 TimQ (min) 67 Table 3.3. Tissue distributions of 1J5ITI delivered by liposomes of varying sizes and composition. Male Wistar rats (225-275 gm) were injected via the tail vein and allowed to recover in metabolic cages. The liposome doses employed contained 0.8 M L trapped volume corresponding to 2.5-5 mg/kg body weight for the extruded systems (see Table 3.2). At 24 hr post injection the tissues indicated were removed and 1 2 5 m assayed. The amount of 125ITI detected is expressed as a percent of the total 125IT1 detected. The recovery of l 2 3ITI was 86±6%. The errors given are standard errors (n=3). EPC : Cholesterol (1 : 1) Tissue V E T 6 0 0 V E T 1 0 0 VET 3 0 SUV B lood 0.2 + .07 0.1 + 0.03 0.3 + 0.1 0.8 + 0.4 L i v e r 33 + 3 41 + 3 46 + 3 45 + 2 Spleen 6 + 1 1 0 + 2 7 + 4 2 + 0.2 Urine 46 + 4 32 + 1 28 + 4 20 + 2 Kidney 0.4 + 0.1 0.25 + 0.1 0.3+0.1 0.43 + 0.1 Carcass 11 + 3 10 + 2 16 + 3 25 + 4 DSPC : Cholesterol (1 : 1) Tissue • V E T 6 0 0 VETioo VET 3 0 SUV B looa 0.4+0.06 0.3 • 0.04 13 + 5 9 + 2 L i v e r 67 + 8 53 + 5 21 + 7 15 + 2 Spleen 1 4 + 3 12 + 4 3 + 0.1 14 + 5 Urine 10 + 3 17 + 5 13 + 2 14 + 2 Kidney 0.2 + 0.08 0.13 + 1 0.9 + 0.2 0.8 + 0, Carcass 9 + 4 1 3 + 2 45 + 7 45 + 5 68 EPC-Cholesterol systems, where 46% of the 12SITI is excreted for the VET 6 0 0s, in contrast to only 20% for the SUVs. Finally, the smaller extruded and sonicated systems display a tendency to deliver 125ITI to the carcass. This is particularly true of the SUV and VET 3 0 DSPC-Cholesterol systems, where 45% of the injected 125ITI is retained in the animal but not localized in the liver, spleen, blood or kidney. 3.3.2 EFFECT OF SURFACE CHARGE ON THE IN VIVO FATE OF FATMLVS 3.3.2.1 Phosphatidylserine Containing Vesicles Work done in several other laboratories (Abra et al., 1984; Richardson et al., 1977) has shown that vesicles containing phosphatidylserine (PS) tend to localize in the lungs, as measured with a lipid label. It was of interest to try this experiment with 125ITI to determine whether contents are also retained. Unsized (non-extruded) FATMLVs were used because previous work (Sharma et al., 1977) showed that lung deposition increases with size. The composition of the vesicles was EPC:EPS:Cholesterol (4:1:5, mol/mol/mol). As a preliminary experiment, a 24 hr time point was chosen as it was likely to reflect equilibrium values, as discussed in Chapter Two. The effect of lipid dose on the tissue distributions is shown in Figure 3.6. The major result is the increase in the liver deposition with increasing dose and a corresponding decrease in the carcass deposition. This indicates that there are sites besides the liver and spleen which have a high affinity but low saturability for these vesicles. It is noteworthy that there is virtually no 125ITI in the lungs at 24 hrs. This contrasts with earlier work (Abra et al., 1984) which reported about 7% deposition in the lungs at 24 hrs (using 4:1:5:0.1 PC/PS/Cholesterol/a-tocopherol, mol/mol/mol) and might reflect the different labeling protocol or dosage (920 mg lipid/kg body weight) used. As a lipid label was used in their studies it might 69 Figure 3.6. Effect of lipid dose on the tissue distribution of 125IT1 entrapped in FATMLVs composed of EPS:EPC:Cholesterol (4:1:5). Male Wistar rats (225-275 gm) were injected via the tail vein and allowed to recover in metabolic cages. At 24 hrs post-injection the tissues indicated were removed and the U5TTl assayed. The symbols correspond to: (•) liver; (A) urine; (o) lung; and (•) carcass. The percent 1 2 5 r n found in the spleen was less than 3 ±0.6% at all doses indicated. The amount of i : i r n detected is expressed as a percent of the total 125ITI detected. The errors given are standard errors (n=3). 60 40. a ce > a U UJ ce 20 J th d 3 6 10 LIPIO DOSE CMG/KG) 70 be that the vesicles are physically trapped in the capillaries of the lungs, where they progressively leak their contents. If that were the case then it would be expected that a time course would show an early peak of 12SITI in the lungs which would progressively decay to zero at 24 hr. This possibility was examined and the results are shown in Figure 3.7. As predicted, an early peak of l 2 5ITI deposition in the lungs is found, although the maximum deposition (3.6%) is very modest. However, it is clear that vesicles are depositing there as the 1 2 3ITI remaining in total blood (<1.5%) is far too low to account for the lung deposition by the residual blood found in the lungs. Presumably, if the dose were increased 50 fold to the level as used by Abra et al., (1984), the values would be similar. The matter was not investigated further. 3.3.2.2 Stearylamine Containing Vesicles After obtaining such a low 125ITI deposition in the lung with vesicles containing EPS at low dosages, it was of interest to determine if the lung deposition could be increased with stearylamine (SA) containing vesicles. Previous work (Jonah et al., 1975) indicated that SA increased lung deposition under some conditions. A 0.2 MITIOI lipid dose was chosen for injection and the results are shown in Figure 3.8 and Table 3.4. It can be seen that, SA incorporation into FATMLVs gives a transient increase in 125ITI deposition in the lungs, relative to control vesicles. This transient increase appears to be primarily at the expense of the liver deposition, which exhibits a corresponding transient decrease in 125ITI. As free 125ITI is not taken up by the liver, this indicates that at least some of the SA containing vesicles initially found in the lung subsequently migrate to the liver without being ruptured. Table 3.4 also shows that SA incorporation increases the final spleen deposition, while it decreases the final carcass deposition. 71 Figure 3.7. Tissue distribution of '"ITI entrapped in EPS:EPC:Cholesterol (1:4:5) FATMLVs after injection of doses corresponding to 32 mg lipid/kg body weight The symbols correspond to: (•) liver; (•) urine; (A) lung; and (o) carcass. The amount of 1 2 j m found in the spleen was less than 4±0.2% at all times indicated. The amount of u 5 r n is expressed as a percent of 1 J 5 m detected. The errors given are standard errors (n=3). 72 Figure 3.8. Tissue distribution of l "rn entrapped in FATMLVs 24 hr after injection of doses corresponding to 6.4 mg/kg. The composition of the FATMLVs was either EPC.Cholesterol (1:1) and is denoted by the open symbols or was EPC:Cholesterol (1:1) containing 5% SA (w/w) and is denoted by the solid symbols. (•) indicates delivery to the liver, and (•) indicates lung. 50 HOURS 73 Table 3.4. Tissue distribution of n 5ITI entrapped in FATMLVs 24 hr after injection of doses corresponding to 2.0 M mol lipid per rat. See Figure 3.8 for details. (PC) denotes FATMLVs composed of 1:1 EPC-Cholesterol while (SA) denotes FATMLVs composed of EPC-Cholesterol (1:1, mol/mol) containing 5% SA (w/w). Values are given as percent U 5ITI recovered. The error indicated is the standard error (n = 3). Time Carcass Carcass Urine Urine Spleen Spleen (hr) PC SA PC SA PC SA 0.5 60.3+ 5.4 45.0± 2.7 0 0 2.2+0.8 12.2+ 1.3 1 29.7+3.8 31.3+1.4 11.2±4.0 9.8+8.2 12.6+3.6 11.0+0.6 2 30.0+ 4.3 18.7+1.0 27.7± 2.1 22.2± 6.6 3.5+1.2 19.9+ 3.0 6 28.6+ 8.7 20.9+ 2.2 35.4± 6.2 34.0+ 5.7 2.4± 1.7 9.0 ± 1.3 24 23.8+0.7 13.0+ 1.8 42.4+ 3.0 40.6+ 8.0 5.3+2.5 14.4± 1.8 74 3.3.3 m VITRO STUDIES 3.3.3.1 Plasma Stability Studies In all of the results presented so far, there has been a large proportion of 125ITI found in the urine (10-50%), implying that vesicles are broken open in the blood subsequent to their injection. As all the liposomes used in these studies contained equimolar cholesterol, it was initially rather suprising to obtain this result as equimolar cholesterol has been reported to stabilize vesicles in blood in vivo (Kirby et cd., 1980). Consequentiy, it was of interest to determine the extent of liposome breakage in vitro, as a comparison to the in vivo findings. As a preliminary experiment, fresh heparinized human plasma was incubated with EPC:Cholesterol VET 1 0 0s (labeled with 14C-cholesterol-oleate to follow the lipid, and 3 H-inulin to follow the vesicle contents). The result is shown in Table 3.5. It can clearly be seen that there is virtually no 3H-inulin leakage throughout the course of the experiment (<6% in 24 hr at 37° C). This is to be contrasted with the in vivo finding that 32% of these vesicles rupture within 24 hr post injection. It is noteworthy that about 15% of the 14C-cholesterol-oleate is lost from the vesicles within 15 min after plasma' addition and yet there is no further loss. In principle, this could be due to either lipid transfer, lipid exchange, or plasma lecithin-cholesterol acyl-transferase (LCAT) activity. However, the matter was not investigated further. 3.3.3.2 Studies with Whole Blood The preceeding experiment demonstrated that the VET 1 0 0 systems used were stable to plasma in vitro, and thus the in vivo results could not be accounted for. However, it could be argued that the lipid concentration used was much higher than the in vivo dose and thus does not reflect the in vivo conditions. Consequently, the next experiment employed V E T 1 0 0 systems composed 75 Table 3.5. Stability of VETi 0 0s composed of EPC:Cholesterol (1:1) in human plasma. Vesicles, labeled with 14C-cholesterol-oleate as a lipid marker and 3H-inulin as a trap marker, were incubated in plasma for the time indicated and then purified on a Ultrogel Ac 34 column (see Methods for details). The percentages listed show the loss of label from the vesicle fraction. Time 3H-Inulin 14C-Cholesterol-Oleate (hr) (% Leaked) (% Removed) 0.25 2 15 0.5 1 16 1 2 14 2 2 16 8 0 16 24 5 13 76 of EPC:Cholesterol (1:1) which contained 12SITI at a high specific activity (1 uCi per M mol lipid) allowing the low dose range to be investigated. Also whole rat blood was used which had been taken immediately before the experiment began, so that vesicle binding and uptake by cells could be monitored. Cellular binding and uptake was estimated for the total cellular fraction (erythrocytes plus leukocytes). The results are shown in Table 3.6. It can be seen that there is less than 5% leakage during the course of the experiment, even at a dose of only 0.1 a mol lipid per ml. Moreover, the cellular fraction, which had been washed 3 times (in buffer containing glucose, CaCl2 and MgCl2 to preserve monocyte activity), shows a maximum uptake of only 1.04 nmol lipid per ml after 1 hr, which was only 0.52% of the total lipid. Thus, uptake by the cellular fraction is negligible under the experimental conditions. It is possible that liposome breakage and binding to cells are calcium dependent processes and the EDTA used for the anticoagulant is interfering with these processes. However, the preceeding experiment with heparinized human plasma makes this possibility less likely. Moreover, a pilot study done (in collaboration with R. Samborski) with cultured human monocytes failed to show significant vesicle uptake under conditions where lipoproteins were actively phagocytosed (data not shown). 3.4 DISCUSSION The principal results presented here concern the inhibition of the initial rapid clearance of liposomes from the rat circulation by employing small sonicated and extruded vesicles, and the different size-dependent tissue distributions of the liposome contents that are observed. Additionally, the ancillary in vitro studies indicate that liposome rupture in vivo is not accounted for by simple plasma induced leakage. Lastiy, the data obtained with FATMLVs having a surface charge show a transitory accumulation of liposomal contents in the lungs, particularly when stearylamine (SA) is 77 Table 3.6. Fate of 125ITI entrapped in VET 1 0 0 systems composed of EPC:Cholesterol (1:1) after incubadon in whole rat blood. Vesicles containing 1 u Ci 125ITI per nmol lipid were incubated in whole fresh rat blood at 40° C, at a concentration of either 0.1 or 0.2 n mol lipid/ml, as indicated. At the indicated times, the cell associated, free, or vesicle entrapped 125ITI was determined as described in Methods. The percentages shown are the percentage of total 123ITI entrapped at time zero. [Lipid] Time 125ITI Leakage Cell Bound (M mol/ml) (minutes) (%) (%) 0.1 15 0.5 0.24 30 1 0.39 45 0 0.72 60 1 0.62 0.2 15 2 0.34 30 4 0.46 45 7 0.35 60 2 0.52 78 used. Each of these areas are discussed in turn. The observation that (larger) liposomes exhibit biphasic clearance kinetics, with a rapid initial phase and a slower second phase, is consistent with previous studies (Senior et al., 1985). However, direct comparisons are difficult due to the fact that these earlier studies employed higher lipid doses, a different lipid composition, a different animal model and an initial time point of 1 hr, which is far too late to discern the first phase decay. The studies presented here, facilitated by the 12SITI label and the cannulation technique, permit a much finer time resolution and show that a rapid clearance phase is dominant at lower doses (4 mg/kg body weight) of the larger VETioo or VET 6 0 0 systems. The dose studies done with VET 1 0 0s suggest that higher doses saturate out the initial rapid clearance phase, while the second, slower phase remains relatively constant. It is possible that the rapid clearance phase represents non-specific binding to the vasculature (including cells of the MPS), whereas the slower clearance phase represents actual uptake by the MPS. Thus the initial, phase saturates the binding sites, while the second phase represents binding sites which become available again after endocytosis. The slope of the second phase would then measure the rate of uptake. The observation that the SUVs and VET 3 0 systems do not experience the rapid initial decay is of interest. The previous discussion leads to the suggestion that vesicle binding is highly size dependent, which could be interpreted as due to retention of the larger vesicles in the microvasculature (presumably in the recticuloendothelial system). It is particularly intriguing to note the large difference in residence times between the VET 1 0 0 and VET 3 0 systems, considering the relatively small difference in size. The enhanced stability of the smaller vesicles is not currently understood, but in light of the preceeding discussion it appears that VET 3 0s and SUVs do not bind as readily to the MPS. This could be related to plasma protein binding and penetration. 79 The different tissue distributions of the contents of vesicles with different sizes are suggestive. The observation that delivery to the "carcass" is significantiy enhanced for the SUVs and VET 3 0s is consistent with the longer residence time in the circulation and consequently increased time to penetrate to other compartments available to the vasculature. Similar results have recentiy been reported elsewhere for SUVs (Senior et al., 1985), where evidence indicating delivery to the bone marrow has been obtained. The results also show that the smaller systems are less "leaky" than the larger systems. The in vitro work presented indicates that this leakage is not a simple plasma induced instability. One hypothesis is that vesicles can rupture while bound to the surface of cells without being endocytosed. The finding that VET 3 0 systems are virtually indistinguishable from the corresponding SUV systems is noteworthy. The fact that the VET 3 0 systems can be prepared easily and reproducibly and exhibit high trapping efficiencies make them quite superior to corresponding SUVs, and are thus the system of choice for long-lived delivery vehicles in vivo. The demonstration of transient lung retention of vesicle contents using FATMLVs with a surface charge leads to several questions. As was mentioned in the Results section, previous work done with lipid labels (Abra et al., 1984) indicated a more permanent retention of label in the lung. In light of the long intracellular retention time of 125ITI demonstrated in Chapter Two, the combined results indicate that the lung associated vesicles have not been endocytosed. Instead, it might be hypothesized that the vesicles are mechanically trapped in the lung capillaries, which are known to be the smallest capillaries in the body. This is consistent with the fact that increasing the size of the vesicle increases the lung retention (Sharma et al., 1977). The vesicles, once trapped in the lungs, could be ruptured, endocytosed by monocytes or alveolar macrophages (perhaps by monocytes becoming alveolar 80 macrophages), or released back into the circulation and metabolized elsewhere. In light of these possiblities, it might be expected that the mechanism by which surface charge enhances lung retention is related to plasma protein binding, which would increase the effective diameter of the vesicle. This possibility is the subject of the next chapter. 4. LIPOSOMAL PLASMA PROTEIN BINDING: SURFACE CHARGE F.FFF.CTS 4.1 INTRODUCTION As discussed in the Introduction, liposomes interact with a wide variety of plasma components. Although plasma protein absorption to liposomes has been investigated for a large variety of proteins (Bonte and Juliano, 1986), the effects on the distribution of vesicles in vivo are not well understood. As there is good reason to believe that plasma protein interactions are important in determining the liposomal fate in vivo, this is an important area to explore. In this context it is of interest to examine liposomal plasma protein binding in whole plasma, using a dose and lipid composition that have been used in vivo. In this way, it may be possible to correlate changes in vesicle biodistribution to changes in plasma protein binding. There have been several investigations on the interaction of purified plasma proteins on cell uptake in vitro, but most of these studies are difficult to extrapolate to the complex in vivo system. In addition, these investigations did not use cholesterol containing vesicles as are used in most drug delivery protocols. There has been one study by Juliano and Lin (1980) which looked at the effect of liposome composition on plasma protein binding in whole plasma or serum, and many basic results were obtained. The work described in this chapter is primarily an extension of their study. However, the lipid dose they used corresponded to approximately 500 mg/kg, which is an order of magnitude higher than that used in this thesis. Consequently, a direct comparison is difficult, because of the possibility that differential binding patterns result from depletion of various plasma components. As was demonstrated in the previous chapter, the addition of charged lipids to EPC:Cholesterol systems results in a transient increase in the percentage of l"ITI found in the lungs (using stearylamine (SA) or phosphatidylserine (PS)). The cause of this accumulation is unclear, but it is possible that liposomal protein binding may be a 81 82 contributing factor. In this context the influence of liposomal surface charge on plasma protein binding is investigated in this chapter, using PS or SA as surface charge modulators. It is shown that alteration of surface charge dramatically alters the amount and type of protein binding. The results also shed insight into factors responsible for liposomal fates in vivo. 4.2 MATERIALS AND METHODS 4.2.1 MATERIALS Outdated human plasma was obtained from the Red Cross in Vancouver, BC. The Folin and Ciocalteu's phenol reagent, 2 N (FC reagent), glycerol, Agarose type V, Factors II and VII deficient plasma, Factor V deficient plasma, Factor IX deficient plasma, Factors VII and X deficient plasma, thromboplastin solution, Russell's viper venom in cephalin solution, normal plasma control, Triton X-100, cholesterol and stearylamine were obtained from Sigma. Activated partial thromboplastin (APT) reagent was obtained from Ortho. Dialysis tubing was from Spectropore. Nitrocellulose BA 85 paper was from Schleicher and Schuell. Chloramine-T was from Fischer. Sodium dodecyl sulfate (SDS), acrylamide, Trizma base, glycine, Coomassie blue R-250, N,N,N',N'-Tetramefhylethylenediamine (TEMED), ammonium persulfate, AG1X10 resin, N,N'-methylene-bis-acrylamide, mercaptoethanol, bromophenol blue, and urea were obtained from Bio-Rad. Nonidet P40 (NP40) was from Aldrich. 2-D Pharmolyte (pH range 3-10) and DEAE Sephacel were from Pharmacia. Thrombin was a generous gift of Ross MacGillivray. goat anti-human albumin, goat anti-human IgG, and rabbit anti-goat IgG (heavy and light chains specific) antisera were from Cappel. Egg PC was isolated from hen egg yolks as described in Chapter Two. Egg PS was synthesized from egg PC as described in Chapter Three. All other chemicals were of analytical grade or better. 83 4.2.2 PREPARATION OF VESICLES MLVs were prepared as described in Chapter Two. Briefly, 50 u, mol lipid of various compositions was dried down from chloroform in a round bottom flask and left under high vaccum for at least two hr. Subsequently, the film was hydrated in lml of 0.145 M NaCl, 20 mM HEPES, pH 7.4 (HBS) by using a marble to agitate the lipid in the flask. 4.2.3 PREPARATION OF HUMAN PLASMA OR SERUMS Only outdated human plasma of type 0+ was used. As pilot studies indicated some variation between different units of plasma, all subsequent studies were done with pooled plasma. Pooled plasma was centrifuged for 1 hour at 10 K rpm in a Sorval centrifuge using a GSA rotor. The residual RBC pellet was discarded. Plasma was stored at 4°C until use. Serum was prepared from the pooled plasma in various ways. Thrombin generated serum was prepared by adding 117 units of thrombin (15 u 1) to 100 ml plasma and incubating at 37° C with gentle agitation for 6 hr in the presence of approximately 4 gm of borosilicate glass chips derived from pasteur pipettes. At the end of the incubation, the resulting serum was decanted off and centrifuged for 1 hour at 10 K rpm in a GSA rotor. The supernatant was stored at 4 ° C until use. Serum was also generated by calcifying plasma. Plasma (100 ml) was warmed to room temperature and various amounts of 2 N CaCl2 was added (to a final concentration as indicated in the Results section). The solution was gently stirred overnight in the presence of about 4 g of borosilicate glass fragments, and the supernatant was decanted and centrifuged in a GSA rotor at 10 K rpm for 1 hr. The serum was stored at 4 ° C until use. Dialyzed plasma preparations were used in one experiment, using either normal plasma or serum made in the presence of 50 mM CaCl2. Aliquots (100 ml) were 84 dialyzed 24 hr against 4 1 of 5 mM HEPES pH 7.4, then 24 hr against 4 1 of HBS. At the end of dialysis 15 M 1 2 M CaCl2 was then added to the plasma preparation, giving a calcium concentration approximately the same as the serum preparation (30 MM). 4.2.4 MEV INCUBATIONS IN PLASMA Unless otherwise mentioned, all incubations were done under identical conditions. 50 ml of plasma or serum was preheated to 37° C for 15 min in a water bath. Subsequendy, 1 ml of MLVs was added (50 M mol lipid in HBS) and incubated for 30 min with occasional agitation. The solution was diluted to 250 ml with HBS and centrifuged for 30 min at 10 K rpm. The pellet was resuspended in 40 ml HBS and centrifuged for 20 min at 15 K rpm in an SS-34 rotor. The pellet was resuspended in 1 ml HBS and centrifuged 5 min in a Fischer microfuge 235B. This last wash was repeated 5 times. The final pellet was resuspended in 1 ml HBS. In some cases the supernatants produced above were saved for analysis. 4.2.5 DETERMINATION OF LIPOSOMAL PROTEIN BINDING Liposome associated protein was quantitated by a modification of the Lowry assay (Lowry et al., 1951), preceded by a delipidation step according to Wessel and Flugge (1984), as follows. Liposomes containing 10-40 M g protein were diluted to 0.5 ml H 20. To this were added 2 ml MeOH, 1 ml CHC13, and 1.5 ml H20 with vortexing after each addition. The two phase system generated after the last addition was centrifuged for 15 min at 3 K rpm. The upper phase was carefully aspirated such that the protein at the interface was left with a slight amount of upper phase. Then 1.5 ml of MeOH was added and the protein was precipitated by centrifugation for 20 min at 6 K rpm. The supernatant was aspirated and the pellet was dried under N 2. 85 The Lowry assay was performed essentially as previously described (Peterson, 1977). To a tube containing a liposomal protein pellet or 0-50 M g BSA standard, 1 ml H 20 was added and vortexed. To this was added 1 ml of copper sulfate solution (made fresh daily by combining stock solutions of 10% Na 2C0 3, 1% CuS04, 2% potassium tartrate/ 10% SDS (w/v), 0.8 M NaOH/ H 20 at a ratio of 1:1:1:1) and the solution was incubated for 10 min at 20° C. Then 0.5 ml of a fresh solution of 1:5 FC reagent: H 20 was added and quickly vortexed. This was left for 30 min then the OD 7 5 0 was read. The BSA standard gave a linear response in the range of 0-40 Mg. Liposomes were assayed for lipid phosphate as previously described in Chapter Two. The quantity of liposome associated protein was expressed as u g protein per M m o l lipid. 4.2.6 GEL ELECTROPHORESIS 4.2.6.1 Buffers and Solutions Stock Solutions Isoelectric Focusing (IEF) Solutions Solution A, IEF Acrylamide:Bis: 28.38 gm acrylamide and 1.62 gm N,N'-methylene-bis-acrylamide were disolved in 100 ml H 2 0, filtered and stored in a dark bottle at 4°C. The solution was used within 1 mo. Solution B, IEF Overlay Buffer: 1 ml pharmolyte (pH range 3-10) was diluted to 100 ml with 8 M urea and frozen in aliquots at - 2 0 ° C until use. Solution C, IEF Sample Buffer: 4 gm NP40, 1 ml pharmolyte (3-10), 2.5 ml mercaptoethanol, and 0.5 mg bromophenol blue were 86 taken up to 50 ml with 9.5 M urea, and aliquots were stored at -20° C until use. SDS-PAGE Solutions Solution D, SDS 6.8 Buffer: 39.4 gm Tris, 2 gm SDS per liter H 20 to pH 6.8 with HCl. Solution E, SDS 8.8 Buffer: 118.2 gm Tris, 2 gm SDS per 500 ml H20, pH to 8.8 with HCl. Solution F, SDS Acrylamide:Bis: 30 gm acrylamide, 0.8 gm N,N'-methylene-bis-acrylamide were dissolved in 100 ml H20, filtered and stored in a dark bottle at 4 ° C until use (within 2 wk). Solution G, 2-D Sample Equilibration Buffer: 10% glycerol (v/v), 5% mercaptoethanol (v/v), 2% SDS (w/v), in 125 mM Tris HCl pH 6.8. Solution H, 1% Bromophenol Blue: 1% bromophenol blue (w/v) in Solution G. Solution I, SDS Sample Buffer: 0.1 ml Solution H in 2 ml Solution G. Solution J, 2-D Agarose: 1 gm agarose was dissolved in 100 ml Solution G at 95° C and cooled to 4 ° C in 5 ml aliquots. Soulution K, SDS Electrode Buffer: 15.15 gm tris, 72 gm glycine, 5 gm SDS in 5 1 H 20 pH = 8.3). Solution L, 10% ammonium persulfate (w/v), made fresh daily. Gel Solutions IEF Gel Solution: 8.25 gm urea, 0.75 ml pharmolyte (3-10), 2 ml Solution A, were taken up in 6 ml H 20 and degassed. Subsequently, 0.3 ml NP40, 30 M 1 Solution L, and 20 M 1 TEMED were mixed in and the solution was used immediately. SDS Gel Solutions: SDS gel solutions of various compositions were 87 used and are referred to according to their acrylamide concentration. In all cases, the solutions were degassed prior to addition of Solution L and TEMED and then used immediately. Their compositions are shown in Table 4.1. 4.2.6.2 Sample Preparation for Gel Electrophoresis Liposomal protein was delipidated according to Wessel and Flugge (1984) as described above, except that up to 2 mg liposomal protein was used, generating a protein pellet. The pellet, or any other protein solution used, was then prepared for SDS-PAGE by adding a sufficient amount of SDS Sample Buffer (Solution I) to give a protein concentration in the range of 1-2 mg/ml. The protein was dissolved in the buffer and the solution was incubated for 3 min at 95° C, and allowed to cool. In order to prepare the sample for isoelectric focusing, 1 ml of the SDS-PAGE sample (1-2 mg/ml protein) was made up to 8 M urea, then added to 2 ml IEF Sample Buffer (Solution C). The samples were frozen at - 2 0 ° C until use. 4.2.6.3 SDS-PAGE SDS-PAGE analysis was performed essentially as described by Laemmli (1970). 30 ml of 7.5% SDS Gel Solution (see above) was made up and immediately poured into a 1.5 mm x 14 cm x 16 cm mold. 2 ml of H 2 0 saturated butanol was layered on top and the gel was allowed to polymerize for 2 hr. The butanol was then removed, the gel surface was washed several times with H 2 0, and then the mold was filled with fresh 3% gel solution with a 10 well comb in place. After allowing the gel to polymerize for 1 hr, the comb was removed and the gel was placed in a tank containing Solution K in both reservoirs. Samples containing 25-150 Mg protein in SDS Sample Buffer were applied at the cathode end and the gel was run at 30 mA constant current for Table 4.1. The composition of SDS-gel solutions. SOLUTION 2 0 % GEL 7 . 5% GEL 3 % GEL F, SDS AC:BIS 19.5 ml 7.5 ml 1 ml E, SDS 8.8 7.5 ml 7.5 ml D, SDS 6.8 5 ml GLYCEROL 3 ml H 20 15 ml 4 ml L, 1 0 % AMMONIUM 150 M 1 300 M 1 100 M 1 PERSULFATE TEMED 15 M 1 10 M 1 5 MI 89 3 to 5 hr, then removed for staining. 4.2.6.4 Two-Dimensional Gel Electrophoresis 2-D electrophoresis was performed essentially as described by O'Farrell (1975), as modified by Anderson and Anderson (1978). The first dimension employed an isoelectric focusing technique. Fresh IEF gel solution (as described above) was poured into 15 cm tubes with an inside diameter of 3 mm. H 2 0 saturated butanol was layered on top and the gels were allowed to polymerize for about 2 hr. Subsequently, the butanol was removed, the gels were washed several times with H 2 0, and then placed into a tank containing 0.01 M H 3 P0 4 in the anode reservoir and 0.03 M NaOH (which had previously been degassed) in the cathode reservoir. Solution C (10 u 1) was applied to the cathode end of each gel, and then this was overlaid with 10 M 1 Solution B. The gels were prerun at constant voltage, as follows: 200 V, 15 min: 300 V 30 min: 400 V 60 min. The overlaying solutions were then removed, following which 30 u 1 protein solution was applied (20-40 Mg protein; see sample preparation above), and then overlaid with 10 u 1 Solution B. The gels were then run for 18 hr at 400 V constant voltage. At the end of the run, the tube gels were either stained (see below) or prepared for the second dimension, by soaking in 5 ml Solution G for 30 min with gentle agitation. The tube gels were then frozen in Solution G in a dry ice-ethanol bath and stored at -20° C until use. The second dimension utilized a nonlinear acrylamide gradient and was performed as follows. The gradient gels were poured at 4°C by employing a linear gradient maker with 100 ml capacity. Fresh 20% SDS Gel Solution (10.75 ml) was poured into the mixing chamber with a stir bar and the top was plugged with a rubber stopper. The 7.5% Gel Solution (22.5 ml) was poured into the other chamber. The connecting tube was opened and a 1.5 mm x 14 cm x 16 cm gel was poured by gravity with constant stirring. The gel was then 90 removed to room temperature, overlaid with H 20 saturated butanol, and allowed to polymerize for 2 hr. Subsequendy, the butanol was removed, the gel surface was washed several times with H20, and then the mold was filled up to 16 cm with 3% Gel Solution. After polymerization for 1 hr, the gel was placed into a tank containing Solution K. in the anode tank. A tube gel was then thawed out and laid on top of the gradient gel such that contact was made between the tube and the stacking gel. It was then sealed into place with several ml of Solution J which had previously been melted in a 95° C bath. After solidification of the agarose, the cathode reservoir was filled with Solution K and the gel was run at constant 30 mA constant current for 5-7 hr. The gel was then removed for staining as described below. 4.2.6.5 Staining Gels were stained either with Coomassie blue or with silver stain. For Coomassie blue staining, gels were left overnight in 200 ml of Staining Solution (2.5 gm Coomassie Blue R 250 in 1 1 of 99% ethanol, mixed 1:1 with 10% acetic acid immediately before use). Gels were destained in solutions containing 99% ethanol: 5% acetic acid in ratios as follows: 200 ml: 300 ml for 1 hr; 150 ml: 350 ml, for 2 hr, repeated two times; 100 ml:400 ml until completely destained. Gels were stored in 5% acetic acid. Silver staining was done with the Bio Rad Siver staining kit, without modification. 4.2.6.6 Gel Densitometry SDS Gels, stained with either Coomassie blue or Silver stain, were dried down between two sheets of cellophane on an LKB gel dryer, and kept flat until use. Densitometry was performed on a Bio Rad model 620 video densitometer, having an integration mode allowing for quantitation of the peaks. 91 4.2.6.7 Immuno-Autoradiography of Gels ("Western Blot") Antibody Purification Rabbit Anti-Goat IgG (heavy and light chain specific) was obtained from Cappel as a lyophilyzed powder containing 5 mg antibody protein in 72.8 mg protein. It was taken up in 2 ml H 20, diluted to 6 ml with sterile PBS, and then 8 ml of saturated (NH4)3S04 was added dropwise with stirring on ice, and left for 1 hr. The pellet was collected by spinning for 10 min at 10 K rpm in an SS-34 rotor, and taken up in 5 ml of Solution L (40 mM Tris HCl, 20 mM NaCl, pH 7.9). The solution was dialyzed overnight against 1 1 Solution L, and then loaded onto a DEAE Sephacel column (1.5 cm x 20 cm) previously equilibrated with solution L. After five 150 drop fractions were collected the IgG was eluted with a linear gradient of NaCl (20-400 mM) in 20 mM Tris HCl pH 7.9, and 60 drop fractions were collected. Aliquots (50 M 1) of each fraction were subjected to SDS-PAGE (7.5% gel) and run with 25 w l of IgG standard (700 u g/ml). Fractions 34 to 42 were lyophilized with a yield of 100 mg (including salt). The powder was taken up in 2 ml H 2 0 and dialized overnight against 100 ml PBS. Iodination of Rabbit Anti-Goat IgG Iodination of IgG was done using Chloramine-T based on the work of Hunter and Greenwood (1962). One ml of the rabbit anti-goat IgG dialysate was added to 1 mCi of carrier free Na125I (10 jul), and then 25 u 1 of chloramine-T solution (4 mg/ml) was added and incubated for 20 min. The solution was applied to a 1 ml column of AG1X10 resin previously washed with 2 ml of PBS, and the column was centrifuged for 20 min at 2000 g, which removed the free 1251. The eluate was collected 92 and yielded 0.3 mCi 1 2 5I/ml. The solution was made up to 0.1% NaN3 > stored at 4°C, and used within one week. Protein Transfer to Nitrocellulose Electrophoretic transfer of SDS-PAGE proteins was based on the work of Towbin et al., (1979). Proteins were subjected to SDS-PAGE as described above (7.5% gel). At the end of the run, the gel was washed in Buffer M (20 mM Tris Acetate, 2 mM EDTA, 0.01% SDS w/v, pH 7.4) and then tranferred on to nitrocellulose by electrophoresis in a Bio Rad Transblot apparatus at 4 ° C in Buffer M at 4.0 mA constant current overnight. The post transfer gel was stained in Coomassie Blue, ensuring that the transfer was complete. Incubation with IgG The protein impregnated nitrocellulose paper was cut into two lane strips, which were placed in 10 cm petri plates and then incubated in 25 ml of "Quench Buffer" containing 2% Carnation Instant Milk (w/v) in "Immunoblot Buffer" (150 mM NaCl, 10 mM Na3P04, 1 mM EDTA, 0.2% Triton X-100 (w/v), 0.0065% NaN3 (w/v)) for 1 hr at room temperature. The buffer was aspirated off and replaced with 25 ml of Quench Buffer containing 3 mg of either Goat Anti-Human IgG or Goat Anti-Human Albumin IgG (from Cappel) and incubated at room temperature for 1 hr with gende shaking. Subsequendy, the paper was washed five times with Immunoblot Buffer, once with "Urea Buffer" (2 M urea, 0.1 M glycine, 1% Triton X-100 v/v), and then once with Immunoblot Buffer, all for five min each. The paper was then incubated with 60 u 1 1251 Rabbit Anti-Goat IgG (prepared as described above) in 25 ml Quench Buffer for 1 hr. Finally, the paper was washed five times in Immunoblot Buffer, once in 93 Urea Buffer, and then once in Immunoblot Buffer for, five min each. The paper was then allowed to air dry and autoradiography was performed using Kodak XAR-5 X-ray film with a four day exposure. 4.2.7 ASSAYS FOR CLOTTING FACTORS Assays for clotting Factors II, V, IX, and X were done on plasma and plasma derived MLVs by employing Sigma reagents and protocols without modification. All liposome and plasma dilutions were done with 0.145 M NaCl, and all incubations were done at 37° C. 4.2.7.1 Factor II The Factor II assay is based on the method of Hjort et al., (1955). Briefly, 0.1 ml of a test plasma or liposome dilution was added to 0.1 n 1 of Sigma "Factor II and Factor X Deficient Plasma Solution" and then 0.1 ml of Sigma "Russell's Viper Venom in Cephalin Solution" was added and the mixture - was incubated for 30 sec. Subsequently, 0.1 ml of 25 mM CaCl2 was added and the time for a firm clot to form was recorded on a stop watch. The log of the clotting time (sec) was plotted against the log of the Factor II activity (%). The standard plasma dilutions between 1:10 to 1:160 yielded a straight line and was used as a standard curve. 4.2.7.2 Factor V This assay is based on the method of Lewis and Ware (1953). 0.1 ml of a test plasma or liposome dilution was added to 0.1 ml of Sigma "Factor V Deficient Plasma Solution", and then 0.1 ml of Sigma "Thromboplastin Solution" was added and the mixture was incubated for 30 sec. Then, 0.1 ml 25 mM CaCl2 was added and the time required to form a firm clot was recorded on a stop watch. The log of the clotting time (sec) was plotted against the log of the 94 Factor V activity. Test plasma dilutions between 1:10 and 1:160 yielded a straight line and this was used as a standard curve to quantitate Factor V in the samples. 4.2.7.3 Factor IX This assay is based on a measurement of the activated partial thromboplastin time, as described in Biggs (1976). Dilutions of plasma (0.1 ml) or liposome samples were added to 0.1 ml Sigma "Factor IX Deficient Plasma Solution", and then 0.1 ml of Ortho APT reagent was added and the mixture was incubated for 3 min. Subsequently, 0.1 ml of 25 mM CaCl2 was added and the time required to form a firm clot was recorded on a stop watch. The log of the clotting time (sec) was plotted against the log of the Factor IX activity (%). Standard plasma dilutions in the range of 1:10 to 1:160 were yielded a straight line and this was used as a standard curve to quantitate samples. 4.2.7.4 Factor X The assay employed in this work is based on the work of Bachmann et cd., (1958) and employs Russel's viper venom to initiate clotting. Aliquots (0.1 ml) of a dilution of plasma or liposomes was added to Sigma "Factor X and Factor VII Deficient Plasma Solution" and then 0.1 ml of Sigma "Russell's Viper Venom in Cephalin Solution" was added and the mixture was incubated for 30 sec. Then 0.1 ml of 25 mM CaCl2 was added and the time required to form a firm clot was recorded on a stop watch. The log of the clotting time (sec) was plotted against the log of the Factor X activity (%). Serial dilutions of test plasma in the range of 1:20 to 1:640 yielded a straight line which was used as a standard curve to quantitate samples. 95 4.3 RESULTS 4.3.1 PRELIMINARY STUDIES In order to study liposomal plasma protein binding in a way that facilitates comparison to the in vivo results presented in Chapter Three, it is important to choose standard experimental conditions that come as close to the in vivo system as possible. Outdated human plasma was chosen as the basic plasma protein preparation because it is readily available and contains all the major plasma components. The only major drawback with plasma is that the free CaCl2 concentration is only about 50 MM, whereas the normal in vivo concentration is about 2.4 mM (for the plasma phase only). This may bias the results to the extent that protein binding is calcium dependent, and can be expected to be important in the interaction with clotting factors. This point is discussed in more detail below. All experiments were done at 37° C for 30 min, at a lipid concentration of 1 Mmol/ml 100% plasma. This concentration is close to that used for i.v. injections in this work, and thus reflects a typical in vivo case. As the total SA concentration in some experiments is rather high (20% the total lipid or 0.2 mM), it is conceivable that the SA could leave the bilayer and form micelles during the course of the experiment However, using the distribution coefficient of stearic acid as an approximate value for SA (Kd=107 when measured between hexane and water at pH 7.3, Smith and Tanford, 1973), the total free SA in solution is roughly 10-100 pM. Under these conditions, it is highly unlikely that SA micelles could be formed, as the critical micellar concentration of SA is 0.25 mM (Mukerjee and Mysels, 1971). According to Juliano and Lin (1980), plasma protein binding is virtually instantaneous at 37° C, so it can be expected that binding has reached equilibrium by 30 min. The vesicles were isolated from plasma by employing repeated washing and centrifugation (7 cycles). No protein could be detected in the supernatant after the 96 fifth wash, thus only tightly bound protein is isolated by this protocol. It is quite likely that proteins bind liposomes in a specific and reversible fashion, and it is possible that they are important in the in vivo fate of vesicles. Such proteins would be removed by this protocol. However, the study of reversible protein binding in whole plasma is much more involved and was not attempted in this work. It is also conceivable that the protein somehow penetrates through the bilayer during the experiment rather than simply adsorbing to the outer lamellae. However, Juliano and Lin (1980) showed that trypsin cleaved the bound polypeptides within 5 min at concentrations as low as 1 ug/ml. Moreover, pilot studies have shown that MLVs do not leak either 3 H - inulin or 6-carboxyflourescein under similar conditions. Such vesicles even maintain a transmembrane electrochemical gradient (M.B. Bally, personal communication). EPC:cholesterol (1:1) MLVs are stable for at least 6 mo at 4°C without significant leakage of contents or fusion of vesicles. Finally, using magnetic separation techniques it has been shown that vesicles as small as 200 nm bind a similar pattern of plasma proteins (CP. Tilcock, et al.„ submitted 1986). Thus it is highly unlikely that proteins are penetrating the bilayer during the course of the experiment. The measurement of liposome associated protein is complicated by the large amount of lipid present (1000 fold excess, by weight), and so a delipidation step must be included. The use of the Wessel and Flugge (1984) procedure prior to the protein assay yielded reliable results. Pilot studies - showed that this procedure gave 100% recoveries of IgG, BSA, and total plasma protein, in the presence of lipid at the same concentration (1 u mol/ml) as used in the experiments presented in this work. Thus it may be expected that the assay gives reliable results for liposome associated plasma protein as well. However, it is possible that a small amount of protein (<1% total plasma protein) is soluble in the chloroform methanol phase and hence escapes detection. 97 The total amount of protein bound to vesicles of various compositions is quantified in Table 4.2. It is at once clear that the amount of protein binding is strongly dependent on the vesicle composition, as the positively and negatively charged vesicles exhibit a 12-15 fold increase over the neutral vesicles. Figure 4.1 shows SDS-PAGE results for three types of vesicles, reflecting the qualitative differences in protein binding among different types of vesicles. Figure 4.2 shows SDS-PAGE results for similar vesicles employing a 7.5-20% acrylamide gradient which gives higher resolution in the low molecular weight range. Several features are immediately apparent. Of the proteins bound to the neutral vesicles, three very high molecular weight species are most prominent. As there are no corresponding major bands in plasma, they must represent minor plasma components or aggregates which resist the sample preparation conditions. The rest of the pattern resembles the plasma standard in a very general way, indicating a nonspecific binding of most plasma components. However confirmation of this conclusion awaits further characterization of the bands. In comparison to the neutral liposomes, the negatively charged vesicles exhibit a markedly different binding pattern. By far the most conspicuous differences are an increase of a rather smeared band running near the IgG heavy chain of the plasma standard, and the attenuation of the high molecular weight bands. As it was impossible to remove the smearing of the dominant band in one dimension, it was inferred that the band could contain multiple components. The gels of proteins associated with the positively charged vesicles are very similar in appearance to gels obtained for the neutral vesicles, except that there is proportionately less of the high molecular weight components. Of all the vesicle types, positively charged vesicles appear to bind proteins with a distribution most similar to normal plasma. In viewing these gels, it is important to bear in mind that they show relative protein binding differences rather than absolute differences, as the various 98 Table 4.2. Plasma protein binding to MLVs: the effect of surface charge on total protein binding. COMPOSITION PROTEIN BOUND (mol/mol) ( M g/ M mol lipid) 1:1:2 EPS:EPC:Cholesterol 40.3 1:1 EPC: Cholesterol 2.6 1:1 EPC:Cholesterol with 9% SA (w/w) 32.0 99 Fig 4.1. SDS-PAGE analysis of MLV associated protein. Plasma proteins which bound to vesicles were run on 7.5% SDS-PAGE as described by Laemmli (1970), and stained with the Coomassie blue. (1) Protein bound to 1:1 EPC:Cholesterol vesicles. (2) Protein bound to 1:1:2 EPS:EPC:Cholesterol vesicles. (3) Protein bound to 1:1 EPC:Cholesterol, 5% SA (w/w) vesicles (P) Normal plasma standard. (M) Sigma Molecular Weight Standards. 29>- m 100 Fig. 4.2 SDS-PAGE analysis of MLV associated protein. Vesicle bound proteins were run on a 7.5%- 20% gradient, and stained with the Bio-Rad silver staining kit. (1) Protein bound to 1:1 EPC: Cholesterol vesicles; (2) Protein bound to 1:1 EPC: Cholesterol with 5% SA (w/w) vesicles; (3) Protein bound to 1:1:2 EPS:EPC:Cholesterol vesicles; (P) Plasma standard; (M) Sigma molecular weight standards. 101 liposome types differ greatly in the amount of total protein they bind. 4.3.2 PARTIAL CHARACTERIZATION OF PROTEINS 4.3.2.1 Symbolic Designation of Proteins In order to understand the complex interaction of liposomes with plasma, it is useful to characterize and identify the individual bound proteins. Consequently, preliminary studies were done with this goal in mind. Due to the enormous number of proteins involved, a symbolic designation system was devised to facilitate discussion. It is illustrated in Figure 4.3 for a typical SDS-PAGE lane. The letters denote the general regions and the numbers refer to the individual proteins in that region. The major protein binding to the negatively charged vesicles (as in Figures 4.1-4.2) is not shown in this Figure, but has been designated F-PS. It should be noted that only the major proteins have been given symbols. There are many more minor bands that are not dealt with, particularly in regions B,G,H and F. In fact the resolution of 1-D SDS-PAGE is far too low to distinguish most of the peaks, as will be seen below. 4.3.2.2 2-D Electrophoresis Although it is a relatively simple procedure to demonstrate the presence of a given protein using Western blot analysis, the sheer number of liposome bound proteins, as well as the enormous number of possible plasma proteins, necessitate that an initial screening method be employed. 2-D electrophoresis, performed according to Anderson and Anderson, (1978), is the technique of choice for the global detection of plasma proteins in complex mixtures. Figure 4.4 is a reproduction from Anderson and Anderson (1977), showing the 2-D map position of some of the major plasma proteins. In a later work (Anderson et al., 1984), a table has been published containing the 2-D map position of 102 Fig. 4.3. Designation of major liposome bound proteins found on SDS-PAGE analysis. 13 — 103 Fig. 4.4. 2-D gel electrophoresis map of human plasma, copied from Anderson and Anderson (1977). This Figure indicates positions of proteins found on 2-D gels which have been identified by either comigration of standards or using immunological techniques. 104 about 40 plasma proteins. By relying on this table, it is possible to tentatively identify many of the major proteins bound to vesicles, as a preliminary screening for Western blot analysis. Figure 4.5 is a 2-D gel of normal plasma. Albumin is by far the most prominent protein and serves as a reference point to compare smaller bands. Comparison with the map in Figure 4.4 allows for a tentative identification of the other major proteins. It should be noted that these gels were done with a 7.5%-20% gradient rather than the 10-20% gradient used by Anderson et al., (1978), in order to allow the higher molecular weight bands to move into the gel. This does not interfere with protein assignment, however. Mention should also be made of the rather large artifacts located at the bottom and edges of the gel. These are due to the ampholines used in the preceeding IEF step which are very difficult to remove. These are straightforward to distinguish from genuine protein bands. In Figure 4.6 a 2-D gel is shown of proteins bound to EPC:cholesterol 1:1 vesicles. It has been silver stained to show the minor bands. The very high resolution of the technique is demonstrated by the separation of all the minor components into distinct regions. The major band maps out in the same position as albumin, as expected. Unfortunately, it appears that the high MW species (designated as Al-3) do not enter the IEF dimension, thus precluding their characterization. The grid line pattern in the background is a 2-D artifact which becomes evident during the silver staining procedure. The 2-D protein binding pattern for 1:1:2 EPS:EPC:Cholesterol vesicles is shown in Figure 4.7. This Figure clearly shows that the F-PS band on the negatively charged vesicles (shown in Figure 4.1) exhibits a charge heterogeneity, and possibly a molecular weight heterogeneity as well. The pattern indicates that it is primarily one species, however. It also demonstrates at a glance that the 105 Fig. 4.5. 2-D gel electrophoresis of human plasma, according to Anderson and Anderson (1977), using a 7.5%-20% acrylamide gradient. Stained with Coomassie Blue. The positions of the molecular weight standards (shown at the left in Kdal), are estimates obtained from a similar gel. 2 9 » 24-106 Fig^ 4.6. 2-D gel electrophoresis of plasma proteins bound to MLVs composed of M EPC: Cholesterol. Stained with the Bio-Rad Silver Staining Kit. •« A C I D I C B A S I C » 107 Fig. 4.7. 2-D gel electrophoresis of plasma proteins bound to MLVs composed of 1:1:2 EPS:EPC:Cholesterol. Stained with the Coomassie Blue. 108 protein is not IgG, although it has a similar MW. The protein above it maps into the albumin region. Figure 4.8 is a similar 2-D gel for 1:1 EPC: Cholesterol vesicles containing 5% SA. Although albumin (E) is by far the most prominent band, this gel shows the enormous number of minor components bound to this vesicle composition. 4.3.2.3 Tentative Protein Identification The SDS molecular weights of each major protein were determined from 1-D SDS-PAGE gels and are given in Table 4.3. By assiduous comparison of the 2-D gels with the map and Table of Anderson et al., (1977, 1984) the tentative identity of many of the proteins was assigned. It should be noted that F-PS could not be identified from these sources and thus is a relatively minor plasma component. Also, the clotting and complement factors are not given in the table but are likely candidates for binding. Of the clotting Factors, II, IX, and X are likely candidates for binding, and have peptide molecular weights of 38, 55.4, and 39 K, respectively (Davie et al., 1979). Factor Va also has a fragment of 50 K which binds lipid (Nelsestuen, personal communication). These peptides would be found in the F and G regions. 4.3.2.4 Identification of Proteins Using "Western" Blotting Given the tentative identities of the liposome associated proteins in Table 4.3, the next experiments were undertaken to positively identify some of the major bands. The Western blot technique (Towbin et al., 1979) allows for positive identification of a protein on a gel, provided that the antibody to the protein is available. Of the proteins in Table 4.3, only antibodies to albumin and IgG were readily available and so these two were chosen for an initial experiment. The results are shown in Figure 4.9. It can be seen that the major 109 Fig. 4.8 2-D gel electrophoresis of plasma proteins bound to MLVs composed of 1:1 EPC: Cholesterol containing 5% SA w/w. Stained with Coomassie Blue. 9 7 » 6 6> 110 Table 4.3 MLV bound plasma proteins were subjected to SDS-PAGE (7.5% gel) and the SDS molecular weights were estimated by co-running Sigma molecular weight standards. By comparison with the SDS molecular weights and 2-D map positions from Anderson et al. (1984), some possible identities of the major bands are indicated in column 3. Protein Designation Measured MW Possible Protein Identity, with SDS-MW (Kdal) Al (245)* A2 (240)* A3 (240)* A3 (222)* B 152 a-2-Macroglobulin, 153.3 CI 122 Ceruloplasmin, 124.3 C2 113 a-Antitrypsin dimer, 108$ C3 99 Plasminogen, 99.4 C4 97 DI 84 D2 80 Transferrin, 77.5 E 66 Albumin, 65.9 F l 55 Fibrinogen B chain, 54.8 F2 54 Gc-Globulin, 53.7 a-Antitrypsin, 53.2 F3 54 IgG, 51.7 (cont.) I l l Tab. 4.3 cont. F-PS 51.6 Factor V, 50f G l 51 Extended fibrinogen g chain, 51.7 G2 49 Fibrinogen g chain, 49.4 G3 39 Haptoglobulin B chain, 40.3 H 34 Apo E, 32.2 11 26 IgG k and 1 chains, 26 12 25 Apo Al , 24.3 13 23 Retinol Binding protein, 21.9 Haptoglobin a2, 18.5 (*) MW could not be determined on a 7.5% gel. However Juliano and Lin (1980) determined the molecular weights of these bands using a different system, and are indicated in parentheses. (+.) SDS-MW was not given, so the true MW is shown instead, (•f) There was no 2D map position for this peptide available. 112 Fig. 4.9. Western blot analysis of plasma proteins bound to: (1) 1:1 EPC:Cholesterol vesicles containing 12% SA; (2) 1:1 EPS:Cholesterol vesicles. Gel A: SDS-PAGE followed by Coomassie blue staining. Gel B: SDS-PAGE followed by western blot analysis using goat anti-human albumin, then 125I-rabbit anti-goat IgG, followed by autoradiography for 4 days. Gel C: SDS-PAGE followed by western blot analysis, using goat anti-human IgG, then 1 2 51-rabbit anti-goat IgG, followed by autoradiograghy for 4 days. A B C 1 2 1 2 1 2 113 band E is albumin, as expected. The IgG heavy chain is also present, but only in trace amounts. It is very difficult to quantitate the amount of protein present. However, it can be seen that the IgG binding is roughly similar for both positively and negatively charged vesicles and so is basically independent of lipid composition. Moreover, the traces of IgG heavy chain present do not account for the large F-PS band, as predicted from the 2-D results. This approach could be used for identifying most of the rest of the major plasma proteins, if the corresponding antibodies were available. 4.3.3 EFFECT OF SURFACE CHARGE ON PROTEIN BINDING 4.3.3.1 Total Protein Binding The preliminary studies described above made it clear that the vesicle surface charge has a large influence on plasma protein binding. It was of interest to characterize this effect more fully. Figure 4.10 shows the effect of varying the surface charge on protein binding. The Figure indicates that the binding minimum is near the neutral point. That this is in fact the case was confirmed by a similar experiment using very small surface charge increments around the zero point (data not shown). As the surface charge is increased at either end of the graph the slope decreases, indicating that either protein saturation of the liposome surface is occurring or that the surface charge effect is approaching a maximum as the charged lipid species approach 100%. 4.3.3.2 Relative Protein Binding The vesicle derived protein from vesicles used in Figure 4.10 was subjected to SDS-PAGE, and the results are shown in Figures 4.11 and 4.12. The gel in Figure 4.11 shows the qualitative changes in protein binding when increasing EPS is added to the vesicle composition. The progressive switch over 114 Fig. 4.10. The effect of vesicle surface charge on the total binding of plasma proteins to MLVs. The cholesterohphospholipid ratio was equimolar in all vesicles. Error bars indicate standard error, with n=3. 3d so AO 3a 2a i a XPS C m o l / m o l p h o s p h o l i p i cO I C 2Q 3 d A-a SO XSA C m o l / m o l p h o o p h o 1 1 p 1 d ) 115 Fig. 4.11. SDS-PAGE analysis of MLV protein binding as a function of vesicle surface charge. Stained with Coomassie Blue. (1) 1:1:2 EPS:EPC:Cholesterol; (2) 3:7:10 EPS:EPC:Cholesterol; (3) 2:8:10 EPS:EPC:Cholesterol; (4) 1:9:10 EPS:EPC:Cholesterol; (5) 1:19:20 EPS:EPC:Cholesterol; (P) Normal plasma standard; (M) Sigma Molecular Weight Standards. M M l 2 3 4 5 P M M 116 Fig. 4.12. SDS-PAGE analysis of MLV protein binding as a function of vesicle surface charge. Stained with Coomassie blue. Vesicles were composed of 1:1 EPC: Cholesterol containing: (1) 0% SA w/w; (2) 3% SA w/w; (3) 6% SA w/w; (4) 9% SA w/w; (5) 12% SA w/w. (P) Plasma protein standard. (M) Sigma Molecular Weight Standards. 117 from albumin to F-PS is evident, as well as the relative reduction in of many of the minor components. The gel in Figure 4.12 shows the effect of increasing the surface charge in the positive direction. Here the progressive loss of the high molecular weight (Al-3) components is evident, relative to the total protein binding. However it can not be determined from this experiment whether this reflects an absolute decrease in the Al-3 components as the amount of protein is increasing (see Figure 4.10). 4.3.3.3 Densitometric Results After demonstrating the influence of surface charge on the total protein binding quantitatively as well as qualitatively, gel densitometry was employed in order to quantitate the effect on the individual peaks. Some representative scans are shown in Figures 4.13 and 4.14. The molecular weights of the major peaks are shown at the tops of the peaks and correspond with the protein designations listed in Table 4.3. The identities of some of the major plasma proteins are shown on the peaks of the plasma standard. Because the number of vesicle associated proteins is greater than the resolution limits of the 1-D SDS-PAGE system, in many cases it is impossible to obtain baseline separations required for accurate peak integration. Consequently, it was neccesary to set the integration limits in such a way as to include multiple peaks in one region. Unfortunately, for many cases this precludes the acquisition of reliable data. Nevertheless, some informative data can be obtained. The integration limits used are shown below the scans, and the region designations they contain correspond to those which have been used in Table 4.3. Clearly, region E contains only one major peak (albumin), and region F in the PS direction contains mostly F-PS. In the SA direction region F contains several proteins. Region D contains only two major proteins, and the 80 K peak (putative transferrin) greatly predominates. Regions C and I, although containing three major bands each, have been followed 118 Fig 4.13. Densitometric scans of MLV bound plasma proteins after SDS-PAGE and Coomassie blue staining. The numbers on the major peaks give the SDS molecular weights. The letters under the scans denote the integration bands used to quantitate the peaks as described in the text (A) Protein bound to 1:1:2 EPS:EPC:Cholesterol vesicles; (B) Protein bound to 1:1 EPC-.Cholesterol vesicles. 2 3 66 I A I B| C JD|E [ F | G | H | I | 119 Fig. 4.14 Densitometric scans of plasma proteins subsequent to SDS-PAGE and Coomassie Blue staining. (A) Proteins bound to 1:1 EPC:Cholesterol containing 9% SA (w/w). The numbers above the peaks give the SDS molecular weights and the letters below the scans denote the integral bands as described in the text (B) Plasma protein standard. The major plasma protein peaks have been denoted as follows: FIB= Fibronectin; MAC= ct-2-Macroglobulin; C3= Complement factor 3; TF=Transferrin; ALB=Albumin; IGG=Immunoglobulin g chains; HP=Haptoglobin; Igl=Immunoglobulin 1 and k chains; A - l = Apo Al . 120 because one band predominates in each (CI or 13, resp.). The data from the other regions could not be used as there were either too many peaks or the intensity was not great enough. The results of the integrations are shown in Figures 4.15-4.17, after normalization for the total protein as determined in Figure 4.10. Several conclusions are immediately evident. In the SA direction it appears that the quantitative increase in protein binding is reflected as an increase for all proteins examined. Unfortunately, there was not enough A region protein to perform an accurate integration for SA containing vesicles but this region might be expected to be an exception. In the PS direction a more interesting result is found. Here it appears that F-PS is competing off all the other proteins examined, except perhaps band C (putative transferrin), which still shows only a marginal increase relative to the SA containing vesicles. The competition is also apparent in region I which mostly contains putative Apo A l (possible HDL binding or Apo Al insertion). This competition is all the more interesting considering that protein binding appears to be essentially instantaneous and irreversible (Juliano and Lin, 1980). Thus the F-PS peptide must compete with the other proteins for the binding sites. These results also point out the dangers of extrapolating binding studies done with purified proteins to the competitive in vivo conditions. 4.3.4 STUDIES WITH DIFFERENT PLASMA PREPARATIONS As was discussed in the Introduction, negatively charged phospholipids are very important cofactors for several steps of the clotting cascade. Thus it would be expected that clotting factors should bind to PS containing vesicles. However, calcium is a required component in the interaction of clotting factors with acidic phospholipids, and citrate treated human plasma contains only approximately 50 u M calcium. Thus it is of interest to investigate the influence of calcium on plasma protein binding to negatively 121 Fig. 4.15. Effect of surface charge on plasma protein binding to MLVs. Densitometric scans of SDS-PAGE gels (Figs. 4.11-12) were integrated over various regions as indicated in (Figs. 4.13-14). The relative integrals were normalized for the total liposomal protein as determined in Fig. 4.10. The x axis gives the liposomal composition as %PS or %SA (percent phospholipid mol/mol in systems containing equimolar cholesterol) The 0 point PC is EPC: Cholesterol 1:1. (A) Integrated over Band E (B) Integrated over Band F. E f f e c t o f S u r f a c e C h a r g e o n t h e B i n d i n g o f B a n d E 50 30 20 10 5 O 13 26 39 %PS P C %SA E f f e c t o f S u r f a c e C h a r g e o n t h e B i n d i n g o f B a n d F 20-r • ~ ~ ™ ~" 1 8 - -IB- -122 Fig. 4.16. Integrals were determined as in Figure 4.15 for: (A) Band C; (B) Band D. E f f e c t o f S u r f a c e C h a r g e o n t h e B i n d i n g o f B a n d C I O T • 50 30 20 10 5 0 13 26 39 %PS PC %SA E f f e c t o f S u r f a c e C h a r g e o n t h e B i n d i n g o f B a n d D 50 30 20 10 5 0 13 26 38 %PS PC %SA__ Fig. 4.17. Integrals were determined as in Figure 4.16 for Band I. E f f e c t of S u r f a c e C h a r g e on the B ind ing of B a n d I 20-1 : — — IB- • fl- -%PS PC %SA 124 charged phospholipids. Of course, such a study is complicated by the fact that calcium also initiates clotting. Nonetheless, initial studies were attempted to investigate the binding of plasma proteins in the presence or absence of calcium. Various amounts of calcium were added to plasma and incubated overnight as described in the Methods. After removing any resultant clot the serum was heated to 37° C and incubated with 1:1:2 EPS: EPC: Cholesterol vesicles at a concentration of 1 n mol/ml. When serum was made with 4 mM calcium, addition of PS vesicles caused the entire sample to form one large clot within 30 min. Use of higher levels of calcium progressively decreased the PS induced clot size until at about 40 mM CaCl2 no clot was formed. Using 40 mM CaCl 2- generated serum, or thrombin-generated serum prepared as in the Methods section, the binding of serum proteins to PS containing vesicles was determined. The results are shown in Table 4.4. It should be noted that the control plasma protein binding value (80.4 n g protein/ y mol lipid) found with this plasma pool was two fold higher than the value reported in Table 4.2 (40.3 y g protein/ ymol lipid), which employed a different pool. It can be seen* that there is much less protein binding in the calcium-generated serum than in normal plasma. This could be due to several causes: 1) The calcium is competing with the protein directiy by binding the PS; 2) clotting factors represent a large fraction of PS bound protein, which is removed by clotting; 3) non clotting factors, which bind PS vesicles, are removed or inactivated during the clotting process. It seems likely that several of these causes may contribute to the result. The value obtained with thrombin-generated serum was in between the calcium value and the control. Thrombin induces Fibrinolysis directly, without activating clotting Factors V, VII, IX, X and XII (See Figure 1.3). However, a portion of the clotting Factors as well as other proteins are co-precipitated with the fibrin clot, and thus thrombin-generated serum is missing many clotting factors. However, it is also. missing calcium, thus indicating that the calcium competition 125 Table 4.4. Quantitation of protein binding to 1:1:2 EPS:EPC:Cholesterol vesicles after incubation with plasma or serum prepared as described in Methods. Plasma Preparation Protein Binding ( u g protein/ u mol lipid) Normal Plasma 80.4 'Thrombin Generated' Serum 17.3 [CaCl2]=50 mM Serum 1.1 Table 4.5. Quantitation of protein binding to 1:1:2 EPS:EPC:Cholesterol vesicles after incubation with dialyzed plasma or serum prepared as described in Methods. Plasma Preparation Protein Binding ( M g protein/ u mol lipid) Normal Plasma 80.4 Dialyzed Plasma 17.3 Dialyzed Serum 7.9 126 accounts for only part of the observed decrease, supporting the possibility that several of the causes noted above are involved. The results given in Table 4.4 are difficult to interpret because several variables are being changed simultaneously. In order to remove the the calcium variable, the following experiment was done. Serum was generated by adding calcium to plasma to a concentration of 30 mM, which is known to activate the entire clotting cascade. Subsequendy the serum or normal plasma was dialyzed against HBS and then calcium was added back to 50 u M (near the control plasma value) as described in the Methods section. The effect of this process on protein binding to PS containing vesicles is shown in Table 4.5. Dialysis of plasma clearly decreases the amount of protein binding to PS vesicles, even without prior clotting. This indicates that one or more small molecular weight species (less than 14 Kdal) increase the binding of protein to PS containing vesicles. The species involved could be anything in plasma except calcium, as the calcium was added back. The Table also shows that PS vesicles bind less protein from dialyzed serum than dialyzed plasma indicating that clotting does remove or inactivate PS binding proteins. As was discussed in the Introduction, activation of the clotting cascade generates several types of carboxy-glutamic acid containing peptides which are involved in calcium bridged PS linkages in the profactors. Therefore, it might be expected that analysis of PS bound protein from dialyzed serum would show an increase in small molecular weight fragments. SDS-PAGE was performed on proteins from dialyzed plasma or serum and the result is shown in Figure 4.18. Indeed, a large number of small molecular weight fragments are found in serum that were not found in plasma. Additionally, there is a decrease in protein B (putative a-2-microglobulin), and some A region proteins. However, most interesting is the enormous attenuation of F-PS in both dialyzed serum and plasma, which both show similar amounts of F-PS. This result, when taken with the changes observed in the total protein binding (Table 4.5) 127 Fig. 4.18. SDS-PAGE analysis of proteins bound to 1:1:2 EPS:EPC:Cholesterol, followed by Silver staining. (P) After incubation with dialyzed plasma (as in Methods). (S) After incubadon with dialyzed serum. (M) Sigma molecular weight standards. M P S M M 128 suggest that dialysis of plasma decreases the binding of F-PS, while activation of clotting primarily affects binding of other proteins. Thus the activation of the clotting cascade does not appear to attenuate the binding of F-PS relative to control. 4.3.5 CLOTTING ASSAYS As the previous experiments do not a allow definitive answer to be given as to the identity of F-PS, it was of interest to determine the clotting factor activity directiy, to allow comparison with the previous results, and determine the influence of PS containing vesicles on plasma. The results are given in Table 4.6. As can be seen, only Factor V and Factor X activities are significantiy affected. Assuming that the values obtained indicate the total Factor binding, then the binding of these proteins can only account for about 0.1% of the total protein bound to PS vesicles. Thus, under these conditions, the binding of active clotting factors is quantitatively insignificant. Conversely, Factor V and Factor X activities are greatiy reduced in the remaining plasma. It is quite likely that a large portion of the clotting factors were removed during the vesicle washing procedure, but this possibility was not checked. It is also possible that F-PS represents a catalytically inactive peptide from a clotting factor. Indeed, the 50 K peptide from Factor V is a likely candidate (G. Nelsestuen, personal communication). This possibility could be tested if an antibody to the 50 K fragment were available. In any case, it is likely that PS containing vesicles bind clotting factors in vivo (resulting in a giant clot as observed above). 4.4 DISCUSSION The primary results of this chapter concern the binding of plasma proteins to liposomes of various compositions. As discussed in the Introduction, there are already many reports in the literature (Morrisett et al., 1977; Bonte and Juliano, 1986) on the liposome plasma protein interactions and thus this work may be considered as an 129 Table 4.6. Quantitation of clotting factor activity after incubation of 1:1:2 EPS:EPC:Cholesterol vesicles in plasma. Clotting factors were assayed in the plasma derived liposome preparation and the concentration of each factor was estimated as described in the methods section. Factor activity in the remaining plasma was also estimated and expressed as % control plasma. Clotting Factor % Control Plasma Liposomal Binding Activity (ng/u mol lipid) II 100% Not detected V 71% 0.7 IX 100% Not detected X 53% 57 130 extension of the previous work. However, due to the complexity of the problem, this work is being presented as an exploratory study only. In order to facilitate discussion, plasma protein binding to the neutral (EPC: Cholesterol, 1:1) vesicles will be considered first, followed by the charged species. The quantity of protein bound to neutral vesicles was relatively low, about 2.6 Mg/ju mol lipid. This compares reasonably well with the value reported by Juliano and Lin (1980), who gave a value of 5.0 M g protein/Mmol lipid for vesicles composed of EPC:Cholesterol (1.12:0.85, mol/mol). Moreover, SDS-PAGE analysis of the bound protein yielded a very similar pattern qualitatively. A comparison of the protein designation system used in this work (Figure 4.3) with the one employed by Juliano and Lin (1980) will show an exact correspondence between the proteins found in the two reports. However, in contrast to most of the earlier studies, the work presented here employed a 2-D gel electrophoresis technique resolve the multitude of plasma proteins. Careful examination of the silver stained 2-D gel shows at least 50 proteins visible with silver stain. In addition, the use of 2-D electrophoresis allows a finer comparison to whole plasma, and particularly the 2-D map positions reported by Anderson et al., (1984). This screening procedure is ideal as a preliminary step in the identification of the proteins, by greatly reducing the number of possible plasma proteins for each band. This also allows for a tentative identity to be given to each band, and clearly sets the stage for positive identification of the peaks using Western Blot analysis. Unfortunately, antibodies to most of these proteins were not commercially available and thus it will be necessary to raise the antibodies prior to the Western analysis. This was not feasible within the context of this work. However, as antibodies were available for albumin and IgG these proteins were identified as a prototype for further work. In examining the protein binding patterns of EPC: Cholesterol (1:1) vesicles with the total plasma protein binding pattern, it is apparent that the vesicles exhibit a 131 relatively random protein binding pattern, in that the tentative identity and relative intensity of most of the bands exactiy reflect the total plasma binding pattern. This leads to the "fly paper" hypothesis for plasma protein binding to neutral vesicles, where the vesicles act as a sticky surface for plasma proteins. As the binding is essentially irreversible (proteins cannot be extracted by high salt, chelating agents or chaotropic agents (Juliano and Lin 1980), and involves naturally hydrophilic (plasma soluble) proteins, it is possible that some of the proteins denature upon absorption to the vesicles, thereby exposing hydrophobic residues. Although this type of analysis appears to be valid for most of the binding pattern, it is noteworthy that some plasma proteins do not appear to be bound to the degree which they are present in plasma. Thus IgG is present on the vesicle surface in only trace amounts whereas it is a major plasma protein. The same is the case with, fibronectin; the gels indicate that only trace amounts of protein with a similar MW bind to vesicles. These results are confirmed by an examination of the work of Juliano and Lin (1980), which shows only traces of Fibronectin binding inspite of the relatively large amount found in plasma. Although it is not clear why these proteins do not adhere to the same extent as albumin or (putative) tranferrin, it is interesting in that IgG and fibronectin are considered to be opsins, and are reputed to be major factors in the clearance of foreign particles (see Introduction). It could be hypothesized that these proteins bind primarily in a reversible manner, such that they are removed during the washing protocol. However, the work of Rossi and Wallace (1983) with puriFied Fibronectin indicated that liposomal Fibronectin binding is irreversible. Thus it appears that the affinity of Fibronectin for vesicles is lower than other plasma proteins (such as albumin). Moreover, the work of Weissmann et al., (1974), has shown that the binding of IgG to non-antigenic vesicles occurs primarily with denatured IgG, and is also irreversible. However, denatured IgG is presumably present in only trace amounts in vivo. It is possible that opsinization process is relatively unimportant in the 132 clearance of simple, non-antigenic liposomes. In this regard the work of Hoekstra et al., (1978,1979) is relevant, as they have shown that the addition of whole serum to liposomes actually inhibits the uptake of vesicles by macrophages in vitro. It is worth noting that most of the proteins which bind neutral vesicles have a negative charge at pH 7.5, and thus the vesicles would acquire a net negative charge. This result is confirmed by the work of Black and Gregoriadis (1976), who demonstrated (using an electrophoretic technique) that initially neutral liposomes migrate towards the anode upon incubation in plasma and even after 6 washings. As it is well known that negatively charged surfaces induce platelet aggregation, it would be very interesting to determine the effect of liposomes on platelet aggregation. Most of the in vitro studies have overlooked this possibly important interaction, through using (platelet depleted) plasma or serum. It was of interest to further characterize the influence of surface charge on liposomal plasma protein binding. This effect was examined as a function of composition and it was determined that the protein binding minimum is found with neutral liposomes. Increasing the charge in either direction results in a rapid increase in protein binding which eventually levels off as the charge is increased even further. With the SA containing vesicles, the results can be compared with those of Juliano and Lin (1980), who gave the value of 31 ug protein/ M mol lipid for vesicles composed of EPC:Cholesterol (1.12:0.85) containing 7.5% SA (w/w). Interpolating the graph in Figure 4.10 gives 30 u g protein/ umol lipid for vesicles composed of EPC:Cholesterol (1:1) containing 7.5% SA (w/w) which agrees remarkably well. For the PS containing vesicles no comparison is possible as they only went up to 10% PS (mol/mol). Although the incorporation of a surface charge into a vesicle results in a similar increase in total protein binding for both positively and negatively charged vesicles, the protein binding patterns are very different (as shown in Figures 4.7-4.8). 133 In the case of the SA containing vesicles, the major protein bound is albumin. As albumin has an isoelectric point (pi) of 4.4 (Putnam 1975) with a corresponding -18 unit charge at pH 7, the gel in Figure 4.8 indicates that plasma protein binding to SA containing vesicles will decrease their net positive charge. This result is fully consistent with the findings of Black and Gregoriadis (1976), who demonstrated that SA containing vesicles actually acquire a net negative charge as a result of plasma protein binding. The gel also shows that, relative to the neutral liposomes, SA containing vesicles bind a large number of minor components with molecular weights in the range of 65-30 K. However, the overall pattern is quite similar to the neutral vesicles and thus they exhibit a relatively random binding of plasma protein. In contrast to the neutral and SA containing vesicles, the vesicles containing large amounts of PS exhibit a binding pattern which greatiy deviates from the pattern produced by total plasma. In particular, the 50.6 K peptide designated F-PS predominates over all other plasma proteins. It is apparent that this protein has a great affinity for PS containing vesicles, as it progressively inhibits the relatively nonspecific binding of the other plasma proteins in a PS dependent fashion. Also, it can readily be seen from Figure 4.7 that F-PS has at least 7 major charge isomers, which could • represent different numbers of sialic acid residues on the peptide (Anderson et al., 1984). Comparison of the protein binding pattern of PS containing vesicles with that of total plasma. control shows that F-PS can only be a relatively minor plasma component as there are no major plasma peptides with a MW of 51.6 K and a pi of about 4.5 (for the center charge isomer). However, using the densitometric results in combination with the protein assays a lower limit can be set for the plasma concentration of F-PS of 18 yg/ml plasma (assuming 100% of the available protein is bound to the vesicles). These parameters greatly reduce the number of plasma peptides which could be F-PS. Because of the well documented role of PS in the clotting cascade (see Introduction) it was hypothesized that F-PS was a peptide 134 from one of the clotting factors. However, evidence for this has been difficult to obtain because F-PS has no clotting factor activity when bound to HBS washed MLVs. Moreover, it appears that activation of the clotting cascade does not greatly reduce F-PS binding to vesicles. Also, the calculated concentration of F-PS is almost two fold higher than the total amount of Factor V in plasma (approximately 10 ug/ml E. Guinto, personal communication). Other possible clotting factors have similar plasma concentrations (for example Factor X has a plasma concentration of approximately 11 ug/ml (Dieijen et al., 1981)). Thus it seems rather unlikely that F-PS is a clotting factor, although the values are close enough to warrant the possiblity. It has been suggested that F-PS could be the 50 K peptide from clotting Factor V (G. Nelestuen, personal communication), as it resists the washing protocol yet has no enzymatic activity in isolation. However, it will be necessary to obtain an antibody to this peptide in order to demonstrate this. It would also be interesting to include 2 mM CaCl2 in the HBS used for isolating vesicles from plasma and observe the effect it has on the protein recovery both quantitatively and qualitatively. It might be expected that more clotting factors would be found under these conditions. In summary, it is clear that a great deal more work will need to be done in this area before definitive answers can be given about the role of plasma proteins in the fate of liposomes in vivo. In particular, it will be necessary to positively identify each of the major proteins (via the Western blot technique), and determine the effect these have on macrophage uptake in vitro, platelet aggregation, and complement activation, all as a function of vesicle composition, size and dose. In doing this, it will be necessary to employ several different in vitro models in addition to the conventional plasma model. For example, it would be very interesting to employ a platelet model and look at the effects of liposomal plasma protein binding on possible vesicle induced platelet aggregation, as a function of [CaCl2] in the mM range. These experiments were beyond the scope of this thesis. 5. CONCLUSIONS The studies presented in this thesis contribute to the research and development of liposomes as drug delivery vehicles. Through an analysis of the existing literature, it became evident that the methods for probing the fate of i.v. injected vesicles were inadaquate to convenientiy and accurately assay the blood clearance and tissue distributions of low doses of lipid. To surmount these difficulties, the synthesis and use of a new vesicle probe (125ITI) is described in the first part of this work. It is shown that 125ITI effectively meets the necessary criteria for a vesicle probe and is superior to all other labels employed in the literature in terms of accuracy, cost, convenience and specific activity. As the use of 125ITI allows for studies employing low, clinically relevant lipid doses, the work presented in Chapter Three investigates the blood clearance and tissue distributions of liposomes following their injection i.v. In doing so several additional techniques have been employed which have not been used in any other in vivo studies in the literature. First, the use of extrusion technology for the convenient production of homogeneous, unilamellar vesicles of various sizes has been applied to the in vivo studies. Second, the development of a cannulation technique for the measurement of the blood clearance times of vesicles has been described which enables time points as short as 1 min post injection to be obtained. This allows precise information to be obtained about the early clearance kinetics of vesicles, effectively extending the time ranges reported in the literature. Thus the combination of 125ITI labeling, extrusion technology, and the cannulation method allow for studies which improved in the accuracy and range of application as compared to currendy employed methodology. After creating this solid technical basis to work from, it was of interest to study in vivo fate of liposomes as a function of vesicle size, dose, composition and time post injection. Although the influence of these parameters have been studied 135 136 many times in previous literature, the development of the new techniques described above warranted their re-investigation. It was shown that the clearance of liposomes was bimodal particularly at low doses, with an alpha phase half life on the order of 20 min followed by a beta phase half life of at least 3 hr. It was found that the relative amount of vesicles cleared in the first phase was strongly dependent on vesicle dose and size. Thus increasing the dose appears to saturate the first phase clearance mechanism, leaving a greater proportion of vesicles for the second phase clearance, and thus increasing the overall blood residence time. It is noteworthy that most of the work presented in the literature employed doses which completely saturate this first phase clearance. It is possible that the first phase clearance represents vesicles which are bound direcdy by the MPS, and saturation of the first phase translates to a saturation of the MPS binding capacity. The percentage of vesicles which are taken up in the first phase is also sharply decreased by decreasing the size of the vesicles, while keeping the injected trap volume constant As decreasing the size corresponds to injecting larger numbers of vesicles, it is possible that the saturation of the MPS depends primarily on the number of particles injected, rather than the total particle volume. However, further experiments need to be done to demonstrate this fully, as it seems unlikely that this explanation could account for the large difference between VETi00 and VET 3 0 systems, which have sizes of 110 nm and 55 nm respectively, (M. Bally, personal communication). In the case of these systems, other factors are probably involved. The tissue distribution data shows that smaller vesicles deposit in the carcass to a larger extent, which is consistent with the longer blood residence time of these systems. The data also show that smaller systems are less leaky than large systems in vivo. Further, the leakage observed in vivo cannot be explained by the rates observed in vitro using EDTA-plasma. Thus there are apparently other mechanisms of liposome lysis operating in vivo, possibly in a calcium dependent fashion. 137 The data also show that the in vivo behavior of VET 3 0 systems is indistinguishable from their sonicated SUV counterparts. This is an important finding because technically VET3 0s are far easier to prepare, and exhibit much higher trapping efficiencies (50%, Hope et al., 1986). The studies done on the tissue distributions of FATMLVs having a surface charge yielded several new findings. First, the use of SA containing FATMLVs gave the largest percentage of vesicles yet reported in the lungs (15%), at lower doses. The rapid decay of 125ITI from the lungs is suggests that the MLVs are not removed from the capillaries, because 125ITI normally has long tissue retention times when taken up by tissues. It is possible that the transient retention of the label in the lungs is due to vesicles which are somehow lodged within the capillaries (it is important to note the basic anatomical fact that the lung capillaries have the smallest diameter of any blood vessel in the body). This being the case, the 125ITI would gradually leak out of the vesicles into the blood and be removed, or the vesicles would eventually be dislodged and removed to other sites intact. Alternatively, it is possible that the vesicles are endocytosed by blood monocytes which temporarily lodge in the lungs. The in vivo behavior of liposomes is likely to be influenced by the binding of plasma proteins. Thus exploratory studies investigating vesicle-plasma interactions were initiated. The preliminary studies demonstrated that addition of a surface charge to a liposome greatiy increased the quantity of plasma protein which bound indicating, for example, that the bound protein might account for the observed lung retention. The qualitative protein binding patterns were investigated and it was shown that a large number of plasma components bind to vesicles. The major proteins were tentatively identified using 2-D electrophoresis techniques and albumin and IgG were positively identified with immuno- autoradiography. The patterns for neutral and SA containing vesicles appeared to exhibit essentially random binding of plasma components while in the PS containing vesicles a minor plasma protein designated F-PS binds preferentially 138 at high PS concentrations. Attempts to identify the F-PS peptide as a clotting peptide were incomplete and requires the use of antibodies specific for the possible peptides. In view of the properties of F-PS it is possible that F-PS is the 50 K peptide from clotting Factor V. The relatively low binding of the major opsins (IgG and fibronectin) in the vesicle protein binding patterns, relative to the total plasma control, is noteworthy (the other major opsin, complement Factor C3B, was not identified on the gel). This finding indicated that the major proteins binding to vesicles are not opsins, and suggests that opsinization may not be important in the clearance of non-antigenic liposomes. The most important net result of the binding of plasma proteins to vesicles may be the negative charge imparted to the vesicle. In particular, it is possible that such vesicles would then bind to platelets in vivo, although this has never been tested. In summary, the work presented in this thesis leads to several studies which should provide important information about the interactions of liposomes with the in vivo system. First, the protein F-PS should be identified, as well as the the other major plasma proteins which bind to vesicles. Presumably this would shed light on the role of plasma proteins in determining the in vivo fate of liposomes. Second, the plasma protein binding studies should be extended to vesicles of various sizes, to see if the increased lifetime of SUV systems in circulation can be correlated to differences in plasma protein binding. This would entail the development of a method for separating smaller vesicles from unbound plasma components, particularly the lipoproteins. Third, a platelet model should be developed to study possible vesicle platelet interactions, and the influence of vesicle size, dose and composition, as well as the effect of plasma protein binding, should be examined. 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