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Role of blood proteins in liposome clearance Chonn, Arcadio 1992

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ROLE OF BLOOD PROTEINS IN LIPOSOME CLEARANCEbyARCADIO CHONNB. Sc. Biochemistry, The University of British Columbia, 1984A THESIS SUBMITI’ED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BIOCHEMISTRYTHE UNIVERSITY OF BRITISH COLUMBIADecember, 1991© Arcadio Chonn, 1991tSignature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of /oHEMI5T1e/The University of British ColumbiaVancouver, CanadaDate c4<. 5J)DE-6 (2/88)Signature(s) removed to protect privacyABSTRACTThe studies presented in this thesis were aimed at understanding the basis for theapparent recognition of liposomes as foreign particles by the reticuloendothelial system.This phenomenon represents a major problem to the development of optimal liposometargeted drug delivery systems. An understanding of the clearance process potentiallywill lead to the design of improved liposome carrier systems that display favorable andpredictable pharmacokinetic properties.The studies fall into two main areas of investigation. The first area involved thestudy of the complement activating properties of liposomes composed of various lipidspecies. This is of interest because the activation of the complement system is known toprovide the effector molecules that lead to opsonization and lysis of foreign particulates.It had not been previously determined whether liposomes composed of simple lipidspecies activate the complement system in the complex biological milieu. The studiespresented here used a complement hemolytic assay to demonstrate that surface charge isa key determinant of complement-activating liposomes. The nature of the charge,whether negative or positive appeared to dictate which pathway of the complementsystem is activated. Negatively charged liposomes activated complement in adependent manner suggesting that activation occurred via the classical pathway.Positively charged liposomes activated complement via the alternative pathway. Neutralliposomes failed to activate complement as measured by the hemolytic assays. Further, itwas shown that unsaturated liposomes were more potent complement activators thansaturated liposomes and that 45 mol% cholesterol promoted complementprotein/liposome interactions. The relation between complement activation and11clearance rate from the circulation was investigated. Although there appeared to be adirect relation between complement activation and clearance for liposomes composed ofvarious anionic phospholipids, the relation appeared to break down for other liposomalsystems. This suggests that other factors play a more important role in liposomeclearance.The second area of investigation was directed at establishing an unambiguous rolefor proteins in mediating the clearance process in vivo. Liposomes were administeredintravenously into CD1 mice and after various times the liposomes were recovered fromthe blood. The amount of total protein binding to the liposomes was quantitated usingthe bicinchoninic acid protein assay, and the proteins associated with the recoveredliposomes were analyzed by SDS-polyacrylamide gel electrophoresis. In order tofacilitate the isolation of liposomes from blood components, a simple and rapidprocedure combining chromatographic and centrifugal methods was developed. This“spin column” procedure enabled the use of large unilamellar liposomes instead ofmultilamellar vesicles, thus making possible the measurement of parameters such as theamount of protein bound/vesicle. From these studies, it was established that the amountof protein bound to the liposomes was inversely related to the circulation half-life of theliposomes. The proteins associated predominantly with very rapidly cleared liposomesconsisted of large amounts of opsonins including C3 fragments and IgG, as well as otherproteins such as apolipoprotein H which as of yet has no recognized opsonic role.Furthermore, these studies demonstrated that the mechanism by which ganglioside GM1prolongs the circulation half-life of liposomes was by reducing the total amount of bloodprotein bound to the liposomes in a relatively non-specific manner. These studiesestablished unambiguously that the apparent differences in liposome clearance behavior111observed in vivo is related to the amount and type of protein associated with theliposomes in vivo. This has important implications for the design of liposomes havingstable properties in the circulation and for the design of biocompatible surfaces.ivTABLE OF CONTENTSAbstract.iiTable of Contents vList of Tables ixList of Figures xAbbreviations xiiAcknowledgements xivDedication xvChapter 1 Introduction 11.1 Liposome Carrier Systems: An Overview 11.2 Liposomes 41.2.1 Classification of liposomes Multilamellar vesicles (MLVs) Large unilamellar vesicles (LUVs) Small unilamellar vesicles (SUVs) 81.3 Fate and Behavior of Liposomes In Vivo 81.3.1 Destabilization of liposomes by plasma components Role of lipoproteins and apolipoproteins Role of other plasma factors involved in lipid metabolism. . Role of complement membrane attack complexes Role of other plasma proteins 151.3.2 Interaction of liposomes with cells Role of immune opsonins Opsonic C3 fragments Anti-phospholipid antibodies Role of other plasma proteins 26v1. Fibronectins . Apolipoproteins Protease inhibitors C-reactive protein 281.3.3 Stabilization of liposomes by plasma factors 291.4 Aspects of Liposome Design Affecting Clearance 301.4.1 Physical aspects of liposomes 311.4.1.1 Vesicle size 311.4.1.2 Surface charge 321.4.2 Chemical properties of liposomes 331.4.2.1 Steric barrier due to an increase in surface hydrophilicity. . 331.4.2.2 Phospholipid structure 341.4.2.3 Cholesterol and a-tocopherol content 351.5 Overall Objectives 36Chapter 2 Activation of the Classical and Alternative Pathways of Complement byLiposomes 392.1 Introduction 392.2 Methods and Materials 422.2.1 Preparation of liposomes 422.2.2 Serum, complement and antiserum 432.2.3 Residual total complement hemolytic assays 442.2.4 Measurement of guinea pig alternative complement pathway activationby liposomes 462.2.5 Measurement of human alternative complement pathway activation byliposomes 472.2.6 Liposome lysis fluorescence assay 472.2.7 Immunoblot analysis of proteins associated with liposomes 482.3 Results 502.3.1 Activation of guinea pig and human complement by PG-containingliposomes 502.3.2 Complement activation by other charged liposomes 552.3.3 Mechanism of complement activation by liposomes 582.4 Discussion 65viChapter 3 Separation of Large Unilamellar Vesicles from Blood Components by a SpinColumn Procedure 693.1 Introduction 693.2 Methods and Materials 713.2.1 Preparation of liposomes 713.2.2 Serum and lipoproteins 723.2.3 Preparation of spin columns 723.2.4 Spin column profiles of liposomes incubated with human serum. . . .733.2.5 Conventional BioGel A15m chromatography of liposome/human serumincubation mixtures 743.2.6 Recovery of liposomes from circulation of CD1 mice 743.2.7 Competitive ELISA for C3 753.2.8 SDS-PAGE analysis of proteins associated with liposomes 763.3 Results 763.3.1 Isolation of large unilamellar liposomes from plasma using BioGelA15m spin columns 763.3.2 Measurement of human C3 bound per liposome 833.3.3 In vivo characterization of murine plasma proteins associated with LUVsover time 863.4 Discussion 89Chapter 4 Association of Blood Proteins with Large Unilamellar Liposomes In Vivo:Relation to Circulation Lifetimes 944.1 Introduction 944.2 Methods and Materials 984.2.1 Preparation of liposomes 984.2.2 In vivo mouse plasma distribution of liposomes 984.2.3 Isolated serum/liposome incubations 994.2.4 Isolation of liposomes from blood components 994.2.5 Measurement of PB values 1004.2.6 SDS-PAGE analysis of proteins associated with liposomes 100vii4.2.7 Heparin-agarose chromatography 1014.2.7 Complement hemolytic assays 1024.3 Results 1024.3.1 In vivo association of murine blood proteins with liposomes exhibitingmarkedly different clearance properties 1024.3.2 Comparison of in vivo and in vitro systems 1074.3.3 Identification of opsonins associated with rapidly cleared liposomes .1084.3.4 Influence of ganglioside GM1 content on the binding of blood proteins toLUVs 1124.4 Discussion 118Chapter 5 Summarizing Discussion 123Bibliography 133viiiLIST OF TABLESTable 1.1. Cellular distribution of complement receptors 22Table 2.1. 45Ca2remaining in the supernatant after incubation of DGVB2buffer orserum with PC:CHOL:PG (35:45:20) FMLVs 61Table 3.1. Amount of C3 associated with various LUVs 85Table 4.1. B values for LUVs recovered from in vitro incubations with isolated humanserum 109Table 4.2 Immunoblot analysis of proteins associated with CL-containing LUVs. . . 114ixLIST OF FIGURESFigure 1.1. Diagram and freeze-fracture electron micrographs of multilamellar andunilamellar vesicles 5Figure 1.2. Complement activation via the classical and alternative pathways 13Figure 1.3. Degradation of C3 and reactivity of its fragments with cellular complementreceptors 21Figure 2.1. Diagramatic representation of complement hemolytic assays 45Figure 2.2. Effect of serum dilution on the sensitivity of the total complement hemolyticassays to detect consumption of guinea pig complement 51Figure 2.3. Effect of PG density on human complement activation by saturated andunsaturated liposomes 53Figure 2.4. Effect of cholesterol on human complement activation by saturated andunsaturated liposomes 54Figure 2.5. Dose response curves of guinea pig serum incubated with LUVs 56Figure 2.6. Dose response curves of human serum incubated with LUVs 57Figure 2.7. Complement activation by liposomes inCa2-depleted guinea pig serum. . 59Figure 2.8. Alternative pathway complement hemolytic activity of human serumincubated with liposomes 60Figure 2.9. Complement-mediated lysis of liposomes containing entrappedcarboxyfluorescein using liposome-treated serum 62Figure 2.10. Immunoblot analysis of proteins associated with PG-containing liposomesafter exposure to human serum 64Figure 3.1. Elution profile of PC:CHOL:CL (35:45:10) LUVs/human serum incubationmixtures chromatographed on conventional columns 77xFigure 3.2. Spin column profiles of liposomes or human serum proteins 79Figure 3.3. Spin column profiles of liposome/human serum incubations 80Figure 3.4. Separation of 100 nm LUVs from VLDL and LDL 81Figure 3.5. SDS-PAGE analysis of the protein content of the spin column fractions.. .82Figure 3.6. Comparison of the protein profile associated with PC:CHOL:CL LUVsisolated using the spin column procedure or conventional chromatographicprocedures 84Figure 3.7. Clearance profile of PC:CHOL and PC:CHOL:DOPS LUVs 87Figure 3.8. Protein profiles of PC:CHOL (55:45) and PC:CHOL:DOPS (35:45:20) LUVsrecovered from mice over time 88Figure 4.1. Plasma clearance of LUVs 104Figure 4.2. Silver-stained nonreducing SDS-PAGE gel of proteins associated withliposomes recovered from the circulation of mice after 2 mm post-injection.. . . 105Figure 4.3. Relation of total amount of protein bound to liposomes and circulation half-life 106Figure 4.4. Comparison of protein profiles of LUVs recovered from the circulation ofmice and from in vitro incubations with isolated serum 110Figure 4.5. Immunoblot analysis of murine opsonins associated with LUVs 111Figure 4.6. Heparin-agarose chromatography of proteins associated with CL-containingLUVs 113Figure 4.7. Effect of ganglioside GM1 and GD1a on human serum protein associationwith liposomes 115Figure 4.8. Inhibition of complement activation by liposomes containing gangliosideGM1 117xiABBREVIATIONSBCA bicinchoninic acidBSA bovine serum albuminC complementCaCI2 calcium chlorideCHOL cholesterolCL cardiolipinDGVB VBS containing 2.5% dextrose and 0.1% gelatinDGVB2 DGVB containing 0.5 mM MgC12and 0.15 mM CaC12DOPA dioleoylphosphatidic acidDOPE dioleoylphosphatidylethanolamineDOPS dioleoylphosphatidylserineDOTAP 1,2-bis(oleoyloxy)-3-(trimethylammonio)propaneDPPC dipalmitoylphosphatidylcholineDPPE dipalmitoylphosphatidylethanolamineDPPG dipalmitoylphosphatidylglycerolEA sheep erythrocytes sensitized with antibodyEDTA ethylenediaminetetraacetic acidEGTA ethylene glycol bis(3-aminoethyl ether)-N, N, N’, N’-tetraaceticacidELISA enzyme-linked immunosorbent assayFMLVs freeze-thawed multilamellar vesiclesHBS HEPES-buffered salinexiiHDL high density lipoproteinsHEPES [4-(2-hydroxyethyl)J-piperazine ethane sulfonic acidIgG immunoglobulin GLDL low density lipoproteinsLUVs large unilamellar vesiclesMgCI2 magnesium chlorideMLVs multilamellar vesiclesPAGE polyacrylamide gel electrophoresisPB protein binding abilityPBS phosphate-buffered salinePA phosphatidic acidPC phosphatidyicholinePE phosphatidylethanolaminePG phosphatidyiglycerolP1 phosphatidylinositolSA stearylamineSDS sodium dodecyl sulphateSM sphingomyelinSUVs small unilamellar vesiclesT112 half-lifeTris tris(hydroxymethyl)aminomethaneTween 20 polyoxyethylene (20) sorbitanVBS veronal-buffered salineVLDL very low density lipoproteinsxiiiACKNOWLEDGEMENTSI would like to acknowledge all of those who have assisted me with the workpresented in this thesis, especially the following people: Sean Semple, for help withcountless spin columns and for his amicable competitive spirit; Dana Masin, for herexpert assistance with the animal experiments; Barb Mui, for the occassional “onesample” phosphate analysis and for the constant supply of HBS; Mike Parr, for his“Freelancing” skills; Dr. Dana Devine, for sharing her expertise in the complement fieldand for making her lab accessible; and, of course, my amazing supervisor, Dr. PieterCullis, for providing seemingly endless support and encouragement, and for criticallyreading this thesis.I feel immensely privileged to have been part of such a dynamic research group atsuch a pivotal period in its transformation into the “Liposome Research Unit”. For this, Iam indebted to Pieter, and to all the friends I have made in Pieter’s group who have madegraduate studies a most enjoyable and worthwhile experience. Mick, Marcel, Tom andKim, thanks for many helpful discussions and for your enthusiastic support over thesepast few years. As well, my sincerest thanks to all of the “Deviners” for putting up withme in the early years. Especially the Issa family, for all the laughs, warmth andhospitality. Lipex Biomembranes, for all the beer, dinners (especially the one atGerards), unforgettable parties and ski trips, many thanks.I would like to acknowledge the financial support of the B. C. Science Council,for providing me with a studentship, and the Medical Research Council and the NationalCancer Institute for funding this research.xivToMom and Dad,andin memory of my brother, WilliexvCHAPTER 1. INTRODUCTION1.1 LIFOSOME CARRIER SYSTEMS. AN OVERVIEWEver since the initial reports describing the ability of liposomes to entrap ions andsmall molecules (Bangham, 1968; Sessa and Weissman, 1968), liposomes have beendeveloped to deliver a wide range of therapeutic or diagnostic agents. These liposomecarrier systems extend from liposome-encapsulated anti-cancer (Mayer et al., 1989;Balazsovits et al., 1989), anti-fungal (Lopez-Berestein et al., 1989; Lopez-Berestein,1988), anti-bacterial (Bakker-Woudenberg et al., 1989, 1988; Tadakuma et al., 1985;Fountain et al., 1985) or anti-viral (Szebeni et al., 1990) pharmaceuticals, andimmunomodulators (Rutenfranz et al., 1990; Debs et al., 1989; Bakouche et al., 1988;Hudson et al., 1988; Fogler and Fidler, 1987) to artificial erythrocytes (Vidal-Naquet etal., 1989; Farmer et al., 1988) and platelets (Baldassare et al., 1985). In recent years,there has been major progress towards the goal of producing an optimized liposomecarrier system. Notably, improved methods for producing liposomes of defined, narrowsize distributions using a wide range of lipid compositions, and improved methods for theefficient trapping of drugs and other macromolecules have been described (Cullis et al.,1989; Hope et al., 1986; Mayer et al., 1986). This has led to the development ofliposome drug delivery systems which display improved therapeutic properties ascompared to the free drug (Mayer et al., 1989). These simple, non-targeted systems willconstitute the first generation of liposomal pharmaceuticals.More sophisticated second generation products will be aimed at targeting tospecific cells or tissue. It is in this area that liposomes exhibit their greatest potential.1Drug delivery to specific sites in vivo would be beneficial in limiting the toxic sideeffects that arise due to systemic delivery of the drug to sensitive tissue not associatedwith the diseased site. Targeted delivery is mediated by the attachment of some form ofhoming device, typically a monoclonal antibody or in some cases, lectins or peptides.Improved protocols for the covalent and noncovalent attachment of proteins to liposomeshave been reported (Loughrey et al., 1990 and 1987; Heath, 1987; Leserman and Machy,1987).Several problems still exist, however, which impede the development ofoptimized liposome carrier systems, especially for targeting applications. A considerableobstacle is the rapid clearance pharmacokinetics exhibited by intravenously administeredliposomal systems. Generally, most liposomes are rapidly sequestered by the free andfixed macrophages of the reticuloendothelial system by as yet poorly definedmechanisms. This has frustrated the development of liposomal systems which targetdrugs to specific tissues other than reticuloendothelial tissues. A compounding problemis the increased permeability of entrapped solutes that liposome membranes exhibit inplasma. Further, an unresolved issue is whether liposomes are capable of extravasatingto the surrounding interstitial tissues and are able to interact with target tissues. Adetailed understanding of the interactions liposomes experience in vivo and the relationbetween such interactions and clearance, drug biodistribution and efficacy is presentlylacking. Clearly, such an understanding must be addressed in order to develop optimizedliposome drug carrier systems.Recently, various liposome compositions that exhibit extended circulationresidence times have been described (Allen and Chonn, 1987; Papahadjopoulos andGabizon, 1987; Gabizon and Papahadjopoulos, 1988; Klibanov et al., 1990). These2findings have infused excitement and optimism in the liposome drug delivery field asnew opportunities are opened for achieving targeting to specific cells in vivo. Forinstance, Gabizon and Papahadjopoulos (1988) have shown that liposomes that arecapable of circulating for longer periods of time, as a result of reduced uptake by thephagocytic cells of the reticuloendothelial system, are able to reach target sites andaccumulate at these sites. There are reports describing improved immunotargeting tospecific cells in vivo using liposomal systems exhibiting extended circulation residencetimes (Maruyama et al., 1990; Klibanov et al., 1991). Immunotargeting of drugs tospecific tissues using liposomes that are capable of extended circulation lifetimes,however, is still only imperfectly designed. For instance, immunoliposomes containingpolyethyleneglycol (5000) exhibit reduced target binding as compared toimmunoliposomes without polyethyleneglycol (5000), probably because of the stericbarrier effect of these hydrophilic polymers (Klibanov et al., 1991). As well, largeliposomes containing polyethyleneglycol (5000) or ganglioside GM1 accumulate tosignificantly greater levels in the spleen compared to liposomes without these molecules(Klibanov et a!., 1991; Liu et al., 1991). An understanding of how these liposomecompositions extend liposome circulation half-lives should provide insight into themechanisms involved in the clearance of liposomes from the circulation.The main focus of this introduction will be to review the plasma factors whichaffect liposome behavior in vivo, as well as several aspects of liposome design whichalter liposome clearance. It will also contain a brief description of liposomes, indicatingthe terminology used in the literature. Finally, the overall objectives of this thesis will beoutlined.31.2 LIPOSOMESLiposomes are closed vesicular structures which spontaneously form when mostspecies of phospholipids are dispersed in excess water. Multilamellar and unilamellarstructures of varying size distributions are obtained (see Figure 1.1). A relevant propertyof these closed vesicular structures is that hydrophilic or lipophilic molecules can betrapped in the aqueous internal compartments or in the lipid membranes, respectively.The physical properties of liposomes and the methods used to generate liposomes forpharmaceutical applications have been described in several excellent reviews (Cullis etal., 1989; Hope et al., 1986).1.2.1 Classification of liposomesOn the basis of their lamellarity and size, liposomes have been classified intothree types: multilamellar vesicles, large unilamellar vesicles and small unilamellarvesicles. Lamellarity concerns the number of internal lamellae sequestered withinmultilamelair vesicles and therefore not exposed to the external medium. Freeze-fracturemicroscopy (Hope et al., 1989), light scattering techniques and trapped volumemeasurements (Hope et al., 1986) are methods used to determine the lamellarity and sizeof the liposome population. Multilamellar vesicles (MLVs)MLVs, first described by Bangham et a!. (1965), are vesicles having amorphology consisting of alternating concentric rings of phospholipid bilayers and4Figure 1.1. Diagram andfreeze-fracture electronmicrographs ofmultilamellar and unilamellar vesicles.MULTI L A IVI E I.. LAP UN IL A MEL LAPVESICLES VESICL.ESDiameter: 0.2 —10 m.- f5aqueous compartments. These are spontaneously formed by mechanical dispersion of alipid film in an aqueous solution. MLVs typically have diameters in excess of 1 urn, andare heterogenous in size (0.2-10 urn) and lamellarity. The ease of preparation of thesevesicles is their major advantage. The heterogeneity in lamellarity and size, however,limits their general use. A major disadvantage, particularly pertinent to the studiesdescribed in this thesis, is that only the outermost lamellae is exposed to theextravesicular environment. This may comprise only 5% or less of the total lipid. Afreezing and thawing procedure can be employed to decrease the number of internallamellae per vesicle (Mayer et al., 1985). Large unilamellar vesicles (LUVs)LUVs are vesicles having a well-defined large internal aqueous compartmentsurrounded by a single lipid bilayer. Their sizes range from 50 to 200 nm in diamter.Early procedures for preparing LUVs commonly employed reverse phase evaporation(Szoka and Papahadjopoulos, 1978) or detergent dialysis (Kagawa and Packer, 1971;Mimms et al., 1981) methods. In reverse phase evaporation, the lipids are firstsolubilized in organic solvents such as diethyl ether; buffer is added to the lipid solutionand a stable emulsion formed by sonicating the mixture; the organic solvent is thenremoved by evaporation under vacuum. The resulting dispersion is a heterogeneousmixture of oligolamellar and unilamellar vesicles which requires low pressure extrusionthrough polycarbonate filters to achieve relatively homogeneous preparations ofunilamellar vesicles. In detergent dialysis, dry lipid or pre-formed vesicles are firstsolubilized in detergent-containing buffer to form mixed micelles. The detergent is then6removed by dialysis, whereby the micelles coalesce and the phospholipids adopt a bilayerconfiguration, resulting in sealed vesicles. The type of detergent employed as well as therate and method of detergent removal determine the type of LUV preparation obtained.Both of these methods have the disadvantage of being dependent on lipid composition.Further, these methods have problems associated with reproducibility, and with removalof residual organic solvents or detergents.The development of an extrusion device, which allows for the rapid production ofLUVs directly from MLVs, has greatly simplified the production of LUVs. This methodinvolves the sequential medium pressure (< 5000 kPa) extrusion of MLVs throughpolycarbonate filters of defined pore sizes (Hope et al., 1985). LUVs produced by thismethod have a relatively narrow size distribution as determined by measurementsemploying light scattering techniques or freeze-fracture microscopy. Using thisextrusion method, LUVs composed of a wide variety of lipid species can be readilyproduced at high concentrations, and in the absence of contaminating detergents ororganic solvents (Hope et al., 1985; Nayar et al., 1989). The studies described in thisthesis largely employ LUVs produced by extrusion through 100 nm pore sized filters.Using this procedure, the population of liposomes is essentially unilamellar for all thelipid compositions studied. These well-defined liposomal systems are ideal for thestudies involving protein/liposome interactions described in this thesis.It is common to refer to LUVs by the methods used to prepare the liposomes. Forexample, REVs would refer to vesicles produced by reverse-phase evaporation (Szokaand Papahadjopoulos, 1978), and LUVETs would refer to vesicles produced by extrusiontechniques (Hope et al., 1985). Small unilamellar vesicles (SUVs)SUVs are vesicles having a small internal aqueous compartment surrounded by asingle lipid bilayer. Their sizes range from 20-50 nm in diameter. SUVs are producedby sonicating MLVs (Huang, 1969) or by French press techniques (Barenholz et al.,1979). In recent years, SUVs have become synonymous with vesicles produced bysonication procedures, regardless of the size distribution attained. This has resulted insome confusion, particularly in studies which use sonicated vesicles to demonstrate thesize dependency of liposome pharmacokinetics. SUVs are composed of highly strained,curved bilayers which can lead to an increase tendency of these vesicles to fuse(Lichtenberg et al., 1981; Wong et al., 1982; Parente and Lentz, 1984). Further, SUVsare more susceptible to attack by phospholipases and high density lipoproteins due totheir highly curved surfaces (Scherphof and Morselt, 1984).1.3 FATE AND BEHAVIOR OF LIPOSOMES IN VIVOGregoriadis and Ryman (1972) first demonstrated that MLVs are cleared rapidlyfrom the circulation of rats in a biphasic manner, with an initial rapid rate followed by aslow rate of elimination. Subsequently, many reports have shown a similar biphasicclearance pattern for MLVs, LUVs and SUVs in various animal models including mice,rats, rabbits, dogs, monkeys and man. The mechanisms for the different phases ofclearance is not clear. Numerous studies (for a review see Gregoriadis, 1988 or Hwangand Beaumier, 1988) have established that liposome clearance results from a combinationof (1) the destabilization of liposome membranes caused by interactions with plasmacomponents, and (2) the adherence and/or internalization of liposomes by various cells,8particularly the resident macrophages of the liver and spleen. In general, a largeproportion (as high as 90% after 1 hr) of the intravenously administered dose ofliposomes is recovered in reticuloendothelial tissues, which include the liver, spleen,lymph nodes, bone marrow and lungs. The biphasic nature of liposome clearance likelyreflects these clearance mechanisms.Whereas the role of various plasma components in affecting the structuralintegrity of liposome membranes has been extensively investigated in vitro and in vivo,the factors mediating the rapid uptake of liposomes by the reticuloendothelial systemremain poorly defined. This section describes our present understanding of the role ofvarious plasma components in influencing liposome behavior in vivo.1.3.1 Destabilization of liposomes by plasma componentsEarly observations regarding liposome stability in the circulation suggest thatleakage of solutes in the circulation may be plasma-dependent. Liposomes which retainsolute on incubation at 37°C for hours in isoosmotic buffer may become leaky in a matterof minutes on incubation at 37°C in the presence of plasma (Gregoriadis, 1973;Gregoriadis and Senior, 1980). Thus, plasma contains a factor or factors which inducebilayer permeability changes leading to the release of entrapped solute. Role of lipoproteins and apolipoproteinsNet transfer of liposomal lipids to plasma lipoproteins has been shown todestabilize liposome membranes resulting in an increased permeability of entrappedsolutes (reviewed extensively by Senior, 1987). Although all lipoprotein species may be9involved, early studies have established that liposome interactions with primarily highdensity lipoproteins (HDL) result in dissolution of the liposome structure and transfer ofliposomal phospholipid to HDL (Tall and Green, 1981; Chobanian et al., 1979;Scherphof et al., 1978; Krupp et al., 1976). Using H- or14C-labeled egg PC liposomescontaining entrapped[I]-albumin, Scherphof et al. (1978) showed that rat, monkey orhuman HDL take up liposomal phospholipid resulting in a massive release of entrapped[5I]-albumin. Liposome-derived phospholipid was found to be associated with HDLafter liposome incubation at 37°C with plasma, isolated HDL, or HDL apolipoproteins(Chobanian et al., 1979).The role of other plasma lipoproteins in liposome membrane destabilization hasalso been suggested. Net transfer of 14C-PC from egg PC SUVs to HDL and to someextent LDL was shown to occur in vivo using a rat model (Tall, 1980). With largeliposome doses, phospholipids are transferred to LDL and probably also VLDL, resultingin the formation of larger less dense particles (Chobanian et al., 1979; Shahrokh andNichols, 1982). The increased liposome membrane permeability induced by interactionswith LDL has recently been demonstrated for anionic liposomes at lipid concentrations aslow as 43 nM phospholipid (Comiskey and Heath, 1990). In the presence of humanLDL, PG:CHOL (67:33) liposomes leaked 73%; whereas in the presence of bovineapolipoprotein A-I or in the presence of human HDL, they leaked only 29 or 25%,respectively.Isolated apolipoproteins have been shown to destabilize liposomes by enhancinglipid movement and leakage of entrapped markers. Several investigators have shown thatapolipoproteins A-I, A-TI, A-TV, B, C and E are transferred to liposome membranes uponplasma incubation in vitro and in vivo (Mendez et al., 1988; Williams and Scanu, 1986;10Nichols et al., 1978; Gong and Nichols, 1980; Guo et al., 1980; Luke et al., 1980; Talland Green, 1981). Incubation of egg PC SUVs with rat apolipoprotein A-I or E resultedin release of entrapped carboxyfluorescein accompanied by structural changes of theliposomes to discs (Guo et al., 1980; Atkinson et al., 1976; Forte et al., 1971). Role ofotherplasma factors involved in lipid metabolismThe observation that liposome membrane permeability is enhanced by otherplasma factors that are present in lipoprotein-free plasma (density> 1.25 gm/mi) has ledto the suggestion that plasma factors other than lipoproteins or apoiipoproteins areinvolved in mediating liposome instability (Tall et al., 1983a, 1983b; Damen et al.,1980). These factors include various lipid transfer proteins which are known to bepresent in lipoprotein-free plasma from many species including human, rabbit, rat andmouse (reviewed by Tall, 1986). Phospholipid transfer proteins are able to mediate thetransfer of a wide variety of phospholipids including PC, SM, PE, PS, PG, PA,galactosylcerebroside, and diacylglycerol (Massey et al., 1985). These plasmaphospholipid transfer proteins greatly enhance the transfer of liposome phospholipid intoHDL resulting in leakage of entrapped carboxyfluorescein (Tall et al., 1983a, 1983b).Lecithin-cholesterol acyltransferase, an acyl-group transferring enzyme which isclosely associated with HDL, has been shown in vitro to destabilize certain liposomes(Jahani and Lacko, 1982). An accumulation of lyso PC, the product of a lecithincholesterol acyltransferase conversion of cholesterol to esterified cholesterol, using PC asa source of fatty acids, has been suggested to cause a disruption of liposome structure andcause leakage of entrapped marker (Marcel, 1982). Role of complement membrane attack complexesActivation of the complement system results in the formation of membrane attackcomplexes which leads to cell lysis (reviewed by Muller-Eberhard, 1986). Assembly ofthe membrane attack complex is initiated after the cleavage of C5 to C5b by either theclassical or the alternative pathway C5 convertase (see Fig. 1.2). Several mechanisms ofgenerating membrane permeability by the membrane attack complex have beenproposed. A “leaky patch” model suggests that local phospholipid disorder induced bymembrane attack complex formation produces membrane permeability (Esser et al.,1979). Another model proposes that a porelike structure, only partially lined with C9polymers, generates membrane permeability (Tschopp, 1984; Amiguet et a!., 1985;Malinski and Nelsestuen, 1989).Early observations suggested that complement proteins may be involved in theplasma-induced destabilization of liposomes resulting in leakage of entrapped contentsinasmuch as decomplemented serum (serum heated at 56°C for 30 mm) reduced the lossof aqueous solutes (Finkeistein and Weissmann, 1979). This study, however, does notexclude the possibility that other heat labile proteins are also inactivated, such as proteinsthat facilitate the PC transfer activity of HDL (Senior et al., 1983).The lytic effect of complement membrane attack complexes on liposomescontaining either naturally occurring lipid antigens such as Forssman antigen (Haxby eta!., 1969, 1968; Kinsky et al., 1969), mammalian ceramides (Inoue et a!., 1971), orbacterial lipopolysaccharides (Kataoka et a!., 1971), or synthetic amphipathic haptens(Uemura and Kinsky, 1972; Six et al., 1973; Okada et al., 1982b) has been welldocumented (reviewed by Kinsky and Nicolotti, 1977; Alving and Richards, 1980).12Figure 1.2. Complement activation via the classical and alternative pathways.The shaded areas denote reactions that occur on membrane surfaces.Alternative Classical3OiOj ntLgenJantlbody + 1::::::::c4:::::.:..:.:.:.:.:::::::::::•:-;•::C,3aC3b C4b::•:•:•::•::::•:•:•:(a::::•:-:-:•::•:•::-:-::-::•:oø:nC5 convertaseC5bC6C7C5b-7membrane Insertton13These compositions require the presence of sensitizing antibodies for classicalcomplement activation (Kinsky, 1972; Alving et al., 1969).The mechanism by which complement causes membrane damage and lysis hasbeen extensively studied using liposome model membrane systems (for reviews, seeAlving and Richards, 1983 and Muller-Eberhard, 1986). The membrane permeability tomacromolecules mediated by the membrane attack complex has recently been studiedusing a well defined system consisting of purified human complement proteins and LUVscomposed of PC or PS containing trapped macromolecules such as bovine trypsininhibitor, thrombin, or glucose-6-phosphate dehydrogenase (Malinski and Nelsestuen,1989). These studies indicate that release of macromolecules from LUVs occurs throughstable lesions with well-defined maximum lesion dimensions of about 10 nm in diameterwhich is produced when only about half of the maximum number of C9 molecules isbound. Release of macromolecules from LUVs follows a one-hit model where a singlemembrane attack complex is responsible for generating a lesion adequate to release allmacromolecules in a single vesicle.Complement damage is also associated with release of phospholipids frommembranes. This phenomenon has been observed with antibody-sensitized sheeperythrocytes (Giavedoni and Dalmasso, 1976), antibody-sensitized tumor cells (Schlageret al., 1978), antibody-sensitized Escherichia coli cells (Inoue et al., 1977), andliposomes (Shin et al., 1978; Kinoshita et al., 1977; Shin et al., 1977). Release ofphospholipid from liposomes is dependent on the dose of complement (Shin et al., 1977),and the amount of phospholipid liberated from Escherichia coli cells is proportional tothe number of complement lesions (Inoue et al., 1977). The release of phospholipid wassuggested to be due to displacement of membrane phospholipids by the C5b-9 complexes14during their insertion into the membrane lipid bilayer (Inoue et al., 1977), and part of thephospholipid removal from liposomes was mediated by C5b-7, and the remainder by C8and/or C9 (Shin et a!., 1977). Role of other plasma proteinsThere are only a few reports of other plasma proteins which induce membranepermeability of liposomes either in vitro or in vivo. Recent studies have shown thatbovine serum albumin purified by cold ethanol extraction induces methotrexate-yaspartate leakage from PG:CHOL and PS:CHOL liposomes (Comiskey and Heath,1990). Leakage in albumin solutions could be substantially reduced by pretreating thealbumin with lipid. As well, Kiwada et a!. (1988) reported that a heat stable plasmafactor which salted out in 50-68% ammonium sulphate (albumin fraction) is involved inthe leakage of carboxyfluorescein loaded into palmitoylglucoside liposomes.C-reactive protein binding to lipid membranes causes agglutination of liposomesand may affect the structural integrity of liposome membranes (Richards et al., 1979,1977). PC:dicetyl phosphate liposomes containing glucose are disrupted by binding ofC-reactive protein, releasing the entrapped glucose (Ohsawa, 1980). Similarly,PC:CHOL:dicetyl phosphate or PC:CHOL:PA liposomes containing[3H]inulin aredisrupted by purified C-reactive protein binding causing release of[3Hjinulin (Pepys etal., 1985).151.3.2 Interaction of liposomes with cellsAs mentioned above, intravenously administered liposomes are readily taken upby the free and fixed macrophages of the reticuloendothelial system. The“reticuloendothelial system” was a term coined by Aschoff (1924) in reference to “a bodyof mesenchymal-derived cells which formed a diffuse system of sessile and wanderingmononuclear macrophages which had the common physiological property of being ableto rapidly ingest and accumulate foreign colloidal and particulate matter” (for anextensive review of the physiology of the reticuloendothelial system see Saba, 1970).Gregoriadis and Ryman (1972) were first to report the rapid clearance from thecirculation and uptake by the liver of protein-containing liposomes.It is not inherently obvious why liposomes, which are composed of naturallyoccuring phospholipids and cholesterol, should be recognized as foreign particles by theimmune system. Reports by Kiwada et al. (1986, 1987) involving perfused rat liversindeed have suggested that liposomes could pass freely through the liver in the absenceof blood components. Uptake by the liver is plasma-dependent. Furthermore, there aresuggestions that serum contains organ-specific opsonins that selectively enhancerecognition of liposomes by macrophages in the specific organs of the reticuloendothelialsystem (Moghimi and Patel, 1988). Although these opsonins have yet to be identified,certain properties of these organ-specific opsonins have been recently described(Moghimi and Patel, 1989a). Liver-specific opsonin is a heat-stable macromoleculewhich on heating or on freezing and thawing exhibits enhanced opsonic activity. Serumalso contains a dialysable factor which inhibits its opsonic activity. The spleen-specificopsonin is a heat-labile macromolecule which is sensitive to freezing and thawing and16requires a dialysable serum co-factor for its optimum opsonic activity on spleenmacrophages.Although the fixed macrophages of the liver and spleen, and to a lesser extent thelung and bone marrow can account for much of the cellular uptake of liposomes,liposome interactions with other cell types have been described. The tissue distributionof liposomes is dependent on vesicle size. For instance, only small liposomes (< 100 nmin diameter) are accessible to the parenchymal cells (hepatocytes) of the liver. For SUVs,up to 95% of the liposomes found in the liver are recovered in the parenchymal cells(Roerdink et al., 1981; Poste et aL., 1982). Intact SUVs labeled with radioactive markersare also taken up by other tissues including intestine, skin, and carcass by an apparentlynon-saturable process (Hwang et al., 1987).There are a few reports which describe the interactions of liposomes with bloodcells. Liposome interactions with blood leukocytes, especially neutrophils andmonocytes, have been reported (Kuhn et al., 1983; Finkelstein et al., 1981; Weissmann etal., 1975). Ultrastructural analyses of leukocytes after incubation with liposomesrevealed that monocytes phagocytose MLVs several-fold more than neutrophils, on a percell basis. Uptake by leukocytes in vitro is linear for 15 mm and is mediated by an activeprocess, being both energy-dependent and surface-dependent, that is saturable anddisplay an affinity constant of 1.1-1.7 mM liposomal lipid. MLVs containing SA, PS orPG have been shown to interact with platelets, causing a transient reduction in thenumber of circulating platelets (Reinish et al., 1988).Reports on the interactions with erythrocytes are fragmentary. It has beenobserved that liposome stability is greater in whole blood than in the serum of rats(Gregoriadis and Davis, 1979) and mice (Kirby et al., 1980a). The presence of17erythrocytes has been suggested to reduce liposomal breakdown by donating cholesterolto the liposomes and thus stabilizing them, or by being involved in interactions withlipoproteins which take precedence over potentially disruptive lipoprotein-liposomeinteractions. Immunotargeting of liposomes to erythrocytes results in elimination of theliposome/erythrocyte complexes by the liver and spleen (Peeters et al., 1988; Agrawal etal, 1987). This process of “erythrophagocytosis” represents a possible mechanism ofliposome clearance. However, there has been no direct evidence that liposomes formstable complexes with erythrocytes. Perhaps for liposomes that exhibit extendedcirculation residence times, these cell interactions become more significant in mediatingliposome elimination.Liposome uptake is for the most part believed to involve opsonic receptors;however, the role of scavenger receptors in mediating the uptake of liposomes by mouseor guinea pig peritoneal macrophages have recently been described (Nishikawa et al.,1990). Macrophages in culture were found to actively incorporate and metabolizePC:CHOL liposomes containing small amounts of acidic phospholipids such as PS, P1, orPA and to store the fatty acyl chains and cholesterol in triacyiglycerol and cholesterylester form in their cytosol. Competition studies using various ligands for the scavengerreceptor showed that acetylated low density lipoprotein, dextran sulfate, or fucoidan wasable to compete for up to 60% of the binding of PS-containing vesicles, and that copperoxidized LDL competed for more than 90% of the vesicle binding. On the other hand,PS-containing vesicles were able to compete for more than 90% of the binding of acetylLDL. These results indicate that acidic phospholipids are recognized by scavengerreceptors on the surface of macrophages and that more than one scavenger receptor existson mouse peritoneal macrophages.18The following sections describe some of the proteins which have been shown toassociate with liposomes and mediate cell interactions. Role of immune opsoninsOur present understanding of the opsonization process has evolved mainly fromstudies using particles that require immunologic coating in order to adhere to phagocyticcells. Wright and Douglas (1903) initially showed that certain serum factors, functioningas “non self” markers, associate with bacteria and enable phagocytic cells to identify thepathogen as foreign cells. Since then, the rapid expansion in the understanding ofimmunoglobulin and complement biochemistry made it apparent that the opsonic serumfactors are IgG and C3; defined the roles of these molecules in the adherence step ofphagocytosis; and led to the concept that plasma membrane receptors, which specificallyrecognize portions of these molecules, are the means by which phagocytic cells recognizeand attach to their surfaces particles coated with these ligands.When particles that normally do not adhere to phagocytic cells are coated withIgG molecules directed against their surfaces, the particles can be recognized andattached by phagocytic cells (Berken and Benacerraf, 1966; Ciccimarra et al., 1975;Huber et al., 1968; Arend and Mannik, 1973; LoBuglio et al., 1967; Phillips-Quagliata etal., 1971; Quie et al., 1968). Attachment is mediated by the Fc portion of the IgGmolecule. Particles coated with F(ab’)2fragments of IgG are not attached, and Fcfragments, but not Fab fragments, of IgG can compete for binding sites on the cell’ssurface and block the attachment of IgG-coated particles (Bamett Foster et al., 1980;Ciccimarra et al., 1975; Haeffner-Cavaillon et al., 1979). This specificity, along with the19finding that the ability of phagocytic cells to bind IgG exhibits saturation kinetics(Alexander et al., 1979; Haeffner-Cavaillon et al., 1979; Yagawa et al., 1979), led to theconcept that these cells bear on their plasma membranes molecules that are Fc receptors.Several investigators have isolated from the plasma membranes of phagocytic cells,molecules that bind specifically to the Fc portion of IgG (Anderson and Grey, 1977;D’Urso-Coward and Cone, 1978; Kulczycki et al., 1980; Loube et al, 1978; Mellman andUnkeless, 1980).Molecules generated by the activation of complement also serve to bind particlesto phagocytic cells (Figure 1.3). The concept that binding of C3-coated particles to thephagocytic cell surface is mediated by specific C3 receptors was supported initially byseveral findings. For instance, binding of complement-coated particles to cells is strictlydependent on the presence of C3, not on other complement components; binding of C3-coated particles can be blocked by either fluid phase C3 or C3b but not by othercomplement components, other C3 fragments, or other serum proteins; and the ability ofphagocytic cells to bind C3b-coated particles can be abolished by treating the cells withtrypsin, suggesting that the recognition site for the C3b molecule is an externallydisposed, trypsin-sensitive plasma membrane protein (Bianco et a!., 1975; Gigli andNelson, 1968; Griffin et al., 1975; Griffin and Griffin, 1979; Lay and Nussenzweig,1968; Lay and Nussenzweig, 1968). These suggestive results have been confirmed bythe isolation and identification of C3 fragment receptors on the surfaces of phagocyticcells (Table 1.1).The understanding of the interactions between liposomes and immune opsonins isimportant in developing optimized liposome technologies. For instance, artificial antigenpresenting cells utilizing antigen-coated liposomes and various immunomodulators have20Figure 1.3. Degradation of C3 and reactivity of its fragments with cellular complementreceptors. Activation of C3 via proteolytic cleavage of the cz-polypeptide chain inducesconformational changes in the nascent C3b that expose a thioester bond linking the -SHgroup of cyteine-lOlO to the y-carboxyl group of glutamine-1013 (Tack et a!., 1980).The exposed thioester bond is susceptible to hydrolysis or alternatively, to nucleophilicattack forming covalent linkages with free -OH or -NH2 groups on the surfaces of avariety of molecules (Law et al., 1981). Once C3b is covalently attached to the surfaceand provided the necessary cofactors are present, Gb is further degraded to the variousforms shown schematically below (reviewed by Becherer et al., 1989). The shaded areasrepresent the fragments of C3 that remain attached to the activator surface.cell SurfaceCHO ReceptorsIcNH iiaaoo occhaln j—COOHL__s__s.J C3HOOCH 76,000 chain 1—NH2CHO03 Convertase1_______________IC3b CR1Factors! + H¶ HCR1, CR2iC3b CR3, CR4plasminC3dg CR2 CR3C3g C3dFactor I and CR1¶j 22,000 1L__s sI 1C3c421Table 1.1. Cellular distribution of complement receptors.Receptor Cellular distributionCR1 erythrocytes, granulocytes, monocytes, macrophages, B-lymphocytes, subset of T-lymphocytes, K cells, glomerularpodocytes, neutrophils, eosinophils, follicular dendriticcellsCR2 B-lymphocytes, epithelial cells, follicular dendritic cells,lymphoid organsCR3 granulocytes, macrophagesCR4 monocytes, neutrophils, macrophages22been recently developed. Inasmuch as C3b and C4b have been shown to potentiate theimmune response by affecting the capacity of antigen presenting cells to trigger antigen-specific T cells (Arvieux et al., 1988), it would be interesting to show whether C3bcoated liposomes could potentiate the immune response of these artificial antigenpresenting cells.Following is a description of reported studies that involve the interactions ofcomplement and IgG with liposomes, and their role in liposome clearance.1 .3.2.1 .1 Opsonic C3 fragmentsTo date, the activation of the complement system by liposomes has beendescribed for only a few specific liposome compositions. These compositions includeliposomes containing haptenated lipids (Okada et al., 1982b), phosphatidylserine withphosphatidylethanolamine (Comis and Easterbrook-Smith, 1986), cardiolipin(Kovacsovics et al., 1985), stearylamine in the presence of galactosyl ceramide(Cunningham et al., 1979), cerebrosides in dimyristoylphosphatidylethanolamine(Michalek et al., 1988) or saturated phosphatidylethanolamine in saturatedphosphatidylcholine liposomes containing cholesterol (Mold, 1989). The emphasis ofthese studies was to use liposomes as model membrane systems to investigate theinteractions of complement with membranes. In general, these studies employ complexand unusual liposome compositions which make it difficult to define fundamentalproperties of complement-activating liposomes. For simpler compositions, such as thosecommonly used in liposome drug delivery formulations, it is not clear whethercomplement activation occurs. Furthermore, it remains to be determined whether23liposomes activate the complement system in the complex biological milieu, and whetherthis leads to C3b deposition.The interactions of C3 fragments with liposomes, moreover, have not beenextensively characterized. This is surprising given the established opsonic role of C3fragments in the clearance of foreign particles. One report has studied in detail theassembly and regulation of human classical complement C3 convertases using purifiedcomplement components and PG-containing SUVs (Thielens and Colomb, 1986). Thisstudy demonstrates in vitro that stable C3 convertases can be assembled on PGcontaining SUVs, and lead to C3 activation and C3b deposition.It has been shown that liposomes coated with complement are taken up morereadily by cultured macrophages. Ingestion of MLVs containing galactosyl ceramide andcoated with 1gM antibodies directed against galactosyl ceramide by cultured mouseperitoneal macrophages is enhanced five to ten fold by addition of guinea pigcomplement (Roerdink et al., 1983). Anti-phospholipid antibodiesAnti-phospholipid antibodies are autoantibodies detected in plasma of patientswith systemic lupus erythematosus, other immunological, neoplastic, or infectivedisorders, and apparently normal people with no evidence of underlying disease (Chenget al., 1989; Lechner, 1987; Alving, 1984; Shapiro and Thiagarajan, 1982; Richards andAlving, 1982) using solid-phase immunoassays in which negatively chargedphospholipids, most commonly CL, are used as the antigen (Harris et al., 1983; Gharaviet al., 1987). Anti-phospholipid antibodies are closely related to lupus anticoagulants in24that the antibodies bind to various anionic phospholipids, CL, PS, P1, or PA, and not tonet neutral PC or PE (Gharvi et at., 1987; Pengo et al., 1987; McNeil et al., 1989).Recently, it has been shown that anti-phospholipid antibody binding to PS or CL affinitycolumns requires a cofactor, apolipoprotein H (also known as 2-glycoprotein I), whichis also present in normal human serum (McNeil et al., 1990; Galli et al., 1990). It hasbeen suggested that anti-phospholipid antibodies are masked in normal human serum andthat apolipoprotein H may play a positive regulatory role (Cheng, 1991). An in depthstudy of the interactions between anti-phospholipid antibodies and regulatory factorswould provide an understanding of the immunopathogenicity of these antibodies tonormal cell membrane lipid components.Antibodies which are capable of recognizing phospholipids on the basis of themembrane structure they form have been described (Rauch et al., 1986). In particular,antibodies against hexagonal phase phospholipids, including natural and synthetic formsof PE, have been characterized as having anticoagulant activity as measured by aprolonged partial thromboplastin time assay.Although the above studies have reported a specific interaction between antiphosphopholipid antibody and anionic phospholipid as shown by competition studies orlittle binding to neutral PC liposomes, one report by Senior et al. (1986) indicates thatnonspecific adsorption of mouse IgG antibodies onto the surface of liposomes composedof equimolar PC and CHOL can be considerable (34-89%).The opsonic role of IgG antibodies in mediating the phagocytic uptake ofliposomes by macrophages has been established in vitro and in vivo. For instance, thecovalent attachment of rabbit IgG to LUVs result in a five-fold increase in liposomeuptake by rat liver macrophages (Kupffer cells) compared to noncoated liposomes25(Derksen et al., 1987). Specific anti-dinitrophenol antibody/dinitrophenylcaproylPEantigen complexes assembled on liposomal surfaces markedly increases the rate andextent of phagocytosis by cultured macrophages (Hsu and Juliano, 1982; Lewis et al.,1980). Similarly, endocytosis of IgG anti-dinitrophenyl liposomes by Fe receptorpositive phagocytic murine tumor cells is enhanced (Leserman et al., 1980). Role of otherplasma proteinsThese proteins may enhance adherence of liposomes to cell membranes. In somecases, they may augment the opsonization process by affecting the function of opsonicreceptors. Suggestive evidence that the association of these proteins with liposomespromote their phagocytic uptake come mainly from in vitro studies involving culturedmacrophages and purified proteins. The association of these proteins with liposomes andtheir role in mediating liposome clearance in vivo are not known.1 . FibronectinsFibronectin (Mr 440 000) is a large extracellular glycoprotein present in normalhuman plasma in concentrations of 0.15-0.55 mg!ml. Purified fibronectin has beenshown to bind to liposomes of various compositions. The binding occurs in the absenceof other proteins, sugars, or divalent cations and results in an extensive aggregation of thevesicles. It was found that fibronectin bound to vesicles containing PC plus either PA,PG or PE. Hsu and Juliano (1980) reported that liposomes coated with purifiedfibronectin augment liposome uptake ten-fold. The extent to which fibronectin enhancesliposome uptake in vivo is not known.26Fibronectin, and other extracellular matrix proteins such as laminin and serumamyloid P component, can enhance the phagocytic activity of peripheral bloodleukocytes (Brown, 1986). This phagocyte-enhancing effect requires direct interaction ofthese proteins with phagocytic cells and occurs through cell surface receptors for thesemolecules. Fibronectin also affects the function of monocyte complement receptors(Pommier et al., 1983; Brown, 1986)The fibronectin receptor has been isolated and characterized by severalinvestigators (reviewed by Ruoslahti, 1988). The receptor belongs to a large family ofcell membrane glycoproteins with sequence homology, known as ‘integrins” (Hynes,1987). These glycoproteins promote several cell/cell and cell/matrix adhesion processesand specifically recognize the amino acid sequence RGD (arginine-glycine-aspartate) inthe extracellular matrix ligands.1 . ApolipoproteinsApolipoproteins A-I (Mr 28 000), A11 (Mr 17 000), AIV (Mr 44 00046 000), B(Mr 350 000550 000), C (Mr 6 000-8 000), and E (Mr 34 000) are transferred toliposome membranes (Guo et al., 1980; Luke et al., 1980; Williams and Scanu, 1986) andperhaps serve as opsonins for the uptake of liposomes by macrophages (Ivanov et al.,1985). Liposomes bearing apolipoprotein E have been shown to efficiently compete for13-VLDL receptors in cultured macrophages (Williams et al., 1987). Apolipoprotein Econtaining liposomes are capable of competing with 3-VLDL for binding toapolipoprotein E receptors on macrophages (Williams et al., 1987). Apolipoproteinshave also been suggested to play a role in the uptake of liposomes by hepatocytes via27apolipoprotein B or apolipoprotein E receptors (Williams et at., 1984; Bisgaier et al.,1989).1 . Protease inhibitorsBlack and Gregoriadis (1976) have shown that human a-2-macroglobulin (Mr750 000) or rat cx-1-macroglobulin interacts with liposomes of all charges imparting a netnegative charge on the liposomes. a-2-macroglobulin has also been shown to associatetightly with MLVs (Juliano and Lin, 1980) and it has been suggested that cx-2-macroglobulin is involved in liposome uptake.1 . C-reactive proteinC-reactive protein (Mr 105 000) is an acute-phase reactant of human serum that isable to activate complement by the classical pathway (Kaplan and Volanakis, 1974;Volanakis and Kaplan, 1974; Siegel et al., 1974; Siegel et al., 1975; Osmand et at.,1975). C-reactive protein belongs to a family of proteins known as pentraxins, that sharethe property of calcium-dependent ligand binding such as phosphoryicholine (Pepys andBaltz, 1983). Interactions between C-reactive protein and positively chargeddimyristoylPC:CHOL:SA liposomes result in both consumption of classical complementcomponents and release of trapped liposomal glucose (Richards et al., 1977). C-reactiveprotein mediated complement consumption also occurs with liposomes containing eggPC and either lysoPC or lysoPE (Volanakis and Narkates, 1981). C-reactive proteinmediated complement consumption and membrane damage are enhanced when theliposomes contain certain ceramides, including galactosyl ceramide, glucosyl ceramide,28but not sphingomyelin. C-reactive protein-dependent glucose release and, to a lesserextent, consumption of hemolytic complement activity are inversely related to the fattyacyl chain length of the liposomal phospholipid (Richards et al, 1979). Phospholipidunsaturation and liposomal cholesterol concentration also influenced both complementconsumption and membrane damage. It was demonstrated that C-reactive protein is ableto enhance macrophage activity. Furthermore, Fe receptors found on monocytes andlymphocytes appear to be important in their interaction with C-reactive protein coatedvesicles (Bama et al., 1984).1.3.3 Stabilization of ilposomes by plasma factorsThere have been a few reports which suggest that there are plasma proteins whichstabilize liposomes in the circulation. For example, Moghimi and Patel (1989b) havesuggested that liposomes composed of saturated phospholipids have no affinity for liver-or spleen-specific opsonins insasmuch as serum fails to enhance their uptake in liver andspleen macrophages, and that these liposomes attract serum dysopsonins which inhibittheir uptake by liver cells.Liu and Huang (1989) have reported that SUVs, but not LUVs composed ofDOPE and oleic acid can be stabilized by a component of human plasma. This factorappears to protect the liposomes from albumin induced leakage, most likely occurring asa result of lipid extraction by albumin. It has been shown, however, that the proteinsassociated with plasma-stabilized DOPE:oleic acid systems do not differ from thosewhich are susceptible to plasma-induced leakage. The polypeptides associated with the29DOPE:oleic acid liposomes could be removed with trypsin digestion and still retaincalcein.1.4 ASPECTS OF LIPOSOME DESIGNAFFECTING CLEARAI’ICEManipulation of liposomal physical and chemical characteristics, such as surfacecharge, vesicle diameter, and chemical composition, has been shown to affect thebiodistribution of liposomes. Much of the early work involving the factors influencingliposome pharmacokinetics has been done using poorly defined liposomal systems andhave resulted in several findings that contradict one another. To date, therefore, theinfluences of these factors in liposome clearance in vivo remain nebulous and theunderlying mechanisms producing these effects are unknown. Attempts have been madeto correlate the in vivo clearance behavior of liposomes to some physical parameter ofliposomes. For example, liposomal half-life in the circulation has been suggested to bedetermined by alterations in plasma-induced bilayer permeability of entrapped solutes(Senior and Gregoriadis, 1982a). This relation, although apparent for neutral SUVsystems, does not hold for charged vesicles or liposomes with larger diameters (Senior etal., 1985). To identify key features of liposome design affecting in vivo liposomeclearance, and to understand the basis for these effects in controlling pharmacokineticproperties are clearly necessary in order to optimize liposomal drug delivery systems.301.4.1 Physical aspects of liposomes1.4.1.1 Vesicle sizeFrom a survey of the numerous in vivo studies involving clearance of liposomes,the trend that larger systems (MLVs and LUVs) are cleared more rapidly than smallerSUVs is readily apparent (Sommerman, 1986 and the references therein; Senior et al.,1985). This size-dependent clearance was clearly demonstrated by Sommerman (1986)who showed that liposomes of various sizes produced by an extrusion technique using600, 100 and 30 nm pore sized filters are cleared at different rates when non-saturatingdoses of liposomes are administered intravenously in rats. Similar results in rats wereobtained by Sato et al. (1986) using liposomes (vesicles produced by reverse-phaseevaporation) having mean diameters of 0.15, 0.22 and 0.43 tm.Correspondingly, tissue accumulation is dependent on liposome size. SUVs areaccessible to the parencymal cells of the liver, whereas larger systems are taken upprimarily by non-parenchymal cells, mostly Kupffer cells (Roerdink et al., 1981; Poste etal., 1982; Spanjer et al., 1986). Further, SUVs composed of equimolar amounts of DSPCand cholesterol (which have a circulation half-life of approximately 16 hr for lipid dosesin excess of 60 mg lipid/kg body weight) accumulate in the bones, presumably by bonemarrow macrophages (Senior et al. 1985). As well, larger systems (d > 200 nm) appearto accummulate at higher levels in the spleen (Klibanov et al., 1990; Liu et al., 1991) andin the lung (Abra et al., 1984; Sharma et al., 1977).311.4.1.2 Surface chargeVesicle surface charge has been found to influence liposome clearance. Ingeneral, liposomes containing net charged phospholipids are cleared more rapidly thanliposomes composed of net neutral phospholipids. Whereas several reports employingrat animal models suggest that negatively charged, PA-, dicetylphosphate- organglioside-containing (Gregoriadis and Neerunjun, 1974) or PS-containing (Juliano andStamp, 1975), MLVs are cleared more rapidly than positively charged, SA-containingMLVs (lipid doses approximately 10-30 mg lipid/kg body weight), studies in mice bySteger and Desnick (1977) indicate that both positively (SA-containing) and negativelycharged (PA-containing) SUVs are both cleared very rapidly with half-lives of 2 and 4mm, respectively (lipid doses not defined).Black and Gregoriadis (1976) showed that neutral liposomes adopt a net negativesurface charge in the presence of blood plasma. The surface charge of inherentlynegative liposomes remain unchanged. The acquisition of a negative charge by neutralliposomes was apparently due to the adsorption ofa2-macroglobulin. Acquisition of anegative surface charge in the presence of plasma regardless of the original charge hasalso been observed by Wilkins and Myers (1966) using latex microspheres and attributedto the adsorption of a plasma component. It has been suggested that the surfaceproperties of the particles affect the steric arrangement of the adsorbed protein in amanner that the fixed macrophages of the reticuloendothelial system will recognize asforeign (Wilkins and Myers, 1966).321.4.2 Chemical properties of liposomes1.4.2.1 Steric barrier due to an increase in surface hydrophilicityRecent studies report the effectiveness of molecules that increase the hydrophilicnature of the liposome surface in prolonging the circulation residence times of liposomes.Such molecules include ganglioside GM1 (Allen and Chonn, 1987; Gabizon andPapahadjopoulos, 1988), amphipathic polyethyleneglycol-PE conjugates (Klibanov et al.,1990; Blume and Cevc, 1990; Senior et al., 1991; Allen et al., 1991), and polysorbate 80,a nonionic surfactant (Kronberg et al., 1990). With respect to the ganglioside molecules,the ability of these molecules to extend the circulation lifetime of liposomes appears to bespecific for the ganglioside GM1 species (Allen and Chonn, 1987; Allen et al., 1989).The mechanism by which these molecules prolong liposome circulation time is yetunknown; however, many researchers have reasonably suggested that these molecules areeffective in prolonging the ability of liposomes to circulate by providing a steric barrierthat inhibits the liposome association of blood opsonins or the liposome interactions withcells (Allen et al., 1989; Klibanov et al., 1991; Senior et al., 1991).Gabizon and Papahadjopoulos (1988) have demonstrated that liposomescontaining ganglioside GM1 accumulate to significantly higher levels in solid tumors thando liposomes without ganglioside GM1. Further, inclusion of ganglioside GM1 does notappear to inhibit the ability of immunoliposomes to attach to target cells(Papahadjopoulos and Gabizon, 1987; Maruyama et al., 1990). These studies suggestthat ganglioside 0M1 provides only a weak steric barrier effect (Klibanov et al., 1991).On the other hand, the inclusion of PE-linked polyethyleneglycol (5000) appears to33diminish the binding efficiency of immunoliposomes to target cells (Klibanov et al.,1991). The inclusion of as little as 0.72 mol% PE-linked polyethyleneglycol (5000)completely abolishes the agglutination of liposomes containing N-biotinaminocaproylPEby streptavidin (Klibanov et al., 1991). These studies strongly support the hypothesisthat polyethyleneglycol (5000) derivatives of PE prolong liposome circulation time byproviding a strong steric barrier that prevents close contact with another liposome or cell.The reduced interaction of liposomes with blood proteins has been suggested byseveral findings. The incorporation of mono-, di-, or trisialogangliosides into theliposome bilayer reduces the leakage of calcein trapped in the aqueous space of SUVs orLUVs in the presence of human plasma (Allen et al., 1985). Similarly, incorporation ofPE-coupled polyethyleneglycol (5000) into liposomes decreases the rate of leakage ofentrapped carboxyfluorescein in the presence of human plasma (Blume and Cevc, 1990).Recently, Senior et al. (1991) used an aqueous two-phase partitioning technique to showthe reduced adsorption of plasma components to liposomes containingmonomethoxypolyethyleneglycol-DPPE. Phospholipid structureSaturated phospholipids in the presence of cholesterol decrease the rate ofclearance of SUVs from the circulation (Gregoriadis and Senior, 1980; Senior andGregoriadis, 1982b). These saturated phospholipids have been shown to decrease thepermeability of liposome entrapped solutes (Senior and Gregoriadis, 1982a and 1982b).The structural integrity of liposomes in serum is enhanced by utilizing etherand/or carbamyl analogs of 1,2-diester phosphatidylcholine (Agarwal et a!., 1986;34Hermetter and Paltauf, 1983; Bali et a!., 1983), or phospholipase-resistant dialkyl analogsof PC (Deshmukh et a!., 1978). Structurally modifying the ester bond in the phospholipidcomponent of liposomes reduces the efflux of entrapped carboxyfluorescein or calceinand lipid transfer to serum proteins.Liposomes composed of polymerizable phospholipids have exhibited someunique aspects in terms of physical stability, permeability properties and interactions withcells (Regen, 1987; Juliano et al., 1985). These liposomes are more stable in plasma thanliposomes composed of “conventional” phospholipids. These liposomes, however, havebeen found to be more rapidly taken up by reticuloendothelial cells (Juliano et al., 1985;Krause et al., 1987). One report demonstrates that these liposomes bind similar proteinsas conventional liposomes (Bonte et a!., 1987). Cholesterol and a-tocopherol contentSolute retention by liposomes introduced into the circulation is enhanced whencholesterol is included in the lipid composition (Papahadjopoulos et a!., 1973; Inoue,1974). This appears to act by reducing the net transfer of phospholipid to HDL incirculating blood (Kirby et al., 1980; Kirby et al., 1980; Gregoriadis and Davis, 1979).As well, cholesterol has been shown to have an inhibitory effect on the uptake ofliposomes (SUVs and REVs containing[‘4C]inulin) by liver and spleen (Pate! et al.,1983) and on the uptake and intracellular degradation of liposomes by cultured Kupffercells (Roerdink et a!., 1989). Cholesterol-free liposomes are cleared more readily thanthe cholesterol-rich (46.6 mol%) liposomes.35Egg PC SUVs containing 15 mol% cc-tocopherol exhibit markedly improvedserum stability properties compared to pure egg PC SUVs or egg PC SUVs containing 37mol% cholesterol (Haiks-Miller et a!., 1985). The tocopherol-containing liposomes wasshown to be stable in the presence of 15% fetal calf serum or apo HDL as measured bycarboxyfluorescein release.1.5 OVERALL OBJECTIVESThe studies described in this thesis are aimed at establishing whether bloodproteins influence the clearance behavior of liposomes in vivo by characterizing the bloodproteins associated with liposomes that have been described to have markedly differentclearance properties. Further, some of the blood protein interactions liposomes encounterin the circulation that influence their clearance behavior are identified. This providesinsight into how various aspects of liposome design influence liposome circulationlifetimes, and will potentially lead to improved design of liposome drug delivery systemsdisplaying favorable and predictable pharmacokinetic properties.Recent studies underline the need to understand the interactions liposomesencounter in the circulation that lead to enhanced clearance rates. Polyethyleneglycolderivatives of PE were found to extend circulation lifetimes of liposomes (Klibanov etal., 1991). The hydrophilic masking of the liposome surface that these polymers provideis believed to diminish the adsorption of plasma proteins responsible for their immunerecognition and consequently, the adsoption to and uptake by phagocytic cells. Thestrong steric barrier that these polymers provide, however, appears to also inhibitimmunoliposome/target cell interactions. Thus, liposomes are in the circulation for36longer periods but are not able to interact with their target cell. On the other hand,ganglioside G extends the circulation lifetime of liposomes by providing only a weaksteric barrier that allows the immunoliposomes to interact with target cells(Papahadjopoulos and Gabizon, 1987; Maruyama et al., 1990). By understanding thespecific mechanisms involved in liposome clearance, and identifying the proteininteractions that enhance liposome uptake by phagocytic cells of the reticuloendothelialsystem that these molecules inhibit, one can better design liposomes which may be ableto specifically inhibit the interactions of these specific opsonins but still allow closeapposition of liposomes and cell membranes. This balance is an important considerationfor designing liposome carrier systems.There are two major objectives to the studies described in the following chapters.The first is to establish the potential of liposomes to activate the complement system inthe complex biological milieu, the effect of lipid composition on the ability of liposomesto activate complement, and the relation between the activation potential of liposomesand liposome clearance in vivo (Chapters 2 and 3). The importance of the complementsystem in providing the effector molecules that leads to enhanced phagocytosis,activation of macrophage, and lysis of foreign particles suggests that the interactions ofliposomes with the complement system, and the aspects of liposome design that affectthis interaction are essential to define. As discussed in Section, previous studieshave failed to clearly define fundamental properties of complement-activating liposomes,such that it is not known whether lipid compositions that are commonly employed inliposome drug delivery formulations are complement-activating.The second main objective is to establish the role of blood proteins in mediatingliposome clearance in vivo. In particular, the amount of associated blood proteins will be37related to the liposome clearance behavior in vivo (Chapter 4). The blood interactionsliposomes encounter in the circulation are undoubtedly more complex than those found invitro employing incubations with isolated plasma or serum. Thus, in order to achieve thisaim, a rapid and convenient method for efficiently recovering LUVs from bloodcomponents was developed (described in Chapter 3). This technique enabled thecharacterization of the in vivo liposome/blood protein interactions employing LUVsexhibiting markedly different clearance properties.38CHAPTER 2 ACTIVATION OF THE CLASSICAL AND ALTERNATIVEPATHWAYS OF COMPLEMENT BY LIPOSOMES2.1 INTRODUCTIONComplement activation by the alternative pathway occurs when C3 interacts withparticulate material such as bacterial cell wall constituents, immune complexes orheterologous erythrocytes (Muller-Eberhard, 1988). The C3b, the activated form of C3,which is particle bound has two possible fates. It may interact with factor B to produce aserine protease which amplifies the activation. Alternatively, the C3b may be bound byfactor H which acts to stop the cascade by providing cofactor activity for the C3binactivating protease, factor I (Molines and Lachmann, 1988). The characteristics of thesurface which promote the binding of one factor over the other are not well understood.Studies of both bacteria and mammalian cells have suggested that sialic acid is animportant regulator of complement activation (Edwards et al., 1982; Lambre et al., 1982).Complement resistant erythrocytes (Pangburn and Muller-Eberhard, 1978) and thecomplement resistant strains of Escherichia coli (Levine et al., 1983; Pluschke et al.,1983) both contain more sialic acid residues than their complement-sensitivecounterparts. These charged residues are thought to influence the binding affinities offactor B and factor H (Kazatchkine et al., 1979; Michalek et al., 1988).Complement activation by the classical pathway occurs when the first componentof complement, Cl, binds either to immunoglobulin or directly to a particle surface. Theantibody-independent activation of Cl has been described for a wide variety ofsubstances including DNA and RNA (Agnello et al., 1969), urate crystals (Giclas et al.,391979), gram-negative bacteria (Cooper and Morrison, 1978), retroviruses (Cooper et al.,1976) and heparin-protamine (Rent et al., 1975). The binding of Cl via the Clq moietyrequires the presence of repeating binding sites in order to bind the multiple heads of theClq molecule (Cooper, 1983). The other factor identified to be important in binding Clqis a negative surface charge (Loos, 1982; Kovacsovics et al., 1985).Liposomes can be used as simple model membranes to study the influence oflipid composition on complement activation. This approach has been used todemonstrate the inhibitory effects of sialic acid on complement activation (Michalek etal., 1988). Liposomes containing gangliosides or erythrocyte glycophorin are relativelyinefficient complement activators while liposomes containing asialoglycolipids orneuraminidase-treated glycophorin fail to inhibit complement activation (Okada et al.,1982a and 1982c). Recent studies using liposomes have demonstrated that sialic acidacts to promote the binding of factor H to C3b (Michalek et al., 1988).The interaction of complement proteins with liposomes also has importantconsequences for the use of liposomes as drug delivery systems. Deposition of opsonicforms of C3 on the liposomal surfaces may contribute significantly to liposome clearancefrom the circulation, a key problem associated with liposomal drug delivery systems.This contribution is suggested by the recent findings of Allen and Chonn (1987) thatliposomes containing sialic acid in the form of gangliosides have significantly extendedcirculation times. An understanding of the complement-activating properties ofliposomes, therefore, may allow the development of liposomal systems which have longbiological half-lives.To date, the activation of the complement system by liposomes has beendescribed for only a few specific lipid compositions. These compositions include40liposomes containing haptenated lipids (Okada et al., 1982b), phosphatidylserine withphosphatidylethanolamine (Comis and Easterbrook-Smith, 1986), cardiolipin(Kovacsovics et a!., 1985), stearylamine in the presence of galactosyl ceramide(Cunningham et a!., 1979), cerebrosides in dimyristoylphosphatidylethanolamine(Michalek et a!., 1988) or saturated phosphatidylethanolamine in saturatedphosphatidyicholine liposomes containing cholesterol (Mold, 1989). From reviewingthese studies, no fundamental property of complement-activating liposomes could bereadily defined because of inconsistencies in the reports and because of the complexsystems studied.The studies described in this chapter establish the membrane properties ofcomplement-activating liposomes. It is demonstrated that surface charge is an essentialcomponent of complement-activating liposomes in human and guinea pig whole serumsystems. The nature of the charge, whether negative or positive, appears to dictate whichpathway of the complement system is activated. Normal human or guinea pig serum wasincubated with liposomes, followed by determining the residual hemolytic activity of theserum as a measure of complement activation. Negatively charged liposomes containingPG, PA, CL, P1 or PS activated complement in aCa2-dependent manner suggestingactivation occurred via the classical pathway. Positively charged liposomes containingSA or DOTAP activated complement via the alternative pathway. Neutral liposomes,PC:CHOL (55:45) and PC:CHOL:DPPE (35:45 :20), failed to activate complement asmeasured by complement hemolytic assays. Immunoblot analysis of the proteinsassociated with PG-containing liposomes showed that C3b and C9 were associated withthese liposomes. Inasmuch as C3b functions as an opsonin, the coating of the liposomes41with C3b may be responsible for the recognition of liposomes by the immune system asforeign particles.2.2 METHODS AND MATERIALS2.2.1 Preparation of liposomesMLVs and freeze-thawed multilamellar vesicles (FMLVs) were preparedaccording to established methods (Hope et al., 1986). Briefly, the appropriate amounts oflipids in chloroform solutions were added to a borosilicate glass test tube and the lipidmixture dried down to a thin film using a stream of nitrogen gas. The lipids were thenplaced under high vacuum for 2 hr to remove residual chloroform. The appropriateamount of buffer was then added to the dried lipids with vigorous vortexing. Theresulting MLV suspension was transferred to cryovials and subjected to five cycles offreezing under liquid nitrogen temperatures and thawing to yield FMLVs.LUVs were prepared by extrusion of FMLVs through 100 nm pore sizedpolycarbonate filters (Nuclepore, Pleasanton, CA) using an extrusion device (LipexBiomembranes, Vancouver, Canada) as previously described (Hope et al., 1985; Hope etal., 1986). Lipids were purchased from the following companies: Egg PC, egg PA, eggPG, bovine liver P1, bovine brain PS, bovine heart CL, and DPPE from Avanti PolarLipids, Pelham, AL; SA and CHOL from Sigma; 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) was a generous gift from Dr. J. R. Silvius (McGillUniversity, Montreal, Canada). These lipids were used without further purification. Thestandard liposome preparations used in the functional complement activation studies42consisted of PC:CHOL (55:45 mol/mol) LUVs extruded through two stacked 100 nmpolycarbonate filters. The average size of these liposomes was 98±23 nm (n=6) asdetermined by quasi-elastic light scattering analysis using unimodal analysis on aNICOMP Model 270 Submicron Particle Sizer (NICOMP Instruments, Santa Barbara,CA). Addition of 20 mol% anionic or cationic lipids did not alter the average sizesignificantly. The liposome suspensions were 100 mM total lipid in isotonic verona!buffered saline (VBS, 10 mM sodium barbital, 145 mM NaC1, pH 7.4).2.2.2 Serum, complement and antiserumGuinea pig serum (Calbiochem Behring, La Jolla, CA) was purchased in alyophilized form, reconstituted in the buffer provided and stored at -70°C. Human serumwas prepared from venous blood from 20 healthy individuals (10 males, 10 females) byimmediately cooling the drawn blood to 0°C using an ice/water bath, centrifuging (2500rpm, 10 mm, 4°C) to pellet the blood cells, pooling the clear plasma into a glass beaker,incubating the pooled plasma at 37°C for 45 mm, and recessing the clot with the aid of aclotting stick. The serum was aliquotted and stored at -70°C. Clq deficient serum waspurchased from Sigma Chemical Co., St. Louis, MO. Purified guinea pig Cl and C2, andhuman Clq, C3 and C9 were purchased from Diamedix, Miami, FL. Trypsin-treated C3was prepared by incubating 50 of 1 mg/ml purified C3 with 5 il of 1 mg/ml Type XIIIL-p-tosylamino-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) at 37°C for 5mm followed by the addition of 5 il of 5 mg/ml soybean trypsin inhibitor (Sigma).Rabbit antisera to human C3d and C9 were purchased from Calbiochem. Peroxidase43conjugated goat anti-rabbit IgG F(ab’)2 fragments were purchased from The JacksonImmunoResearch Laboratories, Bar Harbor, ME.2.2.3 Residual total complement hemolytic assaysLiposome suspensions were serially diluted in dextrose gelatin VBS (DGVB)containing 50% VBS, 2.5% glucose, 0.5 mM MgC12,0.15 mM CaCl2 and 0.1% gelatin(DGVB2+). Fifty !Il of 5% guinea pig serum in DGVB2was incubated with an equalvolume of liposome suspension at 37°C for 1 hr. After the incubation period, themixtures were diluted with 150 l of DGVB2and kept on ice. For studies involvinghuman complement, 50 l of 25% human serum in DGVB2was incubated with an equalvolume of liposome suspension at 37°C for 30 mm. After the incubation period, themixtures were diluted with 150 1il of DGVB2and kept on ice.Residual total complement hemolytic activity of liposome-treated serum wasmeasured according to established methods (Whaley, 1985). The hemolysis ofheterologous erythrocytes sensitized with antibody is complement mediated, involvingactivation via the entire classical pathway. The level of hemolysis is proportional to thelevel of complement present in the serum. A reduction in the complement hemolyticactivity of the serum after incubation with liposomes implies complement consumptionand activation by liposomes had occurred (see Fig. 2.1). Briefly, sheep erythrocytes weresensitized with rabbit antibody to sheep erythrocyte stroma (EA), washed and suspendedat 1.5 x 108 cells/ml in DGVB2. In triplicates, 50 of EA cells was incubated with 50l of liposome-treated serum for 30 mm at 37°C. Samples were then diluted by theaddition of 2 ml of DGVB containing 40 mM EDTA (EDTA-GVB). Unlysed EA cells44Figure 2.1. Diagramatic representation of complement hemolytic assays. Thehemolytic level of antibody-sensitized erythrocytes (EA cells) is directly related to theamount of functional complement present in serum. Upon incubation with serum,complement-activating liposomes will consume complement, resulting in a reduction inthe functional complement hemolytic levels.NonactivatorLiposomesIc3J0C3QC3C3ActivatorLiposomesC3bQc3b0c3b3b43C3bQc3bEAC3bC3bQcab0reduction in lysis levelsCC] available tolyse EA cells isreduced by ilposomesactivating complementC3—C3bEA00a0100% lysls[C] before and afterincubation of serumwIth liposomesIs the same45were pelleted by centrifugation and the amount of hemoglobin released into thesupernatant was quantitated spectrophotometrically at 414 nm.2.2.4 Measurement of guinea pig alternative complement pathway activation byliposomesLiposome suspensions were serially diluted in DGVB containing 0.5 mM MgCI2(Mg-DGVB). Ca2-depleted guinea pig serum was obtained by treating the guinea pigserum with 10% 0.2 M EDTA (pH 7.4) at 37°C for 5 mm, followed by supplementing theserum with 10% 0.25 M MgC12. Fifty d of 5%Ca2-depleted guinea pig serum wasincubated with an equal volume of liposome suspension at 37°C for 1 hr. After theincubation period, the mixtures were diluted with 150 tl of Mg-DGVB and kept on ice.Residual guinea pig alternative pathway complement hemolytic activity wasdetermined in a Ca2-free assay using EAC1,4,2 cells as the target cells (Whaley, 1985).Immediately before performing the hemolytic assays, the EAC1,4,2 cells were assembledas follows: EA cells (1 X i0 cells/ml in DGVB2;1 ml) were incubated with 5000 unitsof guinea pig Cl for 5 mm at 37°C; 5000 units of human C4 were added and theincubation continued for 7 mm at 37°C; 250 units of guinea pig C2 were then added andthe incubation continued for 7 mm at 37°C; finally, the cells were washed andresuspended at 1.5 x 108 cells/ml in Mg-DGVB. In triplicates, 50 l of EAC1,4,2 cellswas incubated with 50 d of liposome-treated serum for 30 mm at 37°C. Samples werethen diluted by the addition of 2 ml of EDTA-GVB. Unlysed EAC1,4,2 cells werepelleted by centrifugation and the amount of hemoglobin released into the supernatantwas quantitated spectrophotometrically at 414 nm.462.2.5 Measurement of human alternative complement pathway activation byliposomesHuman alternative complement pathway activation was determined by incubating500 1 of 20 mM liposomes in EGTA-treated DGVB, containing 0.5 mM MgCI2(MgEGTA-DGVB) with 500 .il of 20% human serum in Mg-EGTA-DGVB at 37°C for 30mm and kept on ice.Residual alternative pathway human complement hemolytic activity wasdetermined according to published methods (Whaley, 1985). By using rabbiterythrocytes, activators of the alternative pathway of human complement, as target cellsin a Ca2+free hemolytic assay, the activation of the alternative pathway of humancomplement by liposomes was assessed. Rabbit erythrocytes were washed andresuspended at 1 x 108 cells/ml in Mg-EGTA-DGVB buffer. In duplicates, 10, 20, 40,60, 80 and 100 il aliquots of the serum/liposome incubation mixtures were incubatedwith 100 tl of rabbit erythrocytes in a final assay volume of 200 d for 1 hr at 37°C. MgEGTA-DGVB was used as the buffer. After the incubation period, 2 ml EDTA-GVBwas added to the samples. Unlysed rabbit erythrocytes were pelleted by centrifugationand the amount of hemoglobin released into the supematant was quantitatedspectrophotometrically at 414 nm.2.2.6 Liposome lysis fluorescence assayLiposomes were prepared as above except that the liposomes were in isotonic 20mM HEPES, pH 7.4, buffer containing 100 mM carboxyfluorescein [purchased fromEastman Kodak and purified according to Weinstein et a!. (1983)] and the external47carboxyfluorescein chromatographically separated using Sephadex G-50 (Sigma)equilibrated with 20 mM HEPES, pH 7.4, 150 mM NaC1 buffer. Liposome-treated serumwas prepared by incubating 200 1il PC:CHOL:PG or PC:CHOL:CL liposomes in VBSwith 1800 l normal human serum at 0°C for 30 mm. One ml aliquots of theliposome/serum incubation mixtures were pipetted into thick walled Beckman 1.5 mlmicroultracentrifuge tubes (Beckman Instruments, Inc., Fullerton, CA) and centrifugedusing the Beckman table microultracentrifuge TL100 for 30 mm, 100,000 rpm at 4°C.The top liposome layer was carefully pipetted off and the clear serum was pooled andstored on ice. The liposome lysis fluorescence assay is detailed elsewhere (Weinstein etal., 1984; Lelkes, 1984). To 2 ml 25% normal human serum or liposome-treated humanserum diluted with DGVB2,5 jtl of carboxyfluorescein-containing PC:CHOL:PG orPC:CHOL:CL liposomes was added and incubated for 30 mm at 37°C. The fluorescenceof the mixture was read using a Perkin Elmer LS5O fluorimeter (Perkin-Elmer Corp.,Norwalk, CT) with the excitation and emission wavelengths set at 492 and 520 nm,respectively. The fluorescence of the total releaseable carboxyfluorescein was measuredby adding 100 10% Triton X-100 to the incubation mixtures.2.2.7 Immunoblot analysis of proteins associated with liposomesTo 2 ml of 100 mM PC:CHOL (55:45) or PC:CHOL:PG (35:45:20) FMLVs, 8 mlof undiluted human serum was added and the mixture incubated at 37°C for 30 mm. Theliposomes were isolated by centrifugation (10,000 x g, 10 mill, 4°C) and washed fivetimes with 50 ml VBS. The final liposome pellet was resuspended in 2 ml VBS.Proteins from the washed liposomes were extracted according to published methods48(Wessel and Flugge, 1984). Briefly, to 1 ml of resuspended liposomes, 4 ml methanol, 1ml chloroform and 1.5 ml distilled water were added with vortexing after each addition.The two phase system generated was separated by centrifugation [20 mm, 3000 rpm,Silencer H-103N Benchtop centrifuge (Western Scientific, Vancouver, Canada)]. Theupper phase was aspirated such that the protein at the interface was left with a slightamount of upper phase. Then 1.5 ml of methanol was added and the protein wasprecipitated by centrifugation (30 mm, 3500 rpm). The supernatant was aspirated and thepellet dried under nitrogen. The dried pellet was resuspended to a concentration of 1mglml in VBS containing 0.5% SDS. Protein concentration was estimated using theBCA protein assay (Pierce, Rockford, IL). Protein separation was performed by SDSPAGE using the automated electrophoresis apparatus, the PhastSystem (Pharmacia,Piscataway, NJ), on precast 7.5% homogenous and 10-15% gradient resolving PhastGels(Pharmacia). Pre-stained SDS-PAGE standards (Diversified Biotech, Newton, MA) wereused to estimate the molecular weights of the proteins. Electrophoretic transfer of theseparated proteins onto nitrocellulose (Nitroplus 2000, Micron Separations, Westboro,MA) was performed using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad,Richmond, CA) according to the instructions of the manufacturers at constant voltage of50 V for 45 mm. Immunoblot analysis was performed by blocking the nitrocellulose blotwith a buffer containing 10 mM Tris, 150 mM NaC1, 3% BSA (Sigma) and 1% normalgoat serum (The Jackson ImmunoResearch Laboratories), pH 7.4 (blocking buffer)overnight at 4°C; incubating 0.1% primary antibody in blocking buffer for 2 hr at roomtemperature; incubating 0.02% peroxidase-labeled secondary antibody in blocking bufferfor 1 hr at room temperature; and finally, developing the labeled bands using 0.3% 4-49chloro-1-naphthol (Sigma) in 10 mM Tris, pH 7.4, 150 mM NaC1 buffer with 0.018%hydrogen peroxide.2.3 RESULTS2.3.1 Activation of guinea pig and human complement by PG-containing liposomesComplement hemolytic assays were used to detect activation of the complementsystem by liposomes. Guinea pig serum was incubated with liposomes composed ofPC:CHOL (55:45) and PC:CHOL:PG (35:45:20) for one hour at 37°C and the residualcomplement hemolytic activity was quantitated. A reduction in the residual complementhemolytic activity of the serum signifies activation of the complement system byliposomes. The detection of this reduction is sensitive to the initial levels of complementpresent in the serum. Preliminary studies, therefore, were performed to determine theoptimal serum concentration for the hemolytic assay to detect complement consumption(Fig. 2.2). Diluting the serum with DGVB2reduced the levels of complement to anoptimal range such that when the complement system was not activated, theconcentration of the complement proteins was nonlimiting and 100% hemolysis of thetarget cells occurred; when the complement system was activated, the concentration ofthe complement proteins was limiting and a reduction in the hemolytic levels occurred.A 1/20 dilution of guinea pig serum was chosen for subsequent hemolysis because thisresulted in an optimal concentration for the detection of changes in functionalcomplement levels involving PC:CHOL:PG (35:45:20) liposomes.50..o0>.‘>C)(‘S0>.0Ea,a,Ea,0E00Figure 2.2. Effect of serum dilution on the sensitivity of the total complementhemolytic assays to detect consumption ofguinea pig complement. Fifty 1il of undiluted(undil) or diluted guinea pig serum in DGVB2was incubated with an equal volume ofDGVB2(11), 20 mM PC:CHOL (55:45) () or 20 mM PC:CHOL:PG (35:45:20) () for1 hr at 37°C. After this incubation period, the mixtures were further diluted with 150 tlDGVB2. The residual complement hemolytic activity of the liposome-treated serumwas determined by the complement hemolytic assays and expressed as a percentage ofthe total hemolytic level determined by lysis of the EA cells with 2 ml distilled water (forexperimental details see “Methods and Materials”).100806040200undil 115 1110 1I15 1120 1/25 1/30 1I40Serum dilution51Figure 2.2 shows that the complement hemolytic activity of guinea pig serumexposed to 1 imol PC:CHOL (55:45) liposomes was the same as serum incubated withDGVB2buffer. Moreover, Figure 2.2 shows that PC:CHOL:PG (35:45:20) liposomesreduce the ability of guinea pig serum to lyse the BA target cells when compared toserum incubations with DGVB2buffer. The detection of this consumption was affectedby the ratio of the complement concentration in the serum to the amount of liposomes inthe incubation mixture (Figs. 2.2 and 2.3).Human complement activation by PG-containing liposomes was alsocharacterized. The hemolytic assays for human serum were optimized essentially as forguinea pig serum and it was found that the optimal dilution of human serum was 1/4.Figure 2.3 shows that increasing the PG content of the liposomes made the liposomesmore potent complement activators. By increasing the surface density of PG, the amountof liposomes required to detect a consumption in the total complement hemolytic activityof human serum was reduced. PG was evidently an important component ofcomplement-activating liposomes as liposomes without PG failed to activate humancomplement (Fig. 2.3). Figure 2.3 also shows that liposomes composed of phospholipidscontaining unsaturated fatty acyl chains are better activators of human complement thanthose containing saturated fatty acyl chains. Less surface charge is required in theunsaturated systems to reduce the functional complement levels.The effect of cholesterol on the complement-activating potential of the liposomesis shown in Figure 2.4. The inclusion of 45 mol% cholesterol in both the saturated andunsaturated liposomal systems provided a more potent complement-activating surface.52:‘1)Figure 2.3. Effect of PG density on human complement activation by saturated andunsaturated liposomes. Liposomes composed of PC (0), PC:PG (9:1) (A), PC:PG (8:2)(o), PC:PG (7:3) (v), PC:PG (6:4) () or PC:PG (5:5) ( at various lipid concentrationswere incubated with an equal volume of 25% human serum in DGVB2+. Their effect onthe residual complement hemolytic activity was determined by the complementhemolytic assays and expressed as a percentage of the hemolytic level of human serumincubated with DGVB2. The PC and PG species are DPPC and DPPG for the saturatedliposomes (panel A), and DOPC and DOPG for the unsaturated liposomes (panel B).100806040200100—VA806040200.001 0.01 0.1Amount of lipid (jmo1)53Figure 2.4. Effect of CHOL on human complement activation by saturated andunsaturated liposomes. Liposomes composed of PC:CHOL (55:45) (0), PC:CHOL:PG(45:45:10) (A), PC:CHOL:PG (35:45:20) (Q), PC:CHOL:PG (25:45:30) (v) orPC:CHOL:PG (15:45:40) (Os) at various liposome concentrations were incubated with25% human serum in DGVB2. Their effect on the residual complement hemolyticactivity was determined by the complement hemolytic assays and expressed as apercentage of the hemolytic level of human serum incubated with DGVB2. The PC andPG species are DPPC and DPPG for the saturated liposomes (panel A), and DOPC andDOPG for the unsaturated liposomes (panel B).:,gQIAmount of Lipid (mo1)0.001 0.01 0.1542.3.2 Complement activation by other charged liposomesAfter the demonstration of the functional activation of the complement system byPG-containing liposomes, it was of interest to determine whether other negativelycharged phospholipids were capable of transforming liposomes into complement-activating membranes. These include CL, P1, PS, and PA, all of which are acidicphospholipids bearing net negative charges at pH 7.4. As shown in Figure 2.5, theliposomes containing these anionic phospholipids, present at 20 mol% of total lipid, werecapable of consuming the complement hemolytic activity of guinea pig serum in a dosedependent manner. Liposomes containing CL were more effective in activating guineapig complement than the other charged phospholipids. This may arise from the fact thatCL bears two negative charges at neutral pH, thus giving rise to a higher surface chargeat the same molar ratio.The importance of surface charge is indicated by the fact that neutral liposomeswere not capable of inhibiting the hemolytic activity of guinea pig serum, even at 50 mMlipid concentrations. Liposomes composed of 20 mol% DPPE (which is net neutral at pH7.4) failed to activate guinea pig complement. Moreover, systems with a positive surfacecharge were capable of consuming guinea pig complement as indicated by the fact thatliposomes containing 20 mol% SA or 20 mol% DOTAP (a non-exchangeable positivelycharged lipid) activated guinea pig complement.Figure 2.6 shows that human complement was activated by similar activatorsurfaces as guinea pig complement and that surface charge is an important determinant.Notably, much less lipid was required to activate human complement.55Figure 2.5. Dose response curves of guinea pig serum incubated with LUVs. A) Netneutral liposomes, PC:CHOL (55:45) (0) and PC:CHOL:DPPE (35:45:20) (0); B)Negatively charged I iposomes, PC:CHOL:PG (35:45:20) (v), PC:CHOL:PA (35:45:20)(), PC:CHOL:PI (35:45:20) (Lx), PC:CHOL:PS (35:45:20) (0) and PC:CHOL:CL(35:45:20) (0); and C) positively charged liposomes, PC:CHOL:SA (35:45:20) (A) andPC:CHOL:DOTAP (35:45:20) (•) were incubated with 5% guinea pig serum inDGVB2. Their effect on the residual complement activity was detennined bycomplement hemolytic assays and expressed as a percentage of the hemolytic level ofguinea pig serum incubated with DGVB2.1 008060Q0L)0VA40200.001 0.01 0.1Amount of Lipid (,imol)156>0000Uz0(ID0Figure 2.6. Dose response curves of human serum incubated with LUVs. A) Netneutral liposomes, PC:CHOL (55:45) (Q) and PC:CHOL:DPPE (35:45:20) (0); B)Negatively charged liposomes, PC:CHOL:PG (35:45:20) (A), PC:CHOL:PA (35:45:20)(Q), PC:CHOL:PI (35:45:20) (a), PC:CHOL:PS (35:45:20) (0) and PC:CHOL:CL(35:45:20) (v); and C) positively charged liposomes, PC:CHOL:SA (35:45:20) (.) andPC:CHOL:DOTAP (35:45:20) () were incubated with 25% human serum in DGVBTheir effect on the residual complement activity was determined by complementhemolytic assays and expressed as a percentage of the hemolytic level of guinea pigserum incubated with DGVB2.100 =Q-O-Q-Q-D=80 A6040201008060402000.0001 0.001 0.01 0.1Amount of Lipid (,imol)572.3.3 Mechanism of complement activation by liposomesBy excluding Ca2+ from the assays, alternative pathway activation can bedistinguished from classical pathway activation because the initiation complex of theclassical pathway, the Clqr2scomplex, requires Ca2. Figure 2.7 shows that thenegatively charged liposomes were not capable of consuming complement in Ca2+depleted serum. This suggests that the negatively charged liposomes do not activate thealternative pathway and must therefore activate the classical pathway. Under similarconditions, incubation of guinea pig serum with positively charged liposomes resulted ina reduction in the serum C3-C9 levels available to lyse the EAC1,4,2 cells (Fig. 2.7).This implies that the positively charged liposomes activated the alternative pathway. Byusing rabbit erythrocytes, activators of the alternative pathway of human complement, astarget cells in the Ca2-free hemolytic assay, the activation of the alternative pathway ofhuman complement by liposomes was detected. Figure 2.8 shows that only the positivelycharged liposomes were capable of reducing the hemolytic activity of human serumunder conditions where only the alternative pathway is functional.To demonstrate that the reduction in residual complement hemolytic activity wasnot due to the liposomes binding Ca2 thereby reducing the Ca2 ion concentration tolevels where the Cl complex is dissociated and not active, binding studies with 45Ca2were done. These studies showed that the liposomes did not significantly deplete theCa2 concentration of the supernatant in the assays (0.15 mM) (Table 2.1).Membrane lysis resulting from activation of the entire complement cascade wasdemonstrated using liposomes containing entrapped carboxyfluorescein (Fig. 2.9). This58Figure 2.7. Complement activation by liposomes in Ca2-depleted guinea pig serum.Liposomes composed of PC:CHOL (55:45) (A), PC:CHOL:PG (35:45:20) (v),PC:CHOL:PI (35:45:20) (), PC:CHOL:SA (35:45:20) (0) and PC:CHOL:DOTAP(35:45:20) (D) were incubated with EDTA-treated, MgLsupplemented guinea pigserum. Their effect on the residual complement activity was determined by a Ca2+freehemolytic assay and expressed as a percentage of the hemolytic level ofCa2+depeletedguinea pig serum incubated with Mg-DGVB.‘—‘ 100>--.->o 80-4-I:____0.001 0.01 0.1Amount of Lipid (/2moI)59—S>..-4->-4-C-)0-4->—0EwI-4--Ca)Ea)0E0C-)Figure 2.8. Alternative pathway complement hemolytic activity of human serumincubated with liposomes. Liposomes composed of PC:CHOL (55:45) (v),PC:CHOL:DPPE (35:45:20) (), PC:CHOL:PG (35:45:20) (A), PC:CHOL:PA (35:45:20)(o) and PC:CHOL:DOTAP (35:45:20) 4 at 20 mM concentrations were incubated withan equal volume of 20% Ca2+depleted human serum. A control mixture of Ca2+depleted human serum and Mg-EGTA-DGVB (0) was included. Various amounts of theliposome/serum mixtures were incubated with rabbit erythrocytes as detailed in “Methodsand Materials” to measure the effect of the liposomes on the residual alternative pathwaycomplement hemolytic activity of the serum.1 00 H80±60-i-40200- —0—— w w -20 40 60 80 100Volume of Serum (1u1)60Table 2.1 45Ca2remaining in the supernatant after incubation ofDGVB2 buffer orserum with PC:CHOL:PG (35:45:20) FMLVs. FMLVs (250 iii) were incubated with anequal volume of either DGVB2 buffer containing a trace amount of 45Ca2 or 5%guinea pig serum in DGVB2buffer containing a trace amount of45Ca2for 1 h at 37°C.The incubation mixtures were then centrifuged (12 000 rpm, 15 mm) to pellet theFMLVs. One hundred 111 of the supernatant was assayed for 45Ca2radioactivity.Concentration of % 45Ca2radioactivity remainingPC:CHOL:PG FMLVs (mM) in the supematantDGVB2 serum20 90.2 98.010 91.0 99.71 90.5 99.80.1 89.9 99.461Figure 2.9. Complement-mediated lysis of liposomes containing entrappedcarboxyfluorescein using liposome-treated serum. Two ml of 25% normal humanserum (Q ) or 25% human serum treated with either PC:CHOL:PG (35:45:20) orPC:CHOL:CL (35:45:10) liposomes ( ) was incubated with 5 tl PC:CHOL:PG(35:45:20) or PC:CHOL:CL (35:45:10) liposomes containing entrappedcarboxyfluorescein for 30 mm at 37eC. The fluorescence due to the releasedcarboxyfluorescein was measured as described in “Methods and Materials” andexpressed as a percentage of the total releaseable carboxyfluorescein determined by theaddition of 100 tl 10% Triton X-100.ci)U,asci)ci)Cci)C)ci,ci)I0D0-oas00iiPC:CHOL:PG PC:CHOL:CL62assay was used to study the effect of pre-adsorbing the serum with anionic liposomes onthe ability of the treated serum to lyse the liposomes. Figure 2.9 shows that incubating10 tmo1 of lipid with 1.0 ml 90% normal human serum for 30 mill at 0°C, followed byseparation of the liposomes by ultracentrifugation, had very little effect on the ability ofthe treated serum to subsequently lyse liposomes. To ascertain whether this lysis wascomplement-mediated, carboxyfluorescein-loaded liposomes were incubated with Clqdeficient human serum. These incubations did not cause an increase in fluorescence overan incubation period of 30 mm.To demonstrate that the consumption of the complement components occurs as aresult of complement activation with the subsequent fixation of the convertedcomponents onto the liposomal surfaces, the proteins associated with the liposomes afterserum incubation were isolated and analyzed by immunoblot analysis. FMLVs wereused in this study since FMLVs were readily isolated from serum by centrifugation.FMLVs activated complement in a similar dose-dependent manner as 100 nm LUVs.Figure 2.10 shows that C3b, the activated form of C3, is the predominant molecularspecies of C3 associated with the complement activating liposomes. C9 was alsodetected on the liposomal membranes suggesting that the entire complement pathwaywas activated and that possibly membrane attack complexes were assembled on thesurfaces of the liposomes. These proteins were not detected in extracts of PC:CHOLliposomes as shown in Figure 2.10. Immunoblot analysis of the proteins with normalrabbit serum did not detect any bands similar to those detected with the specificantibodies.63Figure 2.10. Immunoblot analysis of proteins associated with PG-containingliposomes after exposure to human serum. The proteins associated with PC:CHOL:PG(35:45:20) FMLVs (Lane 2) or with PC:CHOL (55:45) FMLVs (Lane 3) were extracted,electrophoresed on 7.5% homogenous (panel A) and 10-15% gradient (panel B) resolvingSDS-polyacrylamide gels, blotted onto nitrocellulose and probed for the presence ofactivated complement proteins using monospecific antisera to C3d (panel A) or C9 (panelB). Lane A-i is trypsin-treated purified C3 and lane B-i is purified C9.A B123 123C3C3bC9 —•642.4 DISCUSSIONThe studies presented here clearly indicate that membranes bearing a net surfacecharge activate the complement system. Liposomes containing either anionic or cationiclipids reduced the levels of complement present in guinea pig or human serum. Netneutral systems did not affect the functional complement levels.The nature of the surface charge, whether negative or positive, is important indetermining which complement pathway is activated by the liposomes. Under conditionswhere the classical pathway of complement activation was effectively blocked, positivelycharged systems were still able to reduce the C3-C9 complement levels in a dosedependent manner, indicating that positively charged liposomes activated the alternativepathway of complement activation. This is consistent with previous findings that SAcontaining liposomes activate complement via the alternative pathway (Cunningham eta!., 1979) and indicates that guinea pig complement alternative pathway is functional at a1/20 dilution of guinea pig serum. Negatively charged systems, however, did notconsume complement in the absence of Ca2+, suggesting that negatively chargedliposomes activated the complement system via the classical pathway. Further evidenceto support that positively charged liposomes activated the alternative pathway whereasnegatively charged liposomes activated the classical pathway came from measuringresidual alternative pathway human complement hemolytic activity. By using rabbiterythrocytes, activators of the alternative pathway of human complement, as target cellsfor the hemolytic assays, positively charged liposomes were shown to consume thecomplement levels of human serum.65The activation of the classical pathway by negatively charged liposomesapparently occurs in an antibody-independent manner because pre-adsorption of theserum with anionic liposomes had little effect on subsequent complement-mediatedliposome lysis. It is not expected that there be a high titre of antibodies directed againstthe liposomes because these experiments were done in vitro, using isolated guinea pigand human sera. Antibodies to phospholipids, however, have been suggested to bepresent in normal human serum (Alving, 1984). The generality of the activation bynegatively charged phospholipids would suggest that the epitope of the antibody wouldbe the negatively charged phosphate group common to all these anionic phospholipids.PC and PE, however, also possess this phosphate group and thus, one would expect thatPC and PB would be recognized by the antibody and would result in antibody-mediatedcomplement activation. This was not observed. The net negative charge, therefore,directly mediates the liposome-complement interaction in classical pathway activation bynegatively charged liposomes.With regard to the reduction of functional complement levels after incubating theserum with liposomes, a possibility existed that the surface charge facilitated the“mopping up” of plasma proteins due to electrostatic interactions, thereby decreasing thecomplement levels in the serum available to lyse the EA target cells. This mechanism isinconsistent with the findings presented here. First, being able to distinguish the twopathways of complement activation implies specificity in the interactions betweenliposomes and the complement system. Secondly, the interactions between complementand liposomes could be modulated by altering the physical properties of the membrane(for example, increasing membrane hydrocarbon order; Figs. 2.3 and 2.4). Thirdly,immunoblot analysis of the proteins associated with the liposomes after exposure to66serum directly demonstrated that degradative products of the complement proteins wereassociated with anionic liposomal membranes suggesting that the liposomes did representbiologically active surfaces which supported complement activation. Finally, the preadsorption of serum with anionic liposomes at 0°C had little or no effect on thecomplement-mediated lysis of liposomes containing carboxyfluorescein. Consumptionof complement, therefore, was not the result of a general “mopping up” effect.Alternatively, the negatively charged surfaces may bind Ca2, limiting its concentrationin the incubation mixtures to levels where classical pathway activation and the resultantcytolysis of EA cells cannot occur. This was found not to be the case because PGcontaining liposomes did not significantly deplete the supernatant of Ca2.Relatively few studies have used liposomes to model the membrane activation ofthe complement system (Cunningham et al., 1979; Kovacsovics et al., 1985; Comis andEasterbrook-Smith, 1986; Thielens and Colomb, 1986; Michlek et a!., 1988; Mold,1989). Certain inconsistencies were observed in these studies regarding the influence oflipid composition on complement activation that make it difficult to establish thefundamental properties of complement-activating liposomes. For example, Kovacsovicset al. (1985) demonstrated the activation of Cl by liposomes containing CL by [1251]..Clq binding studies; however, liposomes composed of P1 in the same study failed toactivate Cl. Binding of C5b-6 to liposome membranes was shown to require PG or PA;but binding of C5b-6 to PC, PS or P1 was undetectable (Silversmith and Nelsestuen,1986). Liposomes composed of SA have been shown to consume complement by thealternative pathway (Cunningham et a!., 1979); however, these SA-containing systemsrequired the presence of certain glycolipids for C3 conversion. In this study, titrating any67of the anionic or cationic lipids functionally transformed the liposornes into activators ofthe complement system.The activation of the complement system by liposomes was affected by thesurface charge density, as well as the dose of liposomes incubated with the serum. Thismay account for some of the inconsistencies observed by other investigators becausetheir incubations mostly involved only one concentration of liposomes. Their use ofiodinated proteins may also have affected the binding properties and more importantly,the functional activity of the proteins.When liposomes are incubated with serum, complement proteins becomeassociated with the liposomes. Activation of the complement system by PG-containingliposomes resulted in the deposition of activated complement products such as C3b onthe liposome membranes. These products are known to have significant physiologicalroles in the clearance of foreign pathogens. C3b and its degradative products, iC3b andC3dg, have opsonic roles in the clearance of immune complexes. The assembly of theC5b-9 membrane attack complexes results in cell lysis. Inasmuch as these complementcomponents are associated with liposome surfaces, they may play a significant role in theclearance of liposomes from the circulation.In the following chapters, the role of C3b in mediating liposome clearance fromthe circulation will be established first by quantitating the amount of human C3 bound toLUVs of various lipid compositions using an in vitro incubation system (Chapter 3), andsecondly, by determining the relative amounts of murine C3 associated with liposomes invivo (Chapter 4). The amount of C3 bound to the various liposomes will be related totheir apparent circulation lifetimes in vivo.68CHAPTER 3 SEPARATION OF LARGE UNILAMELLAR VESICLES FROMBLOOD COMPONENTS BY A SPIN COLUMN PROCEDURE3.1 INTRODUCTIONWell-defined LUVs are widely used for liposome-based carrier systems (Cullis etal., 1989; Ostro and Cullis, 1989). As with all liposome systems, however, LUVs arerapidly cleared from the circulation, having half-lives ranging from minutes to hours(Gregoriadis, 1988). The interactions liposomes encounter in the circulation are believedto significantly influence their stability in blood via at least two mechanisms. First, theinteractions with lipoproteins, complement and serum albumin have been shown toprimarily result in liposome membrane destabilization leading to contents leakage(Senior, 1987; Scherphof et aL, 1984; Bonte and Juliano; 1986; Kiwada et al., 1988).This has been attributed to a net exchange of lipid to lipoproteins or to the formation ofpores in the membrane in the case of complement proteins. Second, liposomeinteractions with complement, fibronectin and immunoglobulins have been implicated tomediate liposome clearance because liposomes coated with these proteins exhibitenhanced uptake by cultured macrophages (Hsu and Juliano, 1982; Roerdink et al., 1983;Derksen et al., 1987).The factors which mediate opsonization in vivo are poorly understood,particularly for LUV systems. This is because of the difficulty in isolating LUVs fromblood components such as lipoproteins and recovering sufficient amounts to analyze byconventional SDS-PAGE. Much of the early work involved MLVs or sonicated vesicleswhich were recovered from plasma mixtures by ultracentrifugation followed by multiple69washes or by gel filtration chromatography, respectively (Senior, 1987; Bonte andJuliano, 1986; Senior and Gregoriadis, 1984; Kirby et al., 1980). No convenientprocedure for isolating LUVs from plasma components has been available. Such atechnique is important to study the protein interactions LUVs experience in thecirculation. These interactions are likely to differ from those observed for multilamellarand sonicated vesicles as vesicle size affects liposome stability (Juliano and Stamp, 1975;Scherphof and Morselt, 1984).This chapter describes a procedure combining chromatographic and centrifugalmethods for the rapid isolation of LUVs from plasma components. This “spin column”method was used to isolate liposomes from incubation mixtures with human serum ormore significantly, from blood of mice after intravenous administration of liposomes. Inthe latter case the liposomes are exposed to the entire complex biological milieu, i.e. theyare in contact with the immune system, blood cells, coagulation proteins, endothelialcells, blood pressure and physiologic ion (notably Ca2 and Mg2)concentrations. Theapplicability of this spin column procedure in studying protein/liposome interactions isdemonstrated by quantitating the amount of human complement C3 bound per liposomeusing a C3 competitive enzyme-linked immunosorbent assay after incubation with humanserum. Inasmuch as charged liposomes were shown to activate human complementsystem in the previous chapter, the studies described in this chapter are aimed atestablishing a relation between C3 binding and liposome clearance behavior. Theproteins associated with the recovered liposomes were further analyzed usingconventional SDS-PAGE and visualized by silver stain. By isolating circulating LUVsusing the spin column method and characterizing the proteins associated with theirmembranes, this protein fingerprinting approach will expedite identifying protein70interactions which affect liposome stability and clearance in vivo. These studies will bedescribed in Chapter 4.3.2 METHODS AND MATERIALS3.2.1 Preparation of liposomesLUVs were prepared using the extrusion method as described in Section 2.2.1.Liposome suspensions were 20 mM total lipid in isotonic HEPES-buffered saline (HBS;20 mM HEPES, pH 7.4, 145 mM NaC1) sterilized using Syrfil 0.22 m filters(Nuclepore). Size of the extruded vesicles was determined by quasi-elastic lightscattering analysis on a NICOMP Model 270 Submicron Particle Sizer (NICOMPInstruments, Santa Barbara, CA) to be 100 ±30 nm. The liposomes were radiolabelledby incorporating the non-exchangeable, non-metabolizeable marker,[3H]cholesterylhexadecyl ether (10 iCi/30 mol lipid), to follow the biodistribution of the liposomes inmice (Stein et al., 1980) and to quantitate the concentration of the recovered liposomesuspensions. The specific activity of the liposomes was determined by measuring theradioactivity content using standard liquid scintillation counting methods on a HewlettPackard Tri-Carb 2000CA liquid scintillation analyzer and the phospholipid contentusing a colorimetric phosphorus assay (Fiske and Subbarow, 1924). Briefly, thephosphorus assay involved digesting an appropriate aliquot (< 100 tl) of the liposomesuspension in 0.6 ml 70% perchloric acid for 2 h at 180°C; adding to the cooled digest 7ml of ammonium molybdate reagent [0.22%, w/v, ammonium molybdate (BDH,Toronto, Canada) in 2% H2S04,v/v] and 0.75 ml of Fiske-Subbarrow reagent [30 g71sodium bisulfite (BDH), 1 g sodium sulfite (BDH) and 0.5 g 1-amino-2-napthol-4-sulphonic acid (Sigma) in 200 ml water]; incubating the mixture for 15 mm in a boilingwater bath; and spectrophotometrically measuring the absorbance of the solution at awavelength of 815 nm. Lipids were purchased from the following companies: Egg PC,DOPS, DOPA, plant P1 and bovine heart CL from Avanti Polar Lipids, Pelham, AL;CHOL from Sigma; and[3H]cholesteryl hexadecyl ether from Amersham. These lipidswere used without further purification. All liposome compositions are expressed inmolar ratios.3.2.2 Serum and lipoproteinsHuman serum was prepared from venous blood pooled from 20 healthyindividuals (10 males, 10 females) and stored at -70°C (see Section 2.2.2). Purified verylow density lipoproteins and low density lipoproteins were provided by Dr. H. P.Pritchard, Lipoprotein Research Group, Vancouver, Canada.3.2.3 Preparation of spin columnsThe procedure for packing the spin columns is as follows. One ml tuberculinsyringes plugged with glass wool were filled with BioGel A15m, 200-400 mesh size,chromatographic gel (Bio-Rad, Mississauga, Canada) equilibrated with isotonic veronalbuffered saline (VBS, 10 mM sodium barbital, pH 7.4, 145 mM NaCl) and centrifuged(Jouan Centrifuge G4.11; 2000 rpm, 2 mm, 4°C) in 13 x 100 mm borosilicate glassculture tubes. A series of filling and centrifuging were done until the column bed volume72approximated 1.0 ml. A final spin of 2000 rpm for 5 mm assured that the BioGel A15mgel was uniformly packed and that excess buffer was removed.3.2.4 Spin column profiles of liposomes incubated with human serumOne hundred il of the liposome suspension was incubated with 400 itl of poolednormal human serum at 37°C for 30 mm. Aliquots of the liposome/serum incubationmixtures (50 oil) were applied to spin columns and immediately centrifuged (JouanCentrifuge G4.11; 1000 rpm, 1 mm, 4°C). Column fractions were collected in glassculture tubes by applying 50 iI of VBS to the spin columns and centrifuging (1000 rpm,1 mm, 4°C). The liposome content of the column fractions were assayed by determiningthe 3H radioactivity content of the fractions using standard liquid scintillation countingmethods on a Hewlett Packard Tri-Carb 2000CA liquid scintillation analyzer. Proteincontent of the column fractions was determined using the BCA protein assay (PierceChemical Co., Rockford, IL). Briefly, 10 tl of column fractions were incubated with 200tl BCA protein assay reagent in microtiter plates. After an overnight incubation at roomtemperature, the absorbance (540 nm) of the solutions was read using an SLTLabinstruments Austria EAR400AT microtiter plate reader. As controls in all ourexperiments, 50 iil 80% human serum without liposomes was chromatographed usingsimilar spin columns under identical conditions and the column fractions analyzed forprotein content.733.2.5 Conventional BioGel A15m chromatography of liposome/human serumincubation mixturesTwo ml of 100 mM PC:CHOL:CL (35:45:10) LUVs in VBS was incubated with8 ml of normal human serum at 37°C for 30 mm. To isolate the liposomes from humanserum components, the incubation mixture was chromatographed on a 2.5 x 90 cmBioGel A-15m, 100-200 mesh, column pre-equilibrated with VBS buffer at 4°C.Fractions (120 drops/fraction) were collected at a flow rate of 30 ml/h. The columnfractions were analyzed for phospholipid content using a colorimetric phosphorus assay(Fiske and Subbarow, 1924) and for protein content using the BCA protein assay (Pierce,Rockford, IL). Fractions 25-32 were pooled and stored at -20°C.3.2.6 Recovery of liposomes from circulation of CD1 miceTwo hundred iil of the liposome suspension was administered intravenously viathe dorsal tail vein of female CD1 mice (Charles River, St. Constant, Canada; 24-30 g; 4mice/time point). After 2 mm, 30 mm or 1 h post-injection, the mice were anesthesizedwith ether and blood withdrawn via cardiac puncture and collected in ice cold 1.5 mlpolypropylene microcentrifuge tubes (Eppendorf). The blood was immediately cooled to0°C using an ice-water bath to prevent coagulation and centrifuged (12,000 rpm, 2 mm,4°C) to pellet the blood cells. Aliquots of the plasma (50 d) were applied to spincolumns (5 columns/mouse) and immediately centrifuged (Jouan Centrifuge G4.11; 1000rpm, 1 mm, 4°C). Column fractions were collected in glass culture tubes by applying 50itl of VBS to the spin columns and centrifuging (1000 rpm, 1 mm, 4°C). The first twocolumn fractions containing radioactivity (typically fractions 5 and 6, or 6 and 7) were74collected, pooled and concentrated using Centricon 30 microconcentrators (Amicon,Danvers, MA) at 4°C. The radioactivity recoveries from the Centricon 30microconcentrators were typically greater than 95%. The samples were stored at -20°C.3.2.7 Competitive ELISA for C3C3 competitive ELISA was performed on serially diluted isolated liposomesusing the procedure described by Mold (1989). Immulon II microtiter wells (FisherScientific) were coated with 100 tl of 10 .tg/ml purified human C3 in PBS containing0.1% sodium azide and incubated in a 60°C oven overnight. Purified human C3 was agift from Dr. D. V. Devine, The University of British Columbia, Vancouver, Canada, orwas purchased from Calbiochem. The wells were washed thoroughly with distilled waterand blocked for 1 h at room temperature with 200 il of 0.5% BSA (Sigma) in PBS.Purified C3 standards, diluted in 1% Nonidet P-40 in PBS containing lysed liposomes ata concentration of 1 mM lipid, were used to construct a standard curve. Samples andstandards were preincubated for 1 h at room temperature with an equal volume of horseradish peroxidase-conjugated goat anti-human C3 (CalBiochem, San Diego, CA) diluted1/10,000 in PBS, 0.1% BSA and triplicate samples of 100 p1/well of the mixture wereadded to the blocked wells and incubated for 1 h at room temperature. Plates werewashed three times with 200 p1/well PBS, 0.05% Tween 20 and developed with 100p1/well 2, 2’-azino-di-(ethylbenzthiazolin-6-sulfonic acid) (Sigma) substrate [1 mg/ml 2,2’-azino-di-(ethylbenzthiazolin-6.sulfonic acid) and 0.01% hydrogen peroxide incitrate/phosphate buffer, pH 4.6 (Mcllvaine, 1921)]. The absorbance of the solutions was75read using an SLT-Labinstruments Austria EAR400AT microtiter plate reader at awavelength of 540 nm.3.2.8 SDS-PAGE analysis of proteins associated with liposomesProtein separation was performed by SDS-PAGE using the Mini Protean-Ilelectrophoretic apparatus (Bio-Rad) on precast 4-20% gradient Mini Protean-Il gels (BioRad) under nonreducing conditions (Laemmli, 1970). Prestained SDS-PAGE molecularweight standards (Diversified Biotech, Newton, MA) were used to estimate the molecularweights of the proteins. Detection of the proteins was done by an optimized silver stainprocedure (Heukeshoven and Dernick, 1988).3.3 RESULTS3.3.1 Isolation of large unilamellar liposomes from plasma using BioGel A15m spincolumnsInitial studies indicated that coventional BioGel A15m, 100-200 mesh, gelfiltration chromatography was effective in isolating LUVs (extruded through 100 nmpore-sized filters) from human serum components (Fig. 3.1). However, this procedure,requiring lengthy processing times (typically longer than 3 h per separation) andrelatively large sample volumes, was not practical for these studies. Therefore, severalfactors including use of a finer mesh (200-400 mesh) to increase the number ofchromatographic theoretical plates; use of shorter columns, thereby reducing the sample76Figure 3.1. Elution profile of PC:CHOL:CL (35:45:10) LUVslhuman serumincubation mixtures chromatographed on conventional columns. PC:CHOL:CL(35:45:10) LUVslhuman serum incubation mixtures were chromatographed on a 2.5 x 90cm BioGel A-15m, 100-200 mesh, column as described in “Methods and Materials”.The phosphorus content (• ) or the protein content (• ) of the column fractions weredetermined as in “Methods and Materials”.U)14-’Ca)•1-’C00V0.00.(I)0-C0Column Fraction #4 0U)4-’0C)4-’C02 (_)Ca)01a-532100 10 20 30040 50 60 70 80 90 10077volume requirement; and increasing operating pressures, to reduce the separation timewere considered in order to improve this isolation procedure. A rapid method for theisolation of LUVs from plasma components was thus developed using a “spin column”procedure described in Section 3.2.3.The efficiency of separation using this technique is depicted in Figures 3.2 and3.3. Figure 3.2 shows the column profiles of PC:CHOL (55:45) LUVs or of 80% humanserum chromatographed on BioGel A15m 1.0 ml spin columns. Figure 3.3 shows thecolumn profiles of various LUV/80% human serum incubation mixtures. As shown inFigure 3.3, the liposome composition does not affect the elution profile of the liposomes.These profiles are representative of hundreds of columns using LUVs composed of netneutral or anionic lipids. By centrifuging the column in a 13 x 100 mm glass culture tubecarrier, the flow rate through the column was considerably increased. The profiles shownwere obtained using the optimized spin conditions of 1000 rpm for 1 mm. These optimalconditions were determined by varying the rate and duration of centrifugation. Theconditions of 1000 rpm for 1 mm gave the most consistent elution profiles and fractionvolumes (typically 30-40 tl). Spin times longer than 2 mm resulted in channel formationat the top of the column. Column bed volumes averaged 1.00 ± 0.15 ml. The meanliposome recovery in the first 9 fractions was 70%.Elution profiles obtained with purified lipoproteins showed that the liposomeswere effectively resolved from the very low density and low density lipoproteins usingthe spin column procedure (Fig. 3.4).The column fractions were analyzed for their protein content by SDS-PAGEfollowed by silver staining (Fig. 3.5). When 80% serum in VBS was chromatographed,the fractions where the liposomes would elute (fractions 5-7) did not contain any780V>%4-’>4-’0c0Vcx6__0It)4-’A CC)4-’C‘, 00C201Figure 3.2. Spin column profiles of liposomes or human serum proteins. BioGelA15m, 200-400 mesh, 1.0 ml spin columns were calibrated using 50 t1 20 mMPC:CHOL (55:45) LUVs radiolabelled with[3H]cholesteryl hexadecyl ether (0 , 4columns) or 50 tl 80% human serum (D, 4 columns) chromatographed separately underidentical conditions. Column fractions represent the eluant recovered from onecentrifugation (1000 rpm, 1 mm).73000020000100000 00 5 10 15 20 25Column Fraction #79C0>%4-’>4-’00VaSa:Column Fraction #Figure 3.3. Spiii column profiles of liposome/human serum incubations.PC:CHOL:DOPA (35:45:20) (Q), PC:CHOL:DOPS (35:45:20) (A), or PC:CHOL:PI(35:45:20) (0) LUVs containing trace amounts of[H]cholesteryl hexadecyl ether wereincubated with human serum at 37°C for 30 mm. The incubation mixtures were thenchromatographed using the BioGel A15m spin columns as described in HMethods andMaterials”. The open symbols are the radioactive content and the filled symbols are theprotein content of the column fractions.900080007000__6000500040003000200010000534 0U)4-’Ca,4-’C0C.)4-’01020 5 10 15 20 250800I04-’Ca)4-’C0C-)Ca)4-’00Figure 3.4. Separation of 100 nm LUVsfrom VLDL andLDL. BioGel A15m, 200-400mesh, 1.0 ml spin columns were calibrated using 50 i1 20 mM PC:CHOL (55:45) LUVsradiolabelled with[3H]cholesteryl hexadecyl ether (0, 4 columns), 50 tl purified VLDL(El, 4 columns) or 50 il purified LDL (A, 4 columns). VLDL and LDL content weredetected using the BCA protein assay. These were chromatographed separately onsimilar spin columns under identical conditions.15000 0.50__0.40E0• 10000__0.300.2050000.100 0.000 5 10 15 20 25Column Fraction #81Figure 3.5. SDS-PAGE analysis of the protein content of the spin column fractions.The column fractions of chromatographed 50 p1 80% human serum were analyzed fortheir protein content by SDS-PAGE followed by silver staining as described in “Methodsand Materials”. Lane M includes prestained SDS-PAGE molecular weight standards.4 5 6 7 8 9 10 11 M— --200kDa95.582detectable protein. The fractions where the serum proteins started eluting (fraction 9)contained very high molecular weight proteins having mobilities corresponding tomolecular mass greater than 200 kDa.The protein profile of the liposomes isolated using the spin column procedure wascompared to that obtained using the conventional gel filtration column procedure byanalyzing equal amounts of lipid on 4-20% gradient SDS-polyacrylamide gels. Figure3.6 shows that the protein profiles are very similar in type and amount of proteinsassociated with PC:CHOL:CL (35 :45:10) LUVs. PC:CHOL:CL LUVs were usedbecause this composition bound the most complex protein profile of all the anionicliposomes investigated.3.3.2 Measurement of human C3 bound per liposomeAs described in Chapter 2, anionic liposomes activate the complement system viathe classical pathway leading to the liposomal association of C3b. It was suggested thatC3b was one of the plasma proteins which marked the liposomes as foreign particlesbecause C3b is a potent opsonin. The amount of C3b associated with the variousliposomes containing the different anionic phospholipids described in Chapter 2 wasmeasured in order to detemine whether the various liposomes bound different amounts ofC3b, and whether the amount of C3b bound was related to the clearance behavior of theliposomes. As the population of liposomes is essentially unilamellar for all the lipidcompositions employed here, the amount of C3b bound per lipid as a function of lipidcomposition can be best estimated. Using a human C3 competitive ELISA, the amountof C3 associated with the various liposomes was quantitated (Table 3.1). The amount of83Figure 3.6. Comparison of the protein profile associated with PC:CHOL:CL LUVsisolated using the spin column procedure or conventional chromatographicprocedures. PC:CHOL:CL (35:45:10) LUVs recovered using the BioGel A15m, 100-200 mesh, conventional column (Lane A) or the BioGel A15m, 200-400 mesh, spincolumn (Lane B) procedures were analyzed for the proteins associated with theirmembranes by SDS-PAGE analysis as in “Methods and Materials”. Equal amounts oflipid (30 nmol total lipid) were applied to each lane. Lane C is 25 tl of 1/750 dilution ofpooled normal human serum.ABCkDa__ __- e_.J200—95.5 ri r’55-43 ;-_--_ -36I84Table 3.1. Amount of C3 associated with various LUVs.Composition of LUVs Amount of C3 bounda(nmol C3/mmol total lipid)PC:CHOL (55:45) 3.15, 4.61PC:CHOL:PI (35:45:20) 8.76, 9.66PC:CHOL:PG (35:45:20) 13.7, 16.6PC:CHOL:DOPS (35:45:20) 31.5, 19.8PC:CHOL:DOPA (35:45:20) 36.9, 33.1PC:CHOL:CL (35 :45:10) 46.2, 33.9a The LUVs recovered from human serum incubations using the spin columnprocedure were analyzed for C3 content using a C3 competitive ELISA (see“Methods and Materials” for details). The results from two separate experimentsare given.85C3 associated with PC:CHOL:CL (35:45:10) and PC:CHOL:DOPA (35:45:20) LUVs isapproximately 4-10 times greater than for PC:CHOL (55:45), PC:CHOL:PG (35:45:20)or PC:CHOL:PI (35:45:20) LUVs.3.3.3 In vivo characterization of murine plasma proteins associated with LUVs overtimeThe utility of this spin column method in recovering LUVs from blood of miceadministered intravenously is demonstrated using PC:CHOL (55:45) or PC:CHOL:DOPS(35:45:20) liposomes. Figure 3.7 shows the recovery of LUVs from the plasma of CD1mice over a 1 h period. Whereas PC:CHOL:DOPS LUVs are cleared rapidly from theplasma, PC:CHOL LUVs are relatively long-lived in the circulation. The liposomes wereisolated from the blood samples in the absence of chelators or other coagulationinhibitors by cooling the blood samples to 0°C to retard the coagulation process,centrifuging to pellet the blood cells, and separated from the plasma using the BioGelAlSm spin columns. The protein profiles were characterized using SDS-PAGE analysis(Fig. 3.8). Within 2 mm after administration, PC:CHOL LUVs have an associatedprotein composition consisting mainly of albumin and very high molecular weightproteins. Qualitatively, the protein profile associated with circulating PC:CHOL LUVs isnot markedly altered over a 1 h period. The rapidly cleared PC:CHOL:DOPS LUVs, onthe other hand, have a more complex protein profile, both in amount and type ofassociated proteins, than the PC:CHOL LUVs. No protein was detected in these columnfractions when mouse serum alone was chromatographed.860.00EECl)0.E0Na,>00>D-J0.10 10 20 30 40 50 60 70Time (mm)Figure 3.7. Clearance profile of PC:CHOL and PC:CHOL:DOPS LUVs. PC:CHOL(55:45) ( • ) or PC:CHOL:DOPS (35:45:20) ( A ) liposomes were administeredintravenously into CD1 mice and over time aliquots of plasma were counted for 3Hradioactivity to follow the clearance of liposomes from the circulation. Each mousereceived a dose of approximately 4 pmol total lipid in a volume of 200 p1 HBS. Plasmavolume of a mouse was taken to be 5% of body weight.10187Figure 3.8. Protein profiles of PC:CHOL (55:45) and PC:CHOL:DOPS (35:45:20)LUVs recovered from mice over time. The proteins associated with the recoveredliposomes were analyzed using 4-20% gradient SDS-polyacrylamide gel electrophoresisand silver stained as in “Methods and Materials”. Lane A, PC:CHOL, 2 mm; Lane B,PC:CHOL, 30 mm; Lane C, PC:CHOL, 1 h; Lane D, PC:CHOL:DOPS, 2 mm; Lane E,PC:CHOL:DOPS, 30 mm. Lanes A-D represent the proteins associated with 40 nmoltotal lipid; Lane E represents the proteins associated with 20 nmol total lipid.ABODEkDa200—95.5 —55—43—36—-.:883.4 DISCUSSIONThe development of methods to rapidly isolate LUVs from blood components isimportant to understanding the protein/liposome interactions that mediate liposomeleakage and clearance from the circulation. Until now, there has been no satisfactoryprocedure for isolating LUVs from plasma. In this chapter, a procedure which is veryconvenient for the study of protein/liposome interactions that occur in vivo is described.Compared to conventional column chromatographic methods which take about 3-4 h peranalysis (Fig. 3.1 and also Huang, 1969; Reynolds et al., 1983), spin column processingtimes are extremely rapid. From the point of sample application to the collection of theliposome fractions, the isolation procedure takes approximately 6-8 mm. Otheradvantages of this spin column procedure are that many columns can be processed at thesame time (up to 96 depending on the capacity of the centrifuge) and that small samplevolumes can be analyzed. The major advantage in particular to the study of protein-mediated liposome clearance mechanisms, however, is that because processing times arerapid and the procedure can be readily performed at 4°C, the isolation procedure can bedone in the absence of coagulation inhibitors which may affect protein/liposomeinteractions.Good reproducibility in isolating LUVs from blood components using the spincolumn method is obviously necessary for detecting the proteins associated withcirculating liposomes. The results presented here are representative of hundreds of spincolumns; however, some variation in the performance of different batches of BioGelA15m gel has been observed. These variations result in different flow rates and column89fraction volumes. The elution profiles are therefore sometimes different and on oneoccasion resulted in serious tailing in the liposome elution profile. This potentialproblem can be overcome by careful characterization of the elution profiles of the spincolumns using different batches of BioGel A15m gel. Within a particular batch, theelution profiles are very reproducible.Using this spin column procedure a population of PC:CHOL (55:45) LUVs whichremained circulating over a 1 h period has been separated and the associated proteinsanalyzed. Net neutral liposomes bound a number of high molecular weight (> 200 000)proteins which is similar to the in vitro findings of Juliano and Lin (1980) using humanplasma. This similarity is noteworthy since the earlier study involved MLVs isolated byultracentrifugation followed by multiple washes with buffer. This indicates that proteinswhich are associated with the spin column-isolated LUVs are tightly bound. The highmolecular weight proteins as well as albumin are associated with PC:CHOL LUVs withinminutes of intravenous administration, and do not appear to enhance the clearance ofliposomes from the circulation over a 1 h period because the population of liposomesbearing these proteins remain in the circulation over this period.In contrast to the PC:CHOL (55:45) protein profiles, the profile of proteinsassociated with rapidly cleared PC:CHOL:DOPS (35:45:20) LUVs is more complex.The same proteins which are associated with PC:CHOL LUVs are also found associatedwith PC:CHOL:DOPS LUVs; however, more proteins, both in terms of amount and type,are associated with the anionic PC:CHOL:DOPS LUVs. Some of these proteins mayinclude coagulation proteins and complement proteins as it has been reported that PScontaining liposomes activate the coagulation (Juliano and Lin, 1980; Juliano, 1983;Yoshioka et al., 1983; Andersson and Brown, 1981) and complement systems (see90Chapter 2, and Comis and Easterbrook-Smith, 1986). The identification of the proteinsassociated with these liposomes, especially those proteins which are uniquely associatedwith rapidly cleared liposomes, should elucidate proteins which affect liposomeclearance from the circulation.Changes in the protein profiles of the liposomes over time may provide insightinto the proteins affecting liposome stability in the circulation. The finding that specificassociated proteins disappear from the protein profiles over time suggest that liposomesbearing these proteins are cleared more readily. For example, PC:CHOL (55:45) LUVsat 2 mm and 30 mm have a pronounced band, migrating similarly to the 200 000molecular weight standard, associated with their membranes. This band notablydisappears at the 1 h time point. Similarly, a prominent band, migrating with a molecularweight corresponding roughly to 80 000, is associated with the PC:CHOL:DOPS(35:45:20) LUVs at 2 mm but is less intense at 30 mm, although it should be noted thatLane D in Figure 3.8 has twice the amount of total lipid loaded as in Lane E. Similarstudies that characterize the protein profile of liposomes over time, involving morequantitative methods, should be very useful in elucidating the proteins involved inliposome clearance.The PC:CHOL:DOPS (35:45:20) LUVs recovered from the circulation of micehad a more extensive protein profile (Fig. 3.8) than the PC:CHOL:CL (35:45:10) LUVsrecovered from in vitro incubations with human serum (Fig. 3.6), especially with regardto the very high molecular weight proteins (>200 000). This underlines the necessity tostudy the in vivo plasma protein interactions liposomes experience in order to fullyunderstand the protein-mediated clearance behavior of liposomes.91One of the plasma proteins which has been implicated in mediating liposomeuptake is complement component C3, specifically the opsonic form C3b. In Chapter 2,liposomes bearing a net surface charge were shown to be potent activators of thecomplement system resulting in the deposition of C3b molecules onto the liposomemembranes. The results presented in this chapter clearly show that liposomes containingDOPA or CL bind considerably more C3. If liposome clearance behavior is similar inmice and humans, these results suggest a correlation between the amount of C3 boundper liposome and liposome clearance behavior. CL- and DOPA-containing systems,which bind the most C3 (Table 3.1), are cleared very rapidly from the circulation (Julianoand Stamp, 1975; Senior and Gregoriadis, 1982b). PC:CHOL (55:45) and PG- and P1-containing systems are cleared more slowly (Fig. 3.7 and also Kao and Loo, 1980;Gabizon and Papahadjopoulos, 1988) and bind much less C3.In summary, the spin column procedure is a rapid and effective method forrecovering LUVs from incubation mixtures with blood components. This procedurerequires little manipulation of the system; no coagulation inhibitors are necessary and nomultiple washes with buffer are required. The amount of liposomes recovered from thisprocedure is adequate to analyze the protein content of the liposomes by SDS-PAGEfollowed by silver staining. As well, the liposome proteins are readily analyzed usingimmunological methods such as immunosorbent assays. Further, this spin columnisolation procedure allows the use of large unilamellar liposomal systems as opposed tomultilamellar liposomal systems in studies involving protein/membrane interactions. Theadvantage of using LUVs, where all the lamellae are exposed to the extravesicularenvironment, as opposed to MLVs, where only the outermost lamellae is exposed to theextravesicular environment, is clearly demonstrated in the studies quantitating the92amount of liposome-associated C3. The relation between the amount of associated C3and liposome clearance from mouse blood strongly suggests that C3 fragments play arole in liposome clearance. This relation will be further established in vivo in thefollowing chapter. The spin column method will be used to recover LUVs exhibitingmarkedly different clearance properties from the blood of liposome-treated mice. Thelevels of C3 and other opsonins associated with their membranes will be analyzed.93CHAPTER 4 ASSOCIATION OF BLOOD PROTEINS WITH LARGEUNILAMELLAR LIPOSOMES IN VIVO: RELATION TO CIRCULATIONLIFETIMES4.1 INTRODUCTIONA central problem to the use of liposomes and other carriers for drug delivery istheir rapid clearance from the circulation (see Section 1.3). Although the mechanismsinvolved in the in vivo clearance of liposomes from the circulation are poorly understood,various aspects of liposome design are known to strongly influence liposome clearancebehavior. For example, negatively charged liposomes are cleared more rapidly than netneutral or positively charged systems (Juliano and Stamp, 1975). The presence ofsaturated phospholipids (Gregoriadis and Senior, 1980) or equimolar amounts ofcholesterol (Kirby et al., 1980a; Patel et al., 1983; Roerdink et al., 1989) stabilizeliposomes in the circulation and also reduce their uptake by the phagocytic cells of thereticuloendothelial system. Liposomes containing phosphatidylinositol(Papahadjopoulos and Gabizon, 1987), ganglioside GM1 (Allen and Chonn, 1987;Gabizon and Papahadjoupolos, 1988) or PE-linked polyethyleneglycols (Klibanov et a!.,1990; Blume and Cevc, 1990) have been shown to exhibit extended circulation lifetimes.The underlying mechanisms by which these lipids affect liposome clearance are notknown; however, the effects are believed to result from alterations in surface propertieswhich reduce the interactions between liposomes and certain blood components. Forinstance, cholesterol in equimolar amounts has been shown in vitro to completely inhibit94the net transfer of lipids from liposomes to high density lipoproteins, resulting in lesspermeable liposomes (Kirby et al., 1980b).The plasma proteins mediating liposome instability in blood or liposome uptakeby the reticuloendothelial system has been studied extensively using in vitro systems.From these in vitro studies, there appears to be no unambiguous relation between theamount and type of protein bound and liposome clearance behavior. For example, Blackand Gregoriadis (1976) reported that human cc-2-macroglobulin or rat cc-1-macroglobulinwas the only protein associated with liposomes exposed to plasma, and that this proteinimparted a net negative liposome surface charge regardless of the inherent charge of themembrane. In contrast, Juliano and Lin (1980) reported that neutral or positively chargedliposomes bound several plasma proteins including albumin, apolipoprotein Al, IgG, anda group of high molecular weight (> 200 000) proteins. Negatively charged liposomesfailed to bind these high molecular weight components. The amount of proteinassociated with PS-containing liposomes did not significantly differ from that associatedwith PC:CHOL systems (Juliano and Lin, 1980).It has, furthennore, not been unambiguously shown whether blood proteins play arole in mediating liposome instability in the circulation or liposome uptake by phagocyticcells. For instance, the extensive binding of serum proteins to liposomes containingcholesterol do not result in release of entrapped[3H]guanosine monophosphate,indicating that the adsorbed proteins do not alter membrane permeability (Juliano andLin, 1980). Complement, immunoglobulins, and fibronectin have been implicated inmediating liposome clearance because liposomes coated with these purified proteinsenhance liposome uptake by cultured macrophages (Hsu and Juliano, 1982; Roerdink etal., 1983; Derksen et al., 1987). However, the role of liposome-associated proteins in95enhancing phagocytic uptake is uncertain in the presence of excess soluble protein(Torchillin et al., 1980). Whereas liposomes coated with y-globulin caused a moderateincrease in phagocytic uptake by cultured mouse peritoneal macrophages, the presence of5 mg y-globulin/ml in the medium significantly decreased uptake. Similarly, having anexcess of free albumin present in the incubation mixture markedly decreased the uptakeof liposomes precoated with albumin. Using a rat liver perfusion model, Tyrrell et al.(1977) reported that incubations of albumin, y-globulins or heat aggregated y-globulinswith liposomes had no significant effect on liver uptake. On the other hand, a- orglobulins caused a slight increase in uptake of anionic (PC:CHOL:dicetylphosphate;6:2:1) and not cationic (PC:CHOL:SA; 4:1:1) liposomes. Using a similar model, Kiwadaet al. (1986, 1987) showed that liposomes could pass freely through the liver in theabsence of plasma. Uptake by the liver required opsonization by plasma proteins. Thesestudies emphasize the need to characterize protein/liposome interactions under conditionswhich reflect closely in vivo conditions in order to best resolve the role of proteins inmediating liposome instability in the circulation and liposome uptake by thereticuloendothlial system.The extent to which blood proteins interact with liposomes and increase theirclearance in vivo is not known. To date, there have been no studies demonstrating thatliposome clearance in vivo correlates with the amount and type of associated bloodprotein. There are two main reasons for this. First, the large majority of studies on theassociation of plasma proteins with liposomes in vitro have been performed employingmultilamellar systems. Due to the variable lamellarity of liposomes of different lipidcompositions, quantitation of the amount of various proteins associated per liposome hasnot been possible. Second, and more importantly, techniques have not been available for96the isolation of LUVs from blood components recovered in the absence of coagulationinhibitors following the in vivo administration of liposomes.The studies described in this chapter utilize the rapid “spin column” techniquedescribed in the previous chapter to isolate LUVs from the blood of mice treated withLUVs. The proteins that associate with liposomes in the circulation of mice wereanalyzed in order to determine whether bound proteins influence the fate of liposomes invivo. By employing various negatively charged LUVs exhibiting markedly differentclearance properties, it is demonstrated that the amount of total protein binding to theLUVs in vivo is inversely related to the circulation half-life of the LUVs. Protein binding(RB) values in excess of 50 g total protein/mol total lipid were observed for rapidlycleared liposomes such as those containing CL or DOPA; whereas B values of less than20 g total protein/mo! total lipid were observed for circulation stable liposomes such asthose containing 10 mol% gang!ioside GM1. Further, as analyzed by SDS-PAGE, rapidlycleared !iposomes had a more complex profile of proteins associated with theirmembranes than the circulation stable liposomes. The rapid clearance of liposomescontaining high amounts of adsorbed surface protein suggests that these adsorbedproteins include substantial levels of opsonins, leading to rapid uptake by phagocyticcells. This was shown by specific immunoblot analyses to the immune opsonins,fragments of C3 and IgG. These studies also demonstrate that the mechanism by whichganglioside GM1 prolongs the murine circulation half-life of !iposomes is by reducing thetotal amount of blood protein bound to the liposomes in a relatively non-specific manner.974.2 METHODS AND MATERIALS4.2.1 Preparation of ilposomes[3H]cholesteryl hexadecyl ether-labelled LUVs (20 mM total lipid in HBS) wereprepared and characterized as described in Section 3.2.1. The preparation of liposomescontaining ganglioside GM1 was facilitated by extrusion at 65°C. All phospholipids werepurchased from Avanti Polar Lipids, Peiham, AL; cholesterol and ganglioside GM1, fromSigma, St. Louis, MO; and[3H]cholesteryl hexadecyl ether, from Amersham, ArlingtonHeights, IL. Liposome compositions are expressed in molar ratios.4.2.2 In vivo mouse plasma distribution of liposomesTwo hundred tl of the liposome suspension was administered intravenously viathe dorsal tail vein of CD1 mice (female, 6-8 wk old, Charles River, St. Constant,Quebec, Canada). After various times, the mice were sacrificed by overexposure tocarbon dioxide and blood withdrawn and collected in 1.5 ml microcentrifuge tubes(Eppendorf). The blood was immediately cooled to 0°C using an ice-water bath toprevent coagulation and centrifuged (12,000 rpm, 2 mm, 4°C) to pellet the blood cells.Aliquots of plasma were measured for radioactivity content using standard liquidscintillation methods. Plasma volume was assumed to be 5% of total body weight. Forthe 2 mm time point, experiments were repeated at least twice, with a sample size of 4mice.984.2.3 Isolated serum/liposome incubationsLiposomes were prepared as above except that the liposome suspensions were 50mM total lipid in VBS. To 120 ii.! of LUVs, 480 !d of normal human or mouse serumwas added and incubated for 30 mm at 37°C. Normal human serum was prepared fromvenous blood pooled from 20 healthy individuals (10 males, 10 females) and stored at-70°C (refer to Section 2.2.2). Mouse serum was purchased from CedarlaneLaboratories, Hornby, Ontario, Canada.4.2.4 Isolation of liposomes from blood componentsA simple and rapid “spin column” procedure employing BioGel AlSm, 200-400mesh size 1.0 ml chromatography columns was used to isolate liposomes from bloodcomponents as described in Sections 3.2.3 and 3.2.4. Briefly, aliquots of the plasma orserum (50 il) were applied to spin columns and immediately centrifuged (JouanCentrifuge G4.11; 1000 rpm, 1 mm). Column fractions were collected in glass culturetubes by applying 50 iil of VBS to the spin columns and centrifuging (1000 rpm, 1 mm).Column fractions were analyzed for radioactivity content and fractions containingliposomes, typically fractions 5 and 6 from each column, were collected, pooled andconcentrated using Centricon 30 microconcentrators (Amicon, Danvers, MA) at 4°C. Forin vivo experiments, 5 columns/mouse were done. LUVs recovered from 4 mice werepooled. For in vitro experiments, LUVs recovered from 10 columns were pooled. Thesamples were stored at -20°C.994.2.5 Measurement of PB valuesThe liposome associated proteins were efficiently extracted and delipidated usinga procedure described by Wessel and Flugge (1984). To 100 [Li of 1-2 mM LUVs, 400 [Limethanol, 200 p.1 chloroform, and 300 p.1 water were added with vortexing after eachaddition. The two phase system generated after the last addition was centrifuged for 15mm at 3000 rpm (Jouan Centrifuge G4.11). The upper phase was carefully removed suchthat the protein at the interface was left with a slight amount of upper phase. Then 300 [Liof methanol was added and the precipitated protein pelleted by centrifugation for 20 mmat 3000 rpm. The supernatant was discarded and the pellet was dried under a stream ofnitrogen. This delipidation step was required because lipids interfere with most proteinassays (Kessler and Fanestil, 1986). The extracted proteins were resuspended in 1 ml of0.5% SDS in Milli-Q water. Then, 1 ml of micro BCA protein assay reagent (Pierce,Rockford, IL) was added and the mixture incubated at 60°C for 60 mm. After cooling themixture to room temperature, the absorbance of the solution was measuredspectrophotometrically at a wavelength of 562 nm. The A562 was compared to astandard curve that was linear in the range of 0-16 p.g/ml for BSA (Pierce) as the standardprotein. The amount of lipid was calculated from the specific activity of the liposomesuspensions and the volume of liposomes used to extract the proteins.4.2.6 SDS-PAGE analysis of proteins associated with liposomesProtein separation was performed by SDS-PAGE using the Mini Protean-TIelectrophoretic apparatus (Bio-Rad) on precast 4-20% gradient Mini Protean-I! gels (BioRad) under non-reducing conditions (Laemmli, 1970). Prestained SDS-PAGE molecular100weight standards (Diversified Biotech, Newton, MA) or silver stain SDS-PAGEmolecular weight standards (Bio-Rad) were used to estimate the molecular weights of theproteins. Detection of the proteins was done either by a silver stain procedure (Rabilloudet al., 1988) or by electrophoretic transfer of the separated proteins onto nitrocellulose(Nitroplus 2000; Micron Separations, Westboro, MA) using the Mini Trans-BlotElectrophoresis Transfer Cell (Bio-Rad Laboratories) at constant current of 300 mA for60 mm followed by immunoblot analysis using the Enhanced Chemiluminescencewestern blotting detection system (Amersham). The blocking buffer consisted of 10 mMTris, pH 7.6, 150 mM NaC1, 5% dried skim milk powder, and 1% Tween 20 detergent(Sigma). Goat antisera to mouse C3 (Organon Teknika Inc., Scarborough, Canada) wasused at a 1/1000 dilution; peroxidase-conjugated goat anti-mouse IgG and peroxidaseconjugated rabbit anti-goat IgG (both from The Jackson ImmunoResearch Laboratories,Bar Harbor, ME) were used at a 1/5000 dilution. Purified apolipoprotein H (132-glycoprotein I) was purchased from Crystal Chem, Chicago, IL.4.2.7 Heparin-agarose chromatographyPC:CHOL:CL (35:45:10) LUVs were incubated with normal human serum at37°C for 30 mm and the mixture chromatographed on a conventional BioGel A15mcolumn as described in Section 3.2.5. The protein content of the pooled fractions wasdetermined as described in Section 4.2.5 to be 0.8 mg/ml. The liposomes were dialyzedagainst 50 mM Tris, pH 7.4, containing 30 mM NaCl. The serum proteins associatedwith the LUVs were solubilized with 2% octyiglucoside (Sigma) and were fractionatedon a 1.5 x 7 cm heparin-agarose (Sigma) affinity column equilibrated with 50 mM Tris,101pH 7.4, containing 30 mM NaC1. The proteins binding to the heparin-agarose columnwere eluted with 50 mM Tris, pH 7.4, containing 400 mM NaCl. The protein content ofthe column fractions (200 drops/fraction) was determined spectrophotometrically bymeasuring the absorbance at a wavelength of 280 nm, and was analyzed by SDS-PAGE.4.2.7 Complement hemolytic assaysFifty il of 25% normal human serum or 5% guinea pig serum in DGVB2wasincubated with an equal volume of 20 mM liposomes at 37°C for 30 mm. After theincubation period, the mixtures were diluted with 150 [Ii of DGVB2and kept on ice.Complement hemolytic assays were performed as described in Section RESULTS4.3.1 In vivo association of murine blood proteins with liposomes exhibitingmarkedly different clearance propertiesIt has previously been demonstrated that liposomes rapidly bind a complex profileof plasma proteins upon exposure to human whole plasma or serum (Bonte and Juliano,1986; Sommerman, 1986; Juliano and Lin, 1980). It has also previously been observedthat liposomes composed of different acidic phospholipids have markedly differentclearance properties. In particular, liposomes containing PA (Abra and Hunt, 1981) orPS (see Chapter 3) have been shown to have very rapid clearance kinetics; whereasliposomes containing hydrogenated plant P1 (Papahadjopoulos and Gabizon, 1987) or102ganglioside GM1 (Allen and Chonn, 1987; Gabizon and Papahjopoulos, 1988) wereshown to be relatively long-lived in the circulation of mice. A primary objective of thisstudy was to determine whether the apparent differences in liposome clearance behaviorobserved in vivo was related to the amount and type of protein associated with thenegatively charged liposomes in vivo.Figure 4.1 depicts the clearance of liposomes composed of various lipidcompositions from the circulation of CD1 mice over a 2 h period, following intravenousadministration of LUVs at a dose level of 200 mol lipid/kg (approximately 120 mglipid/kg). Three types of behavior in the circulation are evident: short lifetimes (CL-,DOPA-, or DOPS-containing LUVs), intermediate lifetimes (PG or plant P1-containingLUVs), and relatively long lifetimes (gangliosideG1-containing LUVs). At 2 mmpost-injection, the liposomes were recovered from the blood of liposome-treated CD1mice using the spin column procedure. The proteins associated with the recoveredliposomes were then analyzed by SDS-PAGE under non-reducing conditions.Figure 4.2 shows the silver stained gels of the proteins associated with liposomesrecovered at 2 mm post-injection. It is immediately apparent that the amount of proteinassociated with 25 nmol of lipid differs dramatically for different lipid species, with therapidly cleared liposomes having the most associated protein (Fig. 4.2A). Furthermore,liposomes having similar membrane surface charge imparted by different anionicphospholipids can exhibit significantly different protein binding abilities (Figs. 4.2A and4.3). The protein binding ability was measured and related to the circulation half-life ofthe LUVs (Fig. 4.3). It may be observed that B is inversely related to the half-life of theliposomes in the circulation.103Figure 4.1. Plasma clearance of LUVs. Liposomes containing trace amounts of[3HJcholesteryl hexadecyl ether were administered intravenously via the dorsal tail veinof CD1 mice at an approximate dose of 20 mol total lipid per 100 g mouse weight.After various times, the recovery of LUVs in plasma was measured by counting aliquotsof the plasma using standard scintillation methods. Panel A consists of liposomecompositions that are very short-lived: ( 0 ) PC:CHOL:CL (35:45:10), ( D )PC:CHOL:DOPA (35:45:20), and (A ) PC:CHOL:DOPS (35:45:20). Panel B includesliposome compositions that are moderately long-lived or long-lived: ( 0 ) PC:CHOL(55:45), ( • ) PC:CHOL:PG (35:45:20), ( D ) PC:CHOL:plant P1(35:45:20), ( V )SM:PC (4:1), ( • ) SM:PC:GM1 (72:18:10), and ( A ) SM:PG:GM1 (72:18:10).Liposome compositions are expressed in molar ratios. The data points represent theaverage plasma recovery and sample standard deviation from 4 mice.EC00>0C,1001010.10.0510010Time (mm)0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140Time (mm)104Figure 4.2. Silver-stained nonreducing SDS-PAGE gel of proteins associated withliposomes recovered from the circulation of mice after 2 miii post-injection. Theproteins associated with the liposomes were separated electrophoretically on 4-20% SDSPAGE gels and visualized by silver stain. Panel A consists of proteins associated with 25nmol total lipid of LUVs composed of the following: PC:CHOL:CL (35:45:10; Lane 1),PC:CHOL:DOPA (35:45:20; Lane 2), PC:CHOL:DOPS (35:45:20; Lane 3),PC:CHOL:PI (bovine liver; 35:45:20; Lane 4), PC:CHOL:PI (plant; 35:45:20; Lane 5),PC:CHOL:PG (35:45:20; Lane 6), PC:CHOL (55:45; Lane 7), SM:PC:GM1(72:18:10;Lane 8), and SM:PG:GM1(72:18:10; Lane 9). Panel B consists of proteins associatedwith 50 nmol total lipid of SM:PC (4:1; Lane 1) or SM:PC:GM1(72:18:10; Lane 2). Mcontains SDS-PAGE molecular weight standards (myosin, 200 000; 3-galactosidase, 116250; phosphorylase B, 97 400; serum albumin, 66 200; ovalbumin, 45 000; carbonicanhydrase, 31 000, trypsin inhibitor, 21 500; and lysozyme, 14 400).—A1 2 3 4 5 6 7 8kDo -.200— — —B;—95.5 —55-43—36— — aiii105Figure 4.3. Relation of total amount of protein bound to liposomes and circulationhalf-life. Aliquots of the recovered liposomes were delipidated, and the extractedproteins quantitated using the micro BCA protein assay as described in “Methods andMaterials”. The liposomes were composed of ( V ) PC:CHOL:PI (bovine liver,35:45:20), (• ) PC:CHOL:DOPA (35:45:20), (• ) PC:CHOL:CL (35:45:10), (A )PC:CHOL (55:45), (0) PC:CHOL:PI (plant; 35:45:20), () SM:PC (4:1) and (0)SM:PC:GM1(72:18:10).180160 y140ci1204-’04-’100E40ALl200I I I0 40 80 120 160 200 240T112 (mm)106A striking result demonstrating the correlation between clearance rates and theamount of bound protein, as well as the lack of correlation between protein binding andsurface charge is illustrated by the behavior of liposomes containing 20 mol% plant P1 orbovine liver P1. As shown in Figure 4.1, liposomes containing plant P1 exhibit acirculation half-life of approximately 110 mm, whereas liposomes containing the sameamount of bovine liver P1 have a half-life of less than 2 mm, with only 19.6 ± 2.0 % ofinjected dose (n=8) recovered in the blood after 2 mm. This correlates with markeddifferences in protein binding, where the rapidly cleared bovine liver P1 liposomesexhibit B values of 158 g protein/mol lipid as compared to 26 g protein/mol lipid forliposomes containing plant P1.4.3.2 Comparison of in vivo and in vitro systemsThe results to this stage clearly indicate that the amount of blood proteinassociated with liposomes in the circulation dramatically affects liposome clearancebehavior in vivo. In general, liposomes exhibiting very rapid clearance kinetics have thegreatest potential to bind blood proteins. In contrast, liposomes exhibiting extendedcirculation residency times have markedly reduced amounts of associated blood proteins.It should be noted, however, that the in vivo analysis is limited by the amount ofliposomes recovered, especially for rapidly cleared liposomes. In order to furthercharacterize the surface properties of LUV systems in relation to protein binding andknown clearance properties, it is useful to develop an in vitro assay. In this regard it isfirst important to show that the amount of protein bound to various species of LUVs invitro correlates with the amount bound in vivo. Second, it is of interest to compare the107protein profile bound in vivo with that obtained in vitro. The B values determined fromin vitro assays (Table 4.1) were similar to those from in vivo determinations (Fig. 4.3). Itis of interest, however, to note that the profile of bound proteins achieved by employingthe in vitro assay differs considerably from that observed in vivo (Fig. 4.4).4.3.3 Identification of opsonins associated with rapidly cleared liposomesThe rapid clearance of liposomes containing high levels of adsorbed surfaceprotein would suggest that these adsorbed porteins included substantial levels ofopsonins, leading to rapid uptake by phagocytic cells of the reticuloendothelial system.In order to demonstrate this, immunoblot analysis specifc for the immune opsonins, C3and IgG, were performed. As shown in Figure 4.5, higher levels of these opsonins couldbe detected among the more rapidly cleared liposomes such as those containing CL orDOPA.One of the major proteins associated with the rapidly cleared CL- and DOPAcontaining liposomes is a protein with an electrophoretic mobility corresponding to amolecular mass of approximately 50 kDa (Fig. 4.4). An equivalent protein wasrecovered with 10 mol% CL-containing and 20 mol% DOPA-containing LUVs incubatedwith human serum (Fig. 4.4). Earlier work by Sommerman (1986) showed that besidesalbumin, this 50 kDa protein was the major protein bound to PC:CHOL:PS (1:2:1)MLVs. Other investigators have identified a protein of similar molecular weight thatbinds to CL-containing liposomes as apolipoprotein H (McNeil et al., 1990; Galli et al.,1990). As shown in Figure 4.4, purified apolipoprotein H exhibits very similarelectrophoretic mobility as the 50 kDa protein which binds to rapidly cleared LUVs.108Table 4.1. B values for LUVs recoveredfrom in vitro incubations with human serum.Composition of LUVs B (g protein/mol lipid)aPC:CHOL (55:45) 20.8 ± 1.8PC:CHOL:PG (35:45:20) 23.0 ± 2.8PC:CHOL:DOPS (35:45:20) 40.8 ± 2.8PC:CHOL:DOPA (35:45:20) 46.1 ± 3•4PC:CHOL:CL (35:45:10) 101 ±7.1a Values represent average and standard deviation from at least 3 independentdeterminations.109Figure 4.4. Comparison ofprotein profiles of LUVs recoveredfrom the circulation ofmice andfrom in vitro incubations with isolated serum. Using the spin column method,the LUVs, PC:CHOL:CL (35:45:10) or PC:CHOL:DOPA (35:45:20), were isolated from(Lane 1) in vivo 2 mm incubation in mice; (Lane 2) in vitro 2 mm incubation withisolated mouse serum; (Lane 3) in vitro 30 mm incuation with isolated mouse serum; or(Lane 4) in vitro 30 mm incubation with isolated human serum. The proteins associatedwith LUVs (25 nmol total lipid) were electrophoresed on a 4-20% gradient SDS-PAGEgel and detected by silver stain. M represents SDS-PAGE molecular weight standards(refer to Fig. 4.2 for molecular weights) and H represents purified human apolipoproteinH. ( [)represents regions where proteins that are associated with LUVs recovered fromthe circulation of mice differ from those recovered from in vitro incubations with isolatedmouse serum.CL DOPAI II I[-4110Figure 4.5. Immunoblot analysis of murine opsonins associated with LUVs. Theproteins associated with LUVs (25 nmol total lipid) were separated electrophoretically on4-20% SDS-PAGE gels and analyzed by immunoblot analysis specific for mouse C3 ormouse IgG. The lanes contain the following liposome compositions: PC:CHOL:CL(35:45:10; Lane 1), PC:CHOL:DOPA (35:45:20; Lane 2), PC:CHOL:DOPS (35:45:20;Lane 3), PC:CHOL:PI (plant; 35:45:20; Lane 4), PC:CHOL:PG (35:45:20; Lane 5),PC:CHOL (55:45; Lane 6), SM:PC:GM1(72: 18:10; Lane 7), and SM:PG:GM1(72:18:10;Lane 8).12 567834C3/C3b_,12345678IgG .-‘111Further characterization of this protein showed that it has an affinity for heparin asdemonstrated by retention on heparin-agarose columns (Fig. 4.6) and is soluble in thepresence of 3% perchioric acid. These properties are consistent with the identity of this50 kDa protein being apolipoprotein H (Polz et al., 1980; Lee et a!., 1983; Schousboe,1985; Finlayson et al., 1967).Other human serum proteins associated with rapidly cleared PC:CHOL:CL(35:45:10) LUVs were anlayzed by immunoblot analysis employing antisera specific for15 different proteins. As shown in Table 4.2 the large majority of these proteins could bedetected among those bound to the liposomes.4.3.4 Influence of ganglioside GM1 content on the binding of blood proteins to LUVsIncorporation of ganglioside GM1 into SM:PC (4:1) liposomes has been shown toresult in extended circulation residency lifetimes (Allen and Chonn, 1987; Gabizon andPapahadjopoulos, 1988), and it is of interest to examine the mechanism involved. Asshown in Figures 4.2B and 4.3, the amount of protein associated with SM:PC (4:1) LUVsis significantly reduced by inclusion of 10 mol% ganglioside GM1, with a correspondingincrease in circulation lifetime. A particular question concerns whether ganglioside GM1acts to specifically decrease binding of opsonins such as IgG or C3 fragments, or whetherthe ganglioside GM1 effect arises from a non-specific decrease in the binding of all bloodproteins. Using the in vitro system involving incubation of LUVs with human serum, itwas demonstrated that increasing the ganglioside GM1 content of PC:CHOL LUVsprogressively reduced the amount of total protein bound to PC:CHOL LUVs (Fig. 4.7A).The B values were 36.4, 19.6, 15.4, 11.6 and 13.0 g protein/mol lipid for 2, 4, 6, 8 and1120c’J4..ci00Ca).4-’0I-ft.Figure 4.6. Heparin-agarose chromatography of proteins associated with CL-containing LUVs. The proteins associated with PC:CHOL:CL (35:45:10) LUVs weresolubilized with 5% octyiglucoside and were fractionated using a 1.5 x 7 cm heparinagarose column as described in “Methods and Materials”. Proteins having an affinity forheparin were eluted with 50 mM Tris, pH 7.4, containing 400 mM NaC1 (starting atfraction # 10). The second peak contains a protein migrating in the region between 50-55kDa.0.400.300.200.100.000 10 20Column fraction #30113Table 4.2. Immunoblot analysis ofproteins associated with CL-containing LUVs. Theproteins associated with PC:CHOL:CL (35:45:10) LUVs were separatedelectrophoretically using the automated PhastSystem electrophoresis apparatus on 4-15%gradient precast PhastGels (Pharmacia LKB Biotechnology, Piscataway, NJ) andtransblotted onto nitrocellulose and immunoblotted using similar procedures described inSections 2.2.7 and 4.2.6. Antisera to the various human proteins were obtainedcommercially and used at a 1/1000 dilution.Antisera to specific protein Molecular weight Detected (+)/not detected (-)albumin 65 900 +a-1-antitrypsin 53 200 +a-2-macroglobulin 700 000 +C3 185000 +C4 210000 +CS 190000 +C9 70000 +C-reactive protein 95 000 -fibronectin 550 000 +haptoglobin 86 000 +IgG 150000 +1gM 850000 +transferrin 80 000 +vitronectin 69 000 -114Figure 4.7. Effect of ganglioside GMJ and GDJa on human serum protein associationwith liposomes. In Panel A, the proteins associated with LUVs (20 nmol total lipid) wereseparated electrophoretically on 4-20% SDS-PAGE gels and visualized by silver stain.The lanes represent the following liposome compositions: PC:CHOL (55:45; Lane 1);PC:CHOL:GM1(53:45:2; Lane 2); PC:CHOL:GM1(51:45:4; Lane 3); PC:CHOL:GM1(49:45:6; Lane 4); PC:CHOL:GM1(47:45:8; Lane 5); PC:CHOL:GM (45:45:10; Lane6); PC:CHOL:GD1a (53:45:2; Lane 7); PC:CHOL:GD1 (51:45:4; Lane 8); orPC:CHOL:GD (69:45:6; Lane 9). In Panel B, equal amounts of protein (0.5 mg)associated with PC:CHOL:GM1(53:45:2; Lane 1) and PC:CHOL:GM1(45:45:10; Lane2) were loaded per lane. M represents silver stain SDS-PAGE molecular weightstandards (refer to Fig. 4.2 for molecular weights).• :•. •BM 1 2IA1 2 3 4 5 6 7 8 9 M,- —- — — —11510 mol% ganglioside G containing PC:CHOL liposomes, respectively. It is interestingto note that the 2 mol% ganglioside GM1 had a higher B value than PC:CHOL (55:45)LUVs (B value of 23.0 g protein/mol lipid). Furthermore, the decrease in proteinbinding was apparent for all blood serum proteins, suggesting a non-specific effect. Thisis illustrated more clearly in Figure 4.7B where it is shown that when equal amounts oftotal protein are loaded per lane (0.5 mg) the protein profile for PC:CHOL LUVscontaining 2 or 10 mol% ganglioside GM1 are virtually identical.Previous studies have established that other gangliosides, such as gangliosideGD1a, are not effective in prolonging the circulation residency lifetime of liposomes(Allen and Chonn, 1987). As shown in Figure 4.7A, increasing the ganglioside GD1acontent of PC:CHOL (55:45) LUVs promoted the liposome association of serumproteins. Together this illustrates further the significant role of proteins in determiningthe fate of liposomes in the circulation.The reduction in associated blood proteins has significant consequences. Forexample, inhibition of binding of C3 or other complement proteins may result in aninhibition of complement activation, a membrane-requiring process. In Chapter 2, it wasshown using in vitro guinea pig and human systems that liposomes containing anionicphospholipids activate the complement system via the classical pathway, resulting in C3bdeposition onto these liposomes. Using similar in vitro human and guinea pig systems,the ability of ganglioside GM1 to inhibit complement activation was demonstrated. Asshown in Figure 4.8, complement-activating liposomes, PC:CHOL:PG (35 :45 :20), werecapable of reducing the complement hemolytic levels of human or guinea pig serum;however, if 10 mol% ganglioside GM1 was included in 20 mol% PG-containing vesicles,the liposomes were no longer complement activating.116Figure 4.8. Inhibition of complement activation by liposomes containing gangliosideGM1. Complement hemolytic assays were used to determine the residual complementhemolytic activity of the serum after incubation of the serum with 1 imol total lipid ofliposomes composed of PC:CHOL (55:45), PC:CHOL:GM1(45:45:10), PC:CHOL:PG(35:45:20), or PC:CHOL:PG:GM1(25:45:20:10).>4-.0a)Ea)E00ctSD0)a)Human serum Guinea pig serum1174.4 DISCUSSIONLiposomes having similar membrane surface charge imparted by different anionicphospholipids were found to exhibit markedly different clearance behavior in mice. Theresults presented here indicate that this phenomenon is related to the different abilities ofthe various anionic LUVs to interact with blood proteins. In general, LUVs that wererapidly cleared from the circulation (t112 < 2 mm) had the greatest ability to bind bloodproteins (RB> 50 g protein/mol lipid). On the other hand, LUVs exhibiting extendedcirculation lifetimes (‘1/2> 2 h) had significantly reduced amounts of associated bloodproteins (PB < 5 g protein/mol lipid). The protein binding ability of the liposomes fromin vivo experiments was found to be inversely related to circulation half-life. Theseobservations suggest that clearance rate is a function of the amount of protein bound tothe liposomes in the circulation.Liposomes containing equivalent amounts of net negatively chargedphospholipids have different capacities to interact with blood proteins in vivo. Thisinteraction does not appear to be simply electrostatic because liposomes with equivalentmol% net negative charge imparted by various anionic phospholipids have similarsurface potentials. Other factors are important. In the case of the two different sourcesof P1, the fatty acyl chain length and saturation were shown to markedly influence bloodprotein/membrane interactions (see Fig. 4.2). Plant P1 is composed of more saturated[mainly palmityl (16:0) and stearyl (18:0)] fatty acyl groups than bovine liver P1 [mainlystearyl (18:0), linoleyl (18:2) and arachidonyl (20:4)]. The ability of the proteins to insertinto the liposome membrane as a result of membrane defects may be an important118consideration; in which case, these findings suggest that CL or DOPA increases thelikelihood of membrane defects allowing for greater amounts of proteins to insert.The markedly different ability of various anionic LUVs to interact with bloodproteins was also observed using an in vitro system involving liposome incubations withisolated serum. The in vitro findings correlate with the in vivo findings in terms of totalamount of protein bound, indicating that assays for the total amount of serum proteinbinding to LUVs in vitro should be predictive of their clearance behavior in vivo.Noteworthy, however, is the considerable difference in the protein profiles observedwhen employing the in vitro assay compared to that observed in vivo. The profiles of therapidly cleared LUVs recovered from the in vivo assays are more complex and are mostlikely a reflection of the more complex nature of the in vivo system. The proteins whichbind to LUVs in vivo and not in isolated serum may involve coagulation proteins, cell-derived proteins, or cell-mediated cleavage products of blood proteins. This findingunderlines the need to characterize the blood protein interactions liposomes experience invivo in order to best resolve the role of specific proteins in mediating liposome leakageand liposome uptake by the reticuloendothelial system.The rapid clearance of liposomes containing large amounts of surface associatedprotein would suggest that these proteins include substantial levels of opsonins, leadingto rapid uptake by phagocytic cells. Consistent with this hypothesis is the finding thatPC:CHOL:CL (35:45:10) and PC:CHOL:DOPA (35:45:20) LUVs bind the most C3, asdemonstrated by specific immunoblot analysis of the proteins associated with LUVs invivo (see Fig. 4.4). This is in agreement with the previous observation that the amount ofC3 associated with PC:CHOL:CL (35:45:10) and PC:CHOL:DOPA (35:45:20) LUVswas 8-10 times greater than for PC:CHOL (55:45) LUVs after a 30 mm incubation of the119LUVs with isolated human serum (see Chapter 3). By further analyzing the proteinprofiles of the various LUVs recovered from the circulation of mice, it is clear that IgG isalso associated predominantly with rapidly cleared liposomes. This interaction may bespecific for the phospholipids inasmuch as antiphospholipid antibodies have been shownto be present in normal human serum (Alving, 1984), or they may be nonspecific asdemonstrated by Senior et al. (1986).It is noteworthy, however, that the proteins associated with rapidly cleared LUVsinclude several proteins other than the immune opsonins (see Table II). This suggeststhat other proteins may be involved in mediating liposome clearance. The mostprominent protein that is associated with rapidly cleared liposomes is a 50 kDa protein,which has been tentatively identified as apolipoprotein H. Apolipoprotein H has recentlybeen described to have a cofactor role for antiphospholipid antibody binding (McNeil etal., 1990; Galli et al., 1990). Apolipoprotein H has also been shown to have a highaffinity to artificial triglyceride-rich lipid emulsions (Polz et al., 1980) and has beensuggested to be involved in the clearance of triglyceride-rich particles. The observationthat apolipoprotein H strongly interacts with the rapidly cleared CL- or DOPA-containingliposomes suggests that apolipoprotein H may be involved in liposome clearance.Inclusion of ganglioside GM1 reduces the total amount of protein bound to theliposomes in a relatively non-specific manner. A consequence of reducedprotein/liposome interactions is an inhibition of membrane-associated reactions such ascomplement activation (see Fig. 4.8), and a reduction in the association of blood opsonins(see Fig. 4.5). Consequently, gangliosideG1-containing liposomes are capable ofevading liposome recognition and uptake by phagocytic cells. This suggests that animportant property of biocompatible surfaces or of “self’ membranes is that these120membranes are able to inhibit total protein binding, not specifically opsonin binding.This appears to be the mechanism by which ganglioside GM1 extends circulationlifetimes. Furthermore, this predicts that other lipids which inhibit protein/liposomeinteractions in a similar non-specific manner will be useful in prolonging the circulationhalf-life of liposomes.The finding that LUVs containing PA, PS or CL are cleared very rapidly from thecirculation suggests a possible function for these anionic phospholipids in conveying the“non-seW’ signal when exposed on the outer monolayer. Inasmuch as these lipids arenormally not present in the outer monolayer, this function is consistent with thehypothesis that a possible mechanism involved in the recognition of senescent cells isloss of lipid asymmetry resulting in exposure of these acidic phospholipids andsubsequent immune clearance (Schroit et al., 1984; Schlegel et al., 1985; McEvoy et al.,1986; Allen et al., 1988). Schroit et al. (1984) have suggested that PS exposure on theouter monolayer is directly recognized by macrophages. Alternatively, as mentionedabove, enhanced phagocytic recognition and uptake may be mediated by increased bloodprotein interactions promoted by PS exposure.The studies described here demonstrate the usefulness of liposome systems instudying the membrane properties that distinguish “self’ from “non-self, and inidentifying the proteins that enhance clearance. The lipid compositions of the liposomesare readily altered; well-characterized stable large unilamellar liposomes can beemployed in an in vivo assay; and the absence of integral membrane proteins, unlike inbacterial systems, simplifies the analysis of blood proteins associated with the vesicles.It is apparent from these studies that membranes which are recognized as “non-self’ orforeign have the property of supporting increased levels of protein binding. This121increased protein binding results in an increased probability of phagocytic uptake. Thisis strongly indicated by the relation between B values and circulation half-life observedin vivo. Further, this relationship suggests that the amount of protein associated with thevesicles can be used as an indicator of how stable the liposomes will be in the circulation.The B values obtained from LUVs recovered from in vitro incubations with isolatedserum exhibit similar trends as those from LUVs recovered in vivo. The in vitrodeterminations, being simpler and allowing for greater recoveries of LUVs, wouldtherefore be a useful assay for predicting the clearance behavior of liposomes of novelliposome compositions.122CHAPTER 5 SUMMARIZING DISCUSSIONThe studies presented in this thesis were aimed at establishing the role of bloodproteins and in particular, the role of complement proteins, in mediating the clearance ofliposomes in vivo. The relation between the association of these blood proteins andliposome clearance was studied using liposomes exhibiting dramatically differentclearance properties. Initially, it was of interest to determine whether liposomes activatethe complement system when exposed to the complex biological milieu, given thatcomplement activation produces effector molecules that lead to opsonization,macrophage activation and membrane lysis. The interaction between liposomescomposed of relatively simple lipid species and complement proteins had not beenpreviously characterized, especially as it pertains to liposome clearance.In Chapter 2, it was shown that liposomes did activate the complement systemresulting in C3b deposition on liposome surfaces. A study of the membrane surfacerequirements that support complement activation revealed that surface charge was animportant determinant of complement-activating liposomes. Further, the nature of thesurface charge, whether net negative or positive, appeared to dictate which complementpathway, classical or alternative pathway, was activated. The role of surface charge inmediating complement activation, however, was not further investigated in this thesis.The activation of the classical pathway of complement by net negatively chargedliposomes is consistent with previous findings that polyanionic surfaces activate theclassical pathway (refer to Section 2.1).There are at least three possible mechanisms by which negatively chargedphospholipids activate complement. The first model involves the direct adsorption of123Clq molecules to the negatively charged headgroups of the phospholipids. This couldthen lead to conformational changes in Clq that subsequently lead to Cir and Cisactivation. Several findings presented in this thesis support this model of directClq/negatively charged phospholipid interaction. In Chapter 2, it was shown that preadsorption of human serum with anionic liposomes had little effect on subsequentcomplement-mediated liposome lysis. This finding strongly suggested that theconsumption in complement hemolytic levels observed in the complement hemolyticassays was a result of complement activation, and not simply a result of consumption in alimiting serum factor (other than complement proteins) required for complement-mediated hemolysis. Nonspecific adsorption of serum proteins (at 4°C) to the anionicliposomes did not limit a serum factor required for complement activation. Further,several studies have shown that the structure of the phospholipid acyl chains, such asfatty acyl chain length or saturation, markedly affect complement activation (see Fig. 2.3;and also Brulet and McConnell, 1976), suggesting that complement fixation depends onthe lateral mobility of the anionic lipids in the plane of the membrane.Secondly, the adsorption of Clq to the liposome surface could be mediated byantiphospholipid antibodies, which have been described to be present in normal humanserum (refer to Section, leading to the typical classical pathway activation. Asdiscussed in Chapter 2, this possibility seems unlikely.A third possibility is that one or more proteins, which is present in plasma in nonlimiting concentrations, mediate the interaction between Clq and the negatively chargedphospholipids. In this regard, alterations in the membrane composition would affect theamount and type of proteins associating with the liposomes and consequently,complement activation. From the results presented in Chapters 3 and 4, this model seems124likely inasmuch as several proteins have been shown to associate with the liposomes inthe complex biological milieu. Perhaps this is the most interesting possibility in light ofthe finding that one of the predominant proteins associated with anionic liposomes isapolipoprotein H, a cofactor for the binding of antiphospholipid antibodies. A possiblesequence of events, therefore, may be as follows: apolipoprotein H binds to thenegatively charged phospholipids; this leads to the binding of antiphospholipidantibodies, which subsequently leads to classical complement activation. Alternatively,the binding of fibrinogen to these anionic phospholipids could lead to the binding of Clqinasmuch as Clq has been shown to have high-affinity interactions with fibrinogen andfibrin (Entwistle and Furcht, 1988).If a strong correlation between complement activation potential and clearancerates of liposomes could be shown, then this would be the basis for future experimentsdirected at regulating complement activation on liposome surfaces. This could beachieved by reconstituting complement regulatory molecules such as decay acceleratingfactor or homologous restriction factor. It was surmised that a liposome system capableof inherently inhibiting complement activation would be capable of circulating forextended periods since the association of opsonic C3 fragments or membrane attackcomplexes would be inhibited.In Chapters 3 and 4, the amount of C3 associated with liposomes was shown to berelated to the clearance behavior of liposomes in the circulation of mice. For liposomescontaining various anionic phospholipids, it appears that liposome compositions havingthe most membrane-associated C3 were also the most rapidly cleared. Further, inclusionof ganglioside GM1 into liposomes, which extends the circulation half-life of liposomes,was shown to transform classical complement-activating liposomes into non-activators125(see Chapter 4). It was previously demonstrated that ganglioside GM1 inhibits activationof the human alternative pathway by promoting the association of factor H, a solublenegative regulator of the alternative complement pathway, to C3b (Michalek et al., 1988;Okada et a!., 1982). These findings suggest that complement proteins play a significantrole in mediating the clearance of liposomes from the circulation.The relationship between complement activation and liposome clearance rate,however, appeared to break down in at least two instances. First, it was shown inChapter 2 that the inclusion of 45 mol% cholesterol in liposomes containing anionicphospholipids made the liposomes better activators of the complement system asdemonstrated by the complement hemolytic assays; whereas the inclusion of up toequimolar amounts of cholesterol is generally well accepted to stabilize liposomes in thecirculation (see Section Second, neutral systems appear to be very weakactivators of the complement system; yet neutral LUVs composed of saturatedphosphatidylcholines in the absence of cholesterol are cleared very rapidly from thecirculation. At least for these systems, complement does not appear to play a major rolein their clearance from the circulation. Thus, factors other than complement are involvedin mediating liposome clearance.The focus of the work, therefore, switched to the other proteins that associate withrapidly cleared LUVs and long-lived LUVs in order to assess the role of blood proteins inmediating liposome clearance. Given the inconsistencies in previous in vitro reports onthe association of serum proteins with liposomes (primarily MLVs), or on the role ofproteins in enhancing phagocytic uptake by cultured macrophages, it was decided toperform such studies in vivo using a mouse animal model. A significant achievement126central to this approach was the development of a “spin column” procedure to recoverLUVs from blood components (detailed in Chapter 3). By employing this procedure,LUVs could be readily isolated from the blood of mice that had been intravenouslyinjected with LUVs. The method proved to be simple and rapid such that the isolationprocedure could be done in the absence of blood coagulation inhibitors; effective inisolating LUVs from large lipoproteins; and appropriate for small sample volumes.Furthermore, well-defined LUVs could be used instead of MLVs and thus makingpossible the measurement of parameters such as the amount of protein bound per vesicle.In Chapter 4, liposomes having similar membrane surface charge imparted bydifferent anionic phospholipids were found to exhibit markedly different clearancebehavior in mice. The basis for this phenomenon was shown to be the dramaticdifferences in protein binding abilities of the various anionic LUVs. The protein bindingability of the liposomes was found to be inversely related to the circulation half-life.These studies suggest that liposomes that bind large amounts of protein have a greaterprobability of being taken up by the reticuloendothelial system. Conversely, liposomesthat do not bind large amounts of protein exhibit extended circulation lifetimes. It isconcluded therefore that interactions between liposomes and cells, particularlyphagocytic cells, is protein-mediated. The membrane factors that reduce the interactionswith blood proteins will be of importance for designing liposomes which are capable ofextended circulation lifetimes. It will be of interest to see what effects membranecholesterol levels, or fatty acyl chain length and saturation will have on protein bindingabilities of liposomes and their clearance properties.The interactions of blood proteins with liposome membranes in the biologicalmilieu are undoubtedly complex. Thus, to make generalizations on how and why blood127proteins interact with liposome membranes is virtually impossible, especially in light ofthe fact that a variety of blood proteins associate with liposomes in the circulation. Whyblood proteins interact with liposome membranes may be a result of several properties ofthe proteins. For example, size undoubtedly plays a major role in determining the?surface activity” of a protein because larger proteins would have more contact points tointeract with the liposome surface; the charge and charge distribution of proteins arelikely to influence the surface activity because it is known that most of the charged aminoacids reside at the exterior of proteins and therefore, these charged residues must comeinto close proximity with the surface in the process of adsorption. Structural aspects ofthe proteins may also influence the surface activity of the proteins. For example, proteinswhich may readily undergo conformational changes, such as unfolding, are more likely toadsorb to the liposome surfaces. Other properties such as the amphipathic nature of theprotein molecule and the solubility of the proteins in plasma may play a significant rolein the interactions of blood proteins with liposome membranes.The results presented in this thesis indicate that surface charge is a primarymediator of protein/liposome interactions. As discussed in Chapter 4, the majority of theblood proteins, however, do not appear to interact with liposome membranes simply byelectrostatic interactions. Liposomes with equivalent mol% net negative charge impartedby various anionic phospholipids have similar surface potentials and yet the amount ofprotein associated with the various liposomes differ markedly. The ability of the proteinsto insert into the liposome membrane as a result of membrane defects may be animportant consideration. Surface defects would expose hydrophobic regions of themembrane bilayer, allowing for hydrophobic interactions to occur. Consequently, thiswould result in conformational changes in the proteins that would increase the number of128membrane adsorption sites. Furthermore, as previously discussed, the association ofproteins (cofactors) with the liposome membrane could lead to the assembly of multi-component complexes on the surface of the liposomes. An example would be theformation of apolipoprotein H/antiphospholipid antibody complexes. Other secondaryinteractions involve the generation of active fragments as a result of activation ofimmune pathways. An example here would be the generation of active C3b fragments,leading to the covalent attachment of C3b to the membrane surface via a thiolester bond.One further note is that the proteins found to associate with the liposomes mostlikely represent proteins that are tightly bound to the liposome surface. The proteinprofiles observed by isolating the liposomes via the spin column method are in closeagreement with those observed by isolating the liposomes by ultracentrifugation followedby multiple buffer washes. The protein profiles of the liposomes recovered after 2 mm inthe circulation of mice using the spin column procedure have always been veryreproducible, at least qualitatively as determined by SDS-PAGE analysis orquantitatively as determined by B values. This indicates that the proteins are not readilydesorbed or displaced. It has been suggested the this irreversibility in protein binding tothe liposome surface is a result of multiple adsorption sites between proteins and theliposome membrane such that the probability that all the binding sites will be broken atthe same time is highly unlikely (Horbett and Brash, 1987). It should be noted that theproteins that are readily displaceable are probably not recovered with the liposomes usingthe spin column procedure or by ultracentrifugation procedures as a result of dilution ofthe proteins in buffer. The displacement of adsorbed proteins has been observed byseveral investigators and has been termed the “Vroman Effect” (Horbett and Brash,1987). These proteins may also play a role in liposome clearance, either by enhancing129liposome/cell interactions, or by sterically hindering liposome-associated opsonins andcellular receptor interactions and thus, providing a “dysopsonin” effect. It wouldtherefore be of interest to develop methods to enable the characterization of these readilydisplaceable proteins that associate with liposomes.From the studies examining the mechanism by which lipids such as gangliosideGM1 extend the circulation lifetime of liposomes, it was shown that the inclusion ofganglioside GM1 reduces the total protein binding to LUVs in a relatively non-specificmanner. The inclusion of ganglioside GM1 creates a surface that is not interactive withblood proteins; therefore, membrane-associated reactions such as complement activationdo not occur. This has important implications for designing liposomes that haveextended lifetimes. Novel liposome compositions such as liposomes containing PEconjugated polyethyleneglycols have recently been shown to exhibit extended circulationlifetimes. It will be of interest to determine the mechanism by which these moleculesextend the circulation half-life of liposomes, particularly with respect to the inhibition oftotal protein binding.Current theories on how ganglioside GM1 extends the circulation lifetime ofliposomes remain controversial. It is believed that ganglioside GM1, and other moleculessuch as hydrogenated plant PT and PE-conjugated polyethyleneglycols, function byproviding a hydrophilic surface coating that inhibits the membrane insertion of proteins,and by providing a steric barrier such that membrane adsorption sites are masked. ForPE-conjugated polyethyleneglycols, the steric barrier hypothesis appears to be likelybecause coating the liposomes with this polymer has been recently shown to physicallyinhibit the interactions of immunoliposomes with target cells, as well as liposomeagglutination (Klibanov et al., 1991). For ganglioside GM1, however, it is not clear what130properties are important. The effect of ganglioside GM1 appears to be unique to thisganglioside as other gangliosides, such as ganglioside GD1a or GT1b, fail to extendliposome half-lives (Allen and Chonn, 1987). These other gangliosides and glycolipids,such as globosides, share the hydrophilic and steric barrier properties of gangliosideGM1. The uniqueness of ganglioside GM1 may be a result of the conformation that thecarbohydrate moeity (which lies horizontally to the plane of the membrane masking thenegatively charged sialic acid) adopts on the surface of the membrane. Thisconformation appears to be dependent on the presence of the sialic acid becauseneuraminidase treatment of ganglioside GM1 (asialoganglioside GM1) abolishes itsprotective effect. Perhaps the reason that the other gangliosides are not effective isbecause of the exposed negative charges of the sialic acid residues.That the protein binding ability of the liposomes appears to markedly affect theirclearance behavior strongly indicates that in order to achieve effective opsonization orliposome/cell interactions, proteins must be adsorbed on the liposome surfaces. Therapid clearance of liposomes having large amounts of surface associated protein suggeststhat these liposomes contain substantial amounts of proteins that mediate liposome/cellinteractions, such as blood opsonins. It was shown that liposomes binding large amountsof protein also bound large amounts of opsonins, namely C3 and IgG (Chapter 4).The studies described here demonstrate the usefulness of liposome systems incharacterizing the membrane properties that distinguish ‘self” from “non-self”. It wasshown that a property of “non-self’ membranes is a greater capacity to interact withblood proteins. Liposomes are also useful in elucidating novel blood opsonins. Forexample, by using a protein fingerprinting analysis on the proteins associated withrapidly cleared LUVs, it was shown that a 50 kDa protein was associated predominantly131with very rapidly cleared LUVs (Chapter 4). This protein was tentatively identified asapolipoprotein H. Future experiments are aimed at identifying this 50 kDa protein byimmunoblot analysis and by comparing the N-terminal protein sequence. Further,inasmuch as apolipoprotein H has recently been described to function as a cofactor forbinding of antiphospholipid antibodies, and has been suggested to be involved intriglyceride-rich particle clearance, apolipoprotein H may therefore be involved inliposome clearance. Future experiments are aimed at delineating the role ofapolipoprotein H in liposome clearance by determining whether liposomes coated withpurified apolipoprotein H are taken up more readily by cultured macrophages. As well,since apolipoprotein H has been implicated in triglyceride metabolism, futureexperiments will attempt to identify the apolipoprotein H receptor in order to determinespecifically whether this receptor is expressed on phagocytic cells and whether it isinvolved in the opsonization process.The studies presented in this thesis establish that the blood proteins whichassociate with liposomes in the circulation dramatically influence the clearance behaviorof liposomes in vivo. An inverse relation between the amount of protein bound (RB; gprotein/mol lipid) and circulation half-life was shown for liposomes composed of variousanionic phospholipids. This relation suggests that B values can be useful for predictingthe clearance behavior of novel liposome compositions and will surely expedite thedevelopment of an optimized liposome delivery system which circulates for extendedlifetimes.132BIBLIOGRAPHYAbra, R. M., Hunt, C. A. and Lau, D. T. (1984) Liposome disposition in vivo. VI.Delivery to the lung. J. Pharm. Sci. 73:203-206.Agarwal, K., Bali, A. and Gupta, C. M. (1986) Influence of the phospholipid structureon the stability of liposomes in serum. Biochim. Biophys. Acta. 856:36-40.Agnello, V., Can, R. I., Koffler, D. and Kunkel, H. G. (1969) Gel diffusion reactions ofClq with aggregated gamma-globulin, DNA and various anionic substances. Fed. Proc.28:696 (abstract).Agrawal, A. K., Singhal, A. and Gupta, C.M. (1987) Functional drug targeting toerythrocytes in vivo using antibody bearing liposomes as drug vehicles. Biochem.Biophys. Res. Commun. 148:357-361.Alexander, E. L., Titus, J. A. and Segal, D. M. (1979) Human leukocyte Fc (IgG)receptors: quantitation and affinity with radiolabeled affinity cross-linked rabbit IgG. J.Immunol. 123:295-302.Allen, T. M. and Chonn, A. (1987) Large unilamellar liposomes with low uptake intothe reticuloendothelial system. FEBS Lett. 223:42-46.Allen, T. M., Hansen, C. and Rutledge, J. (1989) Liposomes with prolonged circulationtimes: factors affecting uptake of reticuloendothelial and other tissues. Biochim.Biophys. Acta 981:27-35.Allen, T. M., Hansen, C., Martin, F., Redemann, C. and Yau-Young, A. (1991)Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) showprolonged circulation half-lives in vivo. Biochim. Biophys. Acta 1066:29-36.Allen, T. M., Ryan, J. L. and Papahadjopoulos, D. (1985) Gangliosides reduce leakageof aqueous-space markers from liposomes in the presence of human plasma. Biochim.Biophys. Acta 818:205-210.133Allen, T. M., Williamson, P. and Schiegel, R. A. (1988) Phosphatidylserine as adeterminant of reticuloendothelial recognition of liposome models of the erythrocytesurface. Proc. Nat!. Acad. Sci. USA 85:8067-8071.Alving, C. R. (1984) Natural antibodies against phospholipids and liposomes in humans.Biochem. Soc. Trans. 12:342-344.Alving, C. R. and Richards, R. L. (1977) Immune reactivities of antibodies againstglycolipids. II. Comparative properties, using liposomes, of purified antibodies againstmono-, di-, and trihexosyl ceramide haptens. Immunochemistry 14:383-389.Alving, C. R. and Richards, R. L.. (1983) Immunologic aspects of liposomes. InLiposomes. Ostro, M., ed. Marcel Dekker, New York, pp. 209-287.Amiguet, P., Brunner, J. and Tschopp, J. (1985) The membrane attack complex ofcomplement: lipid insertion of tubular and nontubular polymerized C9. Biochemistry24:7328-7334.Anderson, C. L. and Grey, H. M. (1977) Solubilization and partial characterization ofcell membrane Fc receptors. J. Immunol. 118:819-825.Andersson, L. 0. and Brown, J. E.. (1981) Interaction of factor VIlI-von Willebrandfactor with phospholipid vesicles. Biochem. J. 200:161-167.Arend, W. P. and Mannik, M. (1973) The macrophage receptor for IgG: number andaffinity of binding sites. J. Immunol. 110:1455-1463.Arvieux, J., Yssel, H. and Colomb, M. G. (1988) Antigen-bound C3b and C4b enhanceantigen-presenting cell function in activation of human T-cell clones. Immunology65:229-235.Aschoff, L. (1924) Reticuloendothelial system. In Lectures on Pathology. Paul B.Hoeber Inc., New York, pg. 1-33.Atkinson, D., Smith, H. M., Dickinson, J. and Austin, J. P. (1976) Interaction ofapoprotein from porcine high-density lipoprotein with dimyristoyl lecithin. I. Thestructure of the complexes. Eur. J. Biochem. 64:541-547.134Bakker-Woudenberg, I. A., Lokerse, A. F. and Roerdink, F. H. (1989) Antibacterialactivity of liposome-entrapped ampicillin in vitro and in vivo in relation to the lipidcomposition. J. Pharmacol. Exp. Ther. 251:321-327.Bakker-Woudenberg, I. A., Lokerse, A. F. and Roerdink, F. H. (1988) Effect of lipidcomposition on activity of liposome-entrapped ampicillin against intracellular Listeriamonocytogenes. Antimicrob. Agents Chemother. 32:1560-1564.Bakouche, 0., Lachman, L. B., Knowles, R. D. and Kleinerman, E. S. (1988) Cytotoxicliposomes: membrane interleukin 1 presented in multilamellar vesicles. LymphokineRes. 7:445-456.Balazsovits, J. A. E., Mayer, L. D., Bally, M. B., Cullis, P. R., McDonell, M., Ginsberg,R. S. and Falk, R. E. (1989) Analysis of the effect of liposome encapsulation on thevesicant properties, acute and cardiac toxicities, and antitumor efficacy of doxorubicin.Cancer Chemother Pharmacol 23:81-86.Baldassare, J. J., Kahn, R. A., Knipp, M. A. and Newman, P. J. (1985) Reconstitution ofplatelet proteins into phospholipid vesicles: functional proteoliposomes. J. Cliii.Investigation 75:35-39.Bali, A., Dhawan, S. and Gupta, C. M. (1983) Stability of liposomes in circulation ismarkedly enhanced by structural modification of their phospholipid component. FEBSLett. 154:373-377.Bangham, A. D. (1968) Membrane models with phospholipids. Progr. Biophys. Mol.Biol. 18:29-95.Bangham, A. D., Standish, M. M. and Watkins, J. C. (1965) Diffusion of univalent ionsacross the lamellae of swollen phospholipids. J. Mol. Biol. 13:238-252.Barenholzt, Y., Amselem, S. and Lichtenberg, D. (1979) A new method for preparationof phospholipid vesicles (liposomes) - French press. FEBS Lett. 99:210-214.Barna, B. P., Deodhar, S. D., Gautam, S. Yen-Lieberman, B. and Roberts, D. (1984)Macrophage activation and generation of tumoricidal activity by liposome-associatedhuman C-reactive protein. Cancer Res. 44:305-310.135Becherer, J. D., Alsenz, J. and Lambris, J. D. (1989) Molecular aspects of C3interactions and structural/functional analysis of C3 from different species. CurrentTopics Microbiol. Immunol. 153:47-72.Berken, A. and Benacerraf, B. (1966) Properties of antibodies cytophilic formacrophages. J. Exp. Med. 123:119-144.Bianco, C., Griffin, F. M. and Silverstein, S. C. (1975) Studies of the macrophagecomplement receptor. Alteration of receptor function upon macrophage activation. J.Exp. Med. 141:1278-1290.Bisgaier, C. L., Siebenkas, M. V. and Williams, K. J. (1989) Effects of apolipoproteinsA-IV and A-I on the uptake of phospholipid liposomes by hepatocytes. J. Biol. Chem.264:862-866.Black, C. D. V. and Gregoriadis, G. (1976) Interaction of liposomes with blood plasmaproteins. Biochem. Soc. Trans. 4:253-256.Blume, G. and Cevc, G. (1990) Liposomes for the sustained drug release in vivo.Biochim. Biophys. Acta 1029:91-97.Bonte, F. and Juliano, R. L. (1986) Interactions of liposomes with serum proteins.Chem. Phys. Lipids 40:359-372.Bonte, F., Hsu, M. J., Papp, A., Wu, K., Regen, S. L. and Juliano, R. L. (1987)Interactions of polymerizable phosphatidyicholine vesicles with blood components:relevance to biocompatibility. Biochim. Biophys. Acta 900:1-9.Brown, E. J. (1986) The role of extracellular matrix proteins in the control ofphagocytosis. J. Leukoc. Biol. 39:579-591.Brulet, P. and McConnell, H. M. (1976) Lateral hapten mobility and immunochemistryof model membranes. Proc. Nati. Acad. Sci. USA. 73:2977-298 1.Cheng, H. M. (1991) Antiphospholipid antibodies axe masked in normal human serum.Immunol. Today 12:96.136Cheng, H. M., Ngeow, Y. F. and Sam, C. K. (1989) Heat inactivation of serumpotentiates anti-cardiolpin antibody binding in ELISA. J. Immunol. Methods 124:235-238.Chobanian, J. V., Tall, A. R. and Brecher, P. I. (1979) Interaction between unilamellaregg yolk lecithin vesicles and human high density lipoprotein. Biochemistry 18:180-187.Ciccimarra, F., Rosen, F. S. and Merler, E. (1975) Localization of the IgG effector sitefor monocyte receptors. Proc. Nat!. Acad. Sci. USA 72:225-264.Comis, A. and Easterbrook-Smith, S. B. (1986) Inhibition of serum complementhaemolytic activity by lipid vesicles containing phosphatidylserine. FEBS Lett.197:321-327.Comiskey, S. J. and Heath, T. D. (1990) Serum-induced leakage of negatively chargedliposomes at nanomolar lipid concentrations. Biochemistry 29:3626-3631.Cooper, N. R. (1983) Activation and regulation of the first complement component.Fed. Proc. 42:134-138.Cooper, N. R. and Morrison, D. C. (1978) Binding and activation of the first componentof human complement by lipid A region of lipopolysaccharides. J. Immunol. 120:1862-1868.Cooper, N. R., Jensen, F. C., Welsh, R. M. and Oldstone, M. B. A. (1976) Lysis of RNAtumor viruses by human serum: direct antibody-independent triggering of the classicalcomplement pathway. J. Exp. Med. 144:970-984.Cullis, P. R., Mayer, L. D., Bally, M. B., Madden, T. D and Hope, M. J. (1989)Generating and loading of liposomal systems for drug-delivery applications. Adv. DrugDelivery Rev. 3:267-282.Cunningham, C. M., Kingzette, M., Richards, R. L., Alving, C. R., Lint, T. F. andGewurz, H. (1979) Activation of human complement by liposomes: a model formembrane activation of the alternative pathway. J. Immunol. 122:1237-1242.Czop, J. K. (1986) Phagocytosis of particulate activators of the alternative complementpathway: effects of fibronectin. Advances Immunol. 38:361-398.137D’Urso-Coward, M. and Cone, R. E. (1978) Membrane proteins of the P388D1macrophage cell line: isolation of membrane polypeptides that bind to the Fc portion ofaggregated IgG. J. Immunol. 121:1973-1980.Damen, J., Dijkstra, J., Regts, J. and Scherphof, G. (1980) Effect of lipoprotein-freeplasma on the interaction of human plasma high density lipoprotein with egg yolkphosphatidylcholine liposomes. Biochim. Biophys. Acta 620:90-99.Debs, R. J., Duzgunes, N., Brunette, E. N., Fendly, B., Patton, J. and Philip, R. (1989)Liposome-associated tumor necrosis factor retains bioactivity i the presence ofneutralizing anti-tumor necrosis factor antibodies. J. Immunol. 143:1192-1197.Derksen, J. T. P., Morselt, H. W. M., Kalicharan, D., Hulstaert, C. E. and Scherphof, G.L. (1987) Interaction of immunoglobulin-coupled liposomes with rat liver macrophagesin vitro. Exp. CeliRes. 168:105-115.Deshmukh, D. S., Bear, W. D., Wisniewski, H. M. and Brockerhoff, H. (1978) Long-living liposomes as potential drug carriers. Biochem. Biophys. Res. Commun. 82:328-334.Edwards, M. S., Kasper, D. L., Jennings, H. J., Baker, C. J. and Nicholson-Weller, A.(1982) Capsular sialic acid prevents activation of the alternative complement pathway bytype III, group B Streptococci. J. Immunol. 128:1278-1283.Entwistle, R. A. and Furcht, L. T. (1988) Clq component of complement binds tofibrinogen and fibrin. Biochemistry 27:507-5 12.Esser, A. F., Kolb, W. P., Podack, E.R. and Muller-Eberhard, H. J. (1979) Molecularreorganization of lipid bilayers by complement: a possible mechanism formembranolysis. Proc. Nati. Acad. Sci. USA 76:1410-1414.Falcone, D. J. (1986) Fluorescent opsonization assay: binding of plasma fibronectin tofibrin-derivatized fluorescent particles does not enhance their uptake by macrophages. J.Leukoc. Biol. 39:1-12.Farmer, M. C., Rudolph, A. S., Vandegriff, K. D., Hayre, M. D., Bayne, S. A. andJohnson, S. A. (1988) Liposome-encapsulated hemoglobin: oxygen binding propertiesand respiratory function. Biomat. Art. Cells Art. Org. 16:289-299.138Finkeistein, M. C. and Weissmann, G. (1979) Enzyme replacement via liposomes:variations in lipid composition determine liposomal integrity in biological fluids.Biochim. Biophys. Acta 587:202-216.Finkeistein, M. C., Kuhn, S. H., Schieren, H., Wessmann, G. and Hoffstein, S. (1981)Liposome uptake by human leukocytes: enhancement of entry mediated by human serumand aggregated immunoglobulins. Biochim. Biophys. Acta 673:286-302.Fiske, C. H. and Subbarow, Y. (1924) The colorimetric determination of phosphorus. .1.Biol. Chem. 66:375-400.Fogler, W. E. and Fidler, I. J. (1987) Comparative interaction of free and liposomeencapsulated nor-muramyl dipeptide or muramyl tripeptide phosphatidylethanolamine(3H-labelled) with human blood monocytes. mt. J. ImmunopharmacoL 9:141-150.Forte, T., Nichols, A. V., Gong, E. L., Lux, S. and Levy, R. I. (1971) Electronmicroscopic study on reassembly of plasma high density apoprotein with various lipids.Biochim. Biophys. Acta 248:381-386.Foster, D. E. B., Dorrington, K. J. and Painter, R. H. (1980) Structure and function ofimmunoglobulin domains. VIII. An analysis of the structural requirements in humanIgGi for binding to the Fc receptor of human monocytes. J. Immunol. 124:2186-2190.Fountain, M. W., Weiss, S. J., Fountain, A. G., Shen, A. and Lenk, R. P. (1985)Treatment of Brucella canis and Brucella abortus in vitro and in vivo by stableplurilamellar vesicle-encapsulated aminoglycosides. J. Infect. Dis. 152:529-535.Gabizon, A. and Papahadjoupolos, D. (1988) Liposome formulations with prolongedcirculation time in blood and enhanced uptake by tumors. Proc. NatL Acad. Sci. USA85:6949-6953.Galli, M., Comfurius, P., Maassen, C., Hemker, H. C., De Baets, M. H., Van BredaVriesman, P. J. C., Barbui, T., Zwaal, R. F. A. and Bevers, E. M. (1990) Anticardiolipinantibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet335:1544-1547.Gharavi, A. E., Harris, E. N., Asherson, R. A. and Hughes, G. R. V. (1987)Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann.Rheum. Dis. 46:1-6.139Giavedoni, E. B. and Dalmasso, A. P. (1976) The induction by complement of a changein KSCN-dissociable red cell membrane lipids. J. Immunol. 116:1163-1169.Giclas, P. C., Ginsberg, M. H. and Cooper, N. R. (1979) Immunoglobulin Gindependent activation of the classical complement pathway by monosodium uratecrystals. J. Clin. Invest. 63:759-764.Gigli, I. and Nelson, R. A. (1968) Complement dependent immune phagocytosis. I.Requirements for Cl, C4, C2, C3. Exp. Cell. Res. 51:45-67.Gong, E. L. and Nichols, A. V. (1980) Interaction of human plasma high densitylipoprotein HDL2 with synthetic saturated phosphatidylcholines. Lipids 15:86-90.Gregoriadis, G. (1973) Drug entrapment in liposomes. FEBS Lett. 36:292-296.Gregoriadis, G. (1988) Fate of injected liposomes: observations on entrapped soluteretention, vesicle clearance and tissue distribution in vivo. In Liposomes as DrugCarriers: Recent Trends and Progress. Gregoriadis, G., ed. John Wiley and Sons Ltd.,pg. 3-18.Gregoriadis, G. and Davis, C. (1979) Stability of liposomes in vivo and in vitro ispromoted by their cholesterol content and the presence of blood cells. Biochem. Biophys.Res. Commun. 89:1287-1293.Gregoriadis, G. and Neerunjun, E. D. (1974) Control of the rate of hepatic uptake andcatabolism of liposome-entrapped proteins injected into rats: possible therapeuticapplications. Eur. J. Biochem. 47:179-185.Gregoriadis, G. and Ryman, B. E. (1972) Fate of protein-containing liposomes injectedinto rats: an approach to the treatment of storage diseases. Eur. J. Biochem. 24:485-491.Gregoriadis, G. and Senior, J. (1980) The phospholipid component of small unilamellarliposomes controls the rate of clearance of entrapped solutes from the circulation. FEBSLett. 119:43-46.Griffin, F. M., Bianco, C. and Silverstein, S. C. (1975) Characterization of themacrophage receptor for complement and demonstration of its functional independencefrom the receptor for the Fc portion of immunoglobulin G. J. Exp. Med. 141:1269-1277.140Griffin, J. A. and Griffin, F. M. (1979) Augmentation of macrophage complementreceptor function in vitro. I. Characterization of the cellular interactions required for thegeneration of a T-lymphocyte product that enhances macrophage complement receptorfunction. J. Exp. Med. 150:653-675.Guo, L. S. S., Hamilton, R. L., Goerke, J., Weinstein, J. N. and Havel, R. J. (1980)Interaction of unilamellar liposomes with serum lipoproteins and apolipoproteins. J.Lipid Res. 21:993-1003.Haeffner-Cavaillon, N., Dorrington, K. J. and Klein, M. (1979) J. Immunol. 123:1914-1919.Haiks-Miller, M., Guo, L. S. S. and Hamilton, R. L. (1985) Tocopherol-phospholipidliposomes: maximum content and stability to serum proteins. Lipids 20:195-200.Harris, E. N., Gharavi, A. E., Boey, M. L., Patel, B. M., Mackworth-Young, C. G.,Loizou, S. and Hughes, G. R. V. (1983) Anticardiolipin antibodies: detection byradioimmunoassay and association with thrombosis in systemic lupus erythematosus.Lancet 2:1211-1214.Haxby, J. A., Gotze, 0., Muller-Eberhard, H. J. and Kinsky, S. C. (1969) Release oftrapped marker from liposomes by the action of purified complement components. Proc.Nat. Acad. Sci. USA 64:290-295.Haxby, J. A., Kinsky, C. B. and Kinsky, S. C. (1968) Immune response of a liposomalmodel membrane. Proc. Nat. Acad. Sci. USA 61:300-307.Heath, T. D. (1987) Covalent attachment of proteins to liposomes. Methods Enzymol.149:111-118.Hermetter, A. and Paltauf, F. (1983) Interaction between ether glycerophospholipidvesicles and serum proteins in vitro. Biochim. Biophys. Acta 752:444-450.Heukeshoven, J. and Dernick, R. (1988) Improved silver staining procedure for faststaining in PhastSystem development unit. I. Staining of sodium dodecyl sulfate gels.Electrophoresis 9:37-46.141Hope, M. J., Bally, M. B., Mayer, L. D., Janoff, A. S. and Cullis, P. R. (1986)Generation of multilamellar and unilamellar phospholipid vesicles. Chem. Phys. Lipids40:89-107.Hope, M. J., Bally, M. B., Webb, G. and Cullis, P. R. (1985) Production of largeunilamellar vesicles by a rapid extrusion procedure: characterization of size, trappedvolume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812:55-65.Hope, M. J., Wong, K. F. and Cullis, P. R. (1989) Freeze-fracture of lipids and modelmembrane systems. J. Electron Microsc. Tech. 13:277-287.Horbett, T. A. and Brash, J. L. (1987) Proteins and interfaces: current issues and futureprospects. In Proteins at Interfaces: Physicochemical and Biochemical Studies. Brash,J. L. and Horbett, T. A., eds. American Chemical Society, Washington, pg. 1-33.Hostetter, M. K. and Gordon, D. L. (1987) Biochemistry of C3 and related thiolesterproteins in infection and inflammation. Rev. Infect. Dis. 9:97-109.Hsu, M. J. and Juliano, R. L. (1982) Interactions of liposomes with thereticuloendothelial system. II. Nonspecific and receptor-mediated uptake of liposomesby mouse peritoneal macrophages. Biochim. Biophys. Acta 720:411-419.Huang, C. H. (1969) Studies on phosphatidylcholine vesicles: formation and physicalcharacteristics. Biochemistry 8:344-352.Huber, H., Polley, M. J., Linscott, W. D., Fudenberg, H. H. and Muller-Eberhard, H. J.(1968) Human monocytes: distinct receptor sites for the third component ofcomplement and for immunoglobulin G. Science 162:1281-1283.Hudson, M. M., Snyder, J. S., Jaffe, N. and Kleinerman, E. S. (1988) In vitro and in vivoeffect of adriamycin therapy on monocyte activation by liposome-encapsulatedimmunomodulators. Cancer Res. 48:5256-5263.Hwang, K. J. and Beaumier, P. L. (1988) Disposition of liposomes in vivo. InLiposomes as Drug Carriers: Recent Trends and Progress. Gregoriadis, G., ed. JohnWiley and Sons Ltd., pg. 19-36.142Hwang, K., Padki, M. M., Chow, D. D., Essien, H. E., Lai, J. Y. and Beaumier, P. L.(1987) Uptake of small liposomes by non-reticuloendothelial tissues. Biochim. Biophys.Acta 901:88-96.Hynes, R. 0. (1987) Integrins: a family of cell surface receptors. Cell 48:549-554.Inoue, K. (1974) Permeability properties of liposomes prepared fromdipalmitoyllecithin, dimyristoyllecitin, egg lecithin, rat liver lecithin and beef brainsphingomyelin. Biochim. Biophys. Acta 339:390-402.Inoue, K., Kataoka, T. and Kinsky, S. C. (1971) Comparative responses of liposomesprepared with different ceramide antigens to antibody and complement. Biochemistry10:2574-2581.Inoue, K., Kinoshita, T., Okada, M. and Akiyama, Y. (1977) Release of phospholipidsfrom liposomal model membrane damaged by antibody and complement. J. Immunol.119:73-76.Ivanov, V. 0., Preobrazhensky, S. N., Tsibulsky, V. P., Babaev, V. R., Repin, V. S. andSmirnov, V. N. (1985) Liposome uptake by cultured macrophages mediated bymodified low-density lipoproteins. Biochim. Biophys. Acta 846:76-84.Jahani, M. and Lacko, A. G. (1982) Study of the lecithin:cholesterol acyltransferasereaction with liposome and high density lipoprotein substrates. Biochim. Biophys. Acta713:504-511.Juliano, R. L. (1983) In Liposomes. Ostro, M., ed. Marcel Dekker, New York., pg. 53-86.Juliano, R. L. and Lin, G. (1980) The interaction of plasma proteins with liposomes:protein binding and effects on the clotting and complement systems. In Liposomes andImmunobiology. Six, H. and Tom, B., eds. Elsevier, Amsterdam, pg. 49-66.Juliano, R. L. and Stamp, D. (1975) The effect of particle size and charge on theclearance rates of liposomes and liposome encapsulated drugs. Biochim. Biophys. Res.Comm. 63:651-658.143Juliano, R. L., Hsu, M. J. and Regen, S. L. (1985) Interactions of polymerizedphospholipid vesicles with cells: uptake, processing and toxicity in macrophages.Biochim. Biophys. A eta 812:42-48.Kagawa, Y. and Packer, E. (1971) Partial resolution of the enzymes catalyzing oxidativephosphorylation. J. Biol. Chem. 246:5477-5487.Kao, T. J. and T. L. Loo. (1980) Pharmacological disposition of negatively chargedphospholipid vesicle in rats. J. Pharm. Sci. 659:1338-1340.Kaplan, M. H. and Volanakis, J. E. (1974) Interactions of C-reactive protein complexeswith the complement system. I. Consumption of human complement associated with thereaction of C-reactive protein with pneumococcal C-polysaccharide and with the cholinephosphatides, lecithin, and sphingomyelin. J. Immunol. 112:2135-2147.Kataoka, T., Inoue, K., Luderitz, 0. and Kinsky, S. C. (1971) Antibody- andcomplement-dependent damage to liposomes prepared with bacteriallipopolysaccharides. Eur. J. Biochem. 21:80-85.Kazatchkine, M. D., Fearon, D. T. and Austen, K. F. (1979) Human alternativecomplement pathway: membrane-associated sialic acid regulates the competitionbetween B and betalH for cell-bound C3b. J. Immunol. 122:75-81.Kinoshita, T., Inoue, K., Okada, M. and Akiyama, Y. (1977) Release of phospholipidsfrom complement-mediated lesions on the surface structure of Escherichia coli. J.Immunol. 119:73Kinsky, S. C. and Nicolotti, R. A. (1977) Immunological properties of modelmembranes. Annu. Rev. Biochem. 46:49-67.Kinsky, S. C., Haxby, J. A., Zopf, D. A., Alving, C. R. and Kinsky, C. B. (1969)Complement-dependent damage to liposomes prepared from pure lipids and Forssmanhapten. Biochemistry 8:4149-4158.Kirby, C., Clarke, J. and Gregoriadis, G. (1980a) Effect of cholesterol content of smallunilamellar liposomes on their stability in vivo and in vitro. Biochem. J. 186:591-598.144Kirby, C., Clarke, J. and Gregoriadis, G. (1980b) Cholesterol content of smallunilamellar liposomes controls phospholipid to high density lipoproteins in the presenceof serum. FEBS Lett. 111:324-328.Kiwada, H., Miyajima, T. and Kato, Y. (1987) Studies on the uptake mechanism ofliposomes by perfused rat liver. II. An indispensable factor for liver uptake in serum.Chem. Pharm. Bull. 35:1189-1195.Kiwada, H., Obara, S., Nishiwaki, H. and Kato, Y. (1986) Studies on the uptakemechanism of liposomes by perfused rat liver. I. An investigation of effluent profileswith perfusate containing no blood component. Chem. Pharm. Bull. 34:1249-1256.Klibanov, A. L., Maruyama, K., Beckerleg, A. M., Torchilin, V. P. and Huang, L. (1991)Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time ofliposomes depends on the liposome size and is unfavorable for immunoliposome bindingto target. Biochim. Biophys. Acta 1062:142-148.Klibanov, A. L., Maruyama, K., Torchilin, V. P. and Huang, L. (1990) Amphipathicpolyethyleneglycols effectively prolong the circulation time of liposomes. FEBSLett.268:235-237.Kovacsovics, T., Tschopp, J., Kress, A. and Isliker, H. (1985) Antibody-independentactivation of Cl, the first component of complement, by cardiolipin. J. ImmunoL135:2695-2700.Krause, H. 1., Juliano, R. L. and Regen, S. L. (1987) In vivo behavior of polymerizedlipid vesicles. J. Pharm. Sci. 76:1-5.Kronberg, B., Dahlman, A., Carlfors, J., Karisson, J. and Artursson, P. (1990)Preparation and evaluation of sterically stabilized liposomes: colloidal stability, serumstability, macrohage uptake, and toxicity. J. Pharm. Sci. 70:667-671.Krupp, L., Chobanian, A. V. and Brecher, I. P. (1976) The in vivo transformation ofphospholipid vesicles to a particle resembling HDL in the rat. Biochem. Biophys. Res.Commun. 72:1251-1258.Kuhn, S. H., Gemperli, B., Shephard, E. G., Joubert, J. R., Weidemann, P. A. C.,Weissmann, G. and Finkelstein, M. (1983) Interaction of liposomes with humanleukocytes in whole blood. Biochim. Biophys. Acta 762:119-127.145Kulczycki, A., Krause, V., Killion, C. C. and Atkinson, J. P. (1980) Purification of Fcgamma receptor from rabbit alveolar macrophages that retains ligand-binding activity. J.Immunol. 124:2772-2779.Lambre, C. R., Kazatchkine, M. D., Maillet, T. and Thibon, M. (1982) Guinea pigerythrocytes, after their contact with influenza virus, acquire the ability to activate thehuman alternative complement pathway through virus-induced desialation of the cells. J.Immunol. 128:629-634.Law, S. K., Minich, T. M. and Levine, R. P. (1981) Binding reaction between the thirdhuman complement protein and small molecules. Biochemistry 20:7457-7463.Lay, W. H. and Nussenzweig, V. (1968) Receptors for complement of leukocytes. J.Exp. Med. 128:991-1009.Lelkes, P. I. (1983) Methodological aspects dealing with stability measurements ofliposomes in vitro using the carboxyfluorescein assay. In Liposome Technology, Vol. III.Gregoriadis, G., ed. CRC Press, Boca Raton, Fla., p. 225-246.Leserman, L. and Machy, P. (1987) Ligand targeting of liposomes. In Liposomes: fromBiophysics to Therapeutics. Ostro, M. J., ed. Marcell Dekker Inc., New York, pg. 157-194.Leserman, L. D., Weinstein, J. N., Blumenthal, R. and Terry, W. D. (1980) Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells. Proc. Nati. A cad.Sci. USA 77:4089-4093.Levine, R. P., Finn, R. and Gross, R. (1983) Interactions between C3b and cell-surfacemacromolecules. Ann. N. Y. Acad. Sci. 421:235-245.Lewis, J. T., Hafeman, D. G. and McConnell, H. M. (1980) Kinetics of antibody-dependent binding of haptenated phospholipid vesicles to a macrophage-related cell line.Biochemistry 19:5376-5386.Lichtenberg, D., Freire, E., Schmidt, C. F., Barenholz, Y., Felgner, P. L. and Thompson,T. E. (1981) Effect of surface curvature on stability, thermodynamic behavior, andosmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles.Biochemistry 20:3462-2567.146Liu, D. and Huang, L. (1989) Small, but not large, unilamellar liposomes composed ofdioleoylphosphatidylethanolamine and oleic acid can be stabilized by human plasma.Biochemistry 28:7700-7707.Liu, D., Mori, A. and Huang, L. (1991) Large liposomes containing ganglioside GM1accumulate effectively in spleen. Biochim. Biophys. Acta 1066:159-165.LoBuglio, A. F., Cotran, R. S. and Jandl, J. H. (1967) Red cells coated withimmunoglobulin G: binding and sphering by mononuclear cells in man. Science158:1582-1585.Loos, M. (1982) Antibody-independent activation of Cl, the first component ofcomplement. Ann. Immunol. (Paris) 133c:165-179.Lopez-Berestein, G. (1988) Liposomes as carriers of antifungal drugs. Ann. N. Y. Acad.Sci. 544:590-597.Lopez-Berestein, G., Bodey, G. P., Fainstein, V., Keating, M., Frankel, L. S., Zeluff, B.,Gentry, L. and Mehta, K. (1989) Treatment of systemic fungal infections with liposomalamphotericin B. Arch. Intern. Med. 149:2533-2536.Loube, S. R., McNabb, T. C. and Dorrington, K. J. (1978) Isolation of an Fcy-bindingprotein from the cell membrane of a macrophage-like cell line (P388D1) aftersolubilization. J. Immunol. 120:709-715.Loughrey, H. C., Choi, L. S., Cullis, P. R. and Bally, M. B. (1990) Optimizedprocedures for the coupling of proteins to liposomes. J. Immunol. Methods 132:25-35.Loughrey, H., Bally, M. B. and Cullis, P. R. (1987) A non-covalent method of attachingantibodies to liposomes. Biochim. Biophys. Acta 901:157-160.Malinski, J. A. and Nelsestuen, G. L. (1989). Membrane permeability tomacromolecules mediated by the membrane attack complex. Biochemistry 28:61-70.Marcel, Y. L. (1982) Lecithin-cholesterol acyltransferase, and intravascular cholesteroltransport. Adv. Lipid Res. 19:85-136.147Maruyama, K., Kennel, S. J. and Huang, L. (1990) Lipid composition is important forhighly efficient target binding and retention of immunoliposomes. Proc. NatL Acad. Sci.USA 87:5744-5748.Massey, J. B., Hickson-Bick, D., Via, D. P., Gotto, A. M. and Pownall, H. J. (1985)Fluorescence assay of the specificity of human plasma and bovine liver phospholipidtransfer proteins. Biochim. Biophys. Acta 835:124-13 1.Mayer, L. D., Bally, M. B., Hope, M. J. and Cullis, P. R. (1986) Techniques forencapsulating bioactive agents into liposomes. Chem. Phys. Lipids 40:333-345.Mayer, L. D., Tai, L. C., Ko, D. S. C., Masin, D., Ginsberg, R. S., Cullis, P. R. and Bally,M. B. (1989) Influence of vesicle size, lipid composition and drug-to-lipid ratio on thebiological activity of liposomal doxorubicin in mice. Cancer Res. 49:5922-5930.McEvoy, L., Williamson, P. and Schlegel, R. A. (1986) Membrane phospholipidasymmetry as a determinant of erythrocyte recognition by macrophages. Proc. Nat!.Acad. Sci. USA 83:3311-3315.McNeil, H. P., Chesterman, C. N. and Krilis, S. A. (1989) Anticardiolipin antibodiesand lupus anticoagulants comprise separate antibody subgroups with differentphospholipid binding characteristics. Br. J. Haematol. 73:506-513.McNeil, H. P., Simpson, R. J., Chesterman, C. N. and Krilis, S. A. (1990) Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation:2-glycoprotein I (apolipoprotein H). Proc. Nati. Acad.Sci. USA 87:4120-4124.Mellman, I. S. and Unkeless, J. C. (1980) Purification of a functional mouse Fe receptorthrough the use of a monoclonal antibody. J. Exp. Med. 152:1048-1069.Mendez, A. J., He, J. L., Huang, H. S., Wen, S. R. and Hsia, S. L. (1988) Interaction ofrabbit lipoproteins and red blood cells with liposomes of egg yolk phospholipids. Lipids23:961-967.Michalek, M. T., Bremer, E. G. and Mold, C. (1988) Effect of gangliosides onactivation of the alternative pathway of human complement. J. ImmunoL 140:158 1-1587.148Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C. and Reynolds, J. A. (1981)Phospholipid vesicle formation and transmembrane protein incorporation using octylglucoside. Biochemistry 20:833-840.Moghimi, S. M. and Pate!, H. M. (1988) Tissue specific opsonins for phagocytic cellsand their different affinity for cholesterol-rich liposomes. FEBS Lett. 233:143-147.Moghimi, S. M. and Pate!, H. M. (1989a) Differential properties of organ-specificserum opsonins for liver and spleen macrophages. Biochim. Biophys. Acta 984:379-383.Moghimi, S. M. and Patel, H. M. (1989b) Serum opsonins and phagocytosis of saturatedand unsaturated phospholipid liposomes. Biochim. Biophys. Acta 984:384-387.Moilnes, T. E. and Lachmann, P. J. (1988) Regulation of complement. Scand. J.Immunol. 27:127-142.Muller-Eberhard, H. J. (1986) The membrane attack complex of complement. Annu.Rev. Immunol. 4:503-528.Muller-Eberhard, H. J. (1988) Molecular organization and function of the complementsystem. Ann. Rev. Biochem. 57:321-347.Nayar, R., Hope, M. J. and Cullis, P. R. (1989) Generation of large unilamellar vesiclesfrom long-chain saturated phosphatidylcholines by extrusion technique. Biochim.Biophys. Acta 986:200-206.Nichols, A. V., Gong, E. L., Forte, T. M. and Blanche, P. J. (1978) Interaction of plasmahigh density lipoprotein HDL2b (d 1.063-1.100 g/ml) with single-bilayer liposomes ofdimyristoylphosphatidylcholine. Lipids 13:943-950.Nishikawa, K., Arai, H. and Inoue, K. (1990) Scavenger receptor-mediated uptake andmetabolism of lipid vesicles containing acidic phospholipids by mouse peritonealmacrophages. J. Biol. Chem. 265:5226-5231.Nishiya, T. and Ahmed, S. (1990) Circular dichroism study of membrane dynamics:effects of carboxymethyl-chitin. J. Biochem. 107:217-221.Ohsawa, T. (1980) Change in permeability of liposomal membranes mediated by C-reactive protein and its inhibition by cholesterol. Jpn. J. Exp. Med. 50:67-71.149Okada, N., Yasuda, T. and Okada, H. (1982a) Restriction of alternative complementpathway activation by sialosylglycolipids. Nature 299:261-263.Okada, N., Yasuda, T., Tsumita, T. and Okada, H. (1982b) Activation of the alternativecomplement pathway of guinea pig by liposomes incorporated with trinitrophenylatedphosphatidylethanolamine. Immunology 45:115-124.Okada, N., Yasuda, T., Tsumita, T., Shinomiya, H., Utsumi, S. and Okada, H. (1982c)Regulation of glycophorin of complement activation via the alternative pathway.Biochem. Biophys. Res. Commun. 108:770-775.Osmand, A. P., Mortensen, R. F., Siegel, J. and Gewurz, H. (1975) Interactions of C-reactive protein with the complement system. III. Complement-dependent passivehemolysis initiated by CRP. J. Exp. Med. 142:1065-1077.Ostro, M. J. and Cullis, P. R. (1989) Use of liposomes as injectable-drug deliverysystems. Am. J. Hosp. Pharm. 46:1576-1587.Pangburn, M. K. and Muller-Eberhard, H. J. (1978) Complement C3 convertase: cellsurface restriction of betalH control and generation of a restriction on neuraminidasetreated cells. Proc. Nati. Acad. Sci. USA 75:2416-2420.Papahadjopoulos, D. and Gabizon, A. (1987) Targeting of liposomes to tumor cells invivo. Ann. N. Y. A cad. Sci. 507:64-74.Papahadjopoulos, D., Jacobson, K., Nir, S. and Isac, T. (1973) Phase transitions inphospholipid vesicles: fluorescence polarization and permeability measurementsconcerning the effect of temperature and cholesterol. Biochim. Biophys. Acta 3 11:330-348.Parente, R. A. and Lentz, B. E. (1984) Phase behavior of large unilamellar vesiclescomposed of synthetic phospholipids. Biochemistry 23:2353-2362.Pate!, H. M., Tuzel, N. S. and Ryman, B. E. (1983) Inhibitory effect of cholesterol onthe uptake of liposomes by liver and spleen. Biochim. Biophys. Acta 761:142-151.Peeters, P. A., Oussoren, C., Eling, W. M. and Crommelin, D. J. (1988) Immunospecifictargeting of immunoliposomes, F(ab?)2 and IgG to red blood cells in vivo. Biochim.Biophys. Acta 943:137-147.150Pengo, V., Thiagarajan, P., Shapiro, S. S. and Heine, M. J. (1987) Immunologicalspecificity and mechanism of action of IgG lupus anticoagulants. Blood 70:69-76Pepys, M. B. and Baltz, M. L. (1983) Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv.Immunol. 34:141-212.Pepys, M. B., Rowe, I. F. and Baltz, R. L. (1985) C-reactive protein: binding to lipidsand lipoproteins. mt. Rev. Exp. Pathol. 27:83-111.Phillips-Quagliata, J. M., Levine, B. B., Quagliata, F. and Uhr, J. W. (1971)Mechanisms underlying binding of immune complexes to macrophages. J. Exp. Med.133:589-601.Pluschkê, G., Mercer, A., Kusecek, B., Pohi, A. and Achtman, M. (1983) Induction ofbacteremia in newborn rats by Escherichia coli K1 is correlated with only certain 0(lipopolysaccharide) antigen types. Infect. Immun. 39:599-608.Pommier, C. G., Inada, I.; Fries, L. F., Takahashi, T., Frank, M. M. and Brown, E. J.(1983) Plasma fibronectin enhances phagocytosis of opsonized particles by humanperipheral blood monocytes. J. Exp. Med. 157:1844-1854.Poste, G., Bucana, C., Raz, A., Bugeiski, P., Kirsh, R. and Fidler, I. J. (1982) Analysisof the fate of systemically administered liposomes and implications for their use in drugdelivery. Cancer Res. 42:1412-1422.Quie, P. G., Messner, R. P. and Williams, R. C. (1968) Phagocytosis in subacutebacterial endocarditis: localization of the primary opsonic site to Fc fragment. J. Exp.Med. 128:553-570.Rauch, J., Tannenbaum, M., Tannenbaum, H., Ramelson, H., Cullis, P. R., Tilcock, C. P.S., Hope, M. J. and Janoff, A. S. (1986) Human hybridoma lupus anticoagulantsdistinguish between lamellar and hexagonal phase lipid systems. J. Biol. Chem.261:9672-9677.Regen, S. L. (1987) Polymerized liposomes. In Liposomes: from Biophysics toTherapeutics. Ostro, M. J., ed. Marcel Dekker Inc., New York, pg. 73-108.151Reinish, L. W., Bally, M. B., Loughrey, H. C. and Cullis, P. R. (1988) Interactions ofliposomes and platelets. Thromb. and Haemost. 60:518-523.Rent, R., Ertel, N., Eisenstein, R. and Gewurz, H. (1975) C activation by interaction ofpolyanions and polycations. I. Heparin-protamine induced consumption of complement.J. Immunol. 114:120-124.Reynolds, J. A., Nozaki, Y. and Tanford, C. (1983) Gel-exclusion chromatography onS1000 Sephacryl: application to phospholipid vesicles. Anal. Biochem. 130:471-474.Richards, R. L., Gewurz, H., Osmand, A. P. and Alving, C. R. (1977) Interactions of C-reactive protein and complement with liposomes. Proc. Nati. Acad. Sci. USA 74:5672-5676.Richards, R. L., Gewurz, H., Siegel, J. and Alving, C. R. (1979) Interactions of C-reactive protein and complement with liposomes. II. Influence of membranecomposition. J. Immunol. 122:1185-1189.Roerdink, F. H., Regts, J., Handel, T., Sullivan, S. M., Baldeschwieler, J. D. andScherphof, G. L. (1989) Effect of cholesterol on the uptake and intracellular degradationof liposomes by liver and spleen; a combined biochemical and y-ray perturbed angularcorrelation study. Biochim. Biophys. Acta 980:234-240.Roerdink, F., Dijkstra, J., Hartman, G., Bolscher, B. and Scherphof, G. (1981) Theinvolvement of parenchymal, Kupffer and endothelial liver cells in the hepatic uptake ofintravenously injected liposomes: effects of lanthanum and gadolinium salts. Biochim.Biophys. Acta 677:79-89.Roerdink, F., Wassef, N. M., Richardson, E. C. and Alving, C. R. (1983) Effects ofnegatively charged lipids on phagocytosis of liposomes opsonized by complement.Biochim. Biophys. Acta 734:33-39.Ruoslahti, E. (1988) Fibronectin and its receptors. Ann. Rev. Biochem. 57:375-413.Rutenfranz, I., Bauer, A. and Kirchner, H. (1990) Interferon gamma encapsulated intoliposomes enhances the activity of monocytes and natural killer cells and hasproliferative effects on tumor cells in vitro. Blut 61:30-37.152Saba, T. M. (1970) Physiology and physiopathology of the reticuloendothelial system.Arch. Intern Med. 26:1031-1053.Sato, Y., Kiwada, H. and Kato, Y. (1986) Effects of dose and vesicle size on thepharmacokinetics of liposomes. Chem. Pharm. Bull. 34:4244-4252.Scherphof, G. and Morselt, H. (1984) On the size-dependent disintegration of smallunilamellar phosphatidylcholine vesicles in rat plasma: evidence of complete loss ofvesicle structure. Biochem. J. 221:423-429.Scherphof, G. L., J. Damen and J. Wilschut. (1984) Interactions of liposomes withplasma proteins. In Liposome Technology, Vol. III. Gregoriadis, G., ed. CRC Press Inc.,Boca Raton, Fla., pg. 205-224.Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Disintegration ofphosphatidyicholine liposomes in plasma as a result of interaction with high-densitylipoproteins. Biochim. Biophys. Acta 542:296-307.Schiager, S. I., Ohanian, S. H. and Borsos, T. (1978) Identification of lipids synthesizedand released by tumor cells under attack by antibody and complement. J. Iminunol.120:1644-1650.Schiegel, R. A., Prendergast, T. W. and Williamson, P. (1985) Membrane phospholipidasymmetry as a factor in erythrocyte-endothelial cell interactions. J. Cell Physiol.123:215-218.Schwendener, R. A., Lagocki, P. A. and Rahman, Y. E. (1984) The effects of chargeand size on the interaction of unilamellar liposomes with macrophages. Biochim.Biophys. Acta 772:93-101.Senior, J. (1987) Fate and behavior of liposomes in vivo: a review of controllingfactors. Crit. Rev. Ther. Drug Carrier Syst. 3:123-193.Senior, J. and Gregoriadis, G. (1982a) Is half-life of circulating liposomes determinedby changes in their permeability? FEBSLett. 145:109-114.Senior, J. and Gregoriadis, G. (1982b) Stability of small unilamellar liposomes in serumand clearance from the circulation: the effect of the phospholipid and cholesterolcomponents. Life Sci. 30:2123-2136.153Senior, J. and Gregoriadis, G.. (1984) In Liposome Technology, VoL III. (G.Gregoriadis, ed.). CRC Press Inc., Boca Raton, Fla., pp 264-282.Senior, J., Crawley, J. C. W. and Gregoriadis, G. (1985) Tissue distribution ofliposomes exhibiting long half-lives in the circulation after intravenous injection.Biochim. Biophys. Acta 839:1-8.Senior, J., Delgado, C., Fisher, D., Tilcock, C. and Gregoriadis, G. (1991) Influence ofsurface hydrophilicity of liposomes on their interaction with plasma protein andclearance from the circulation: studies with poly(ethylene glycol)-coated vesicles.Biochim. Biophys. A cta 1062:77-82.Senior, J., Gregoriadis, G. and Mitropoulos, K. A. (1983) Stability and clearance ofsmall unilamellar liposomes: studies with normal and lipoprotein-deficient mice.Biochim. Biophys.Acta 760:111-118.Senior, J., Waters, J. A. and Gregoriadis, G. (1986) Antibody-coated liposomes: therole of non-specific antibody adsorption. FEBS Lett. 196:54-58.Sessa, G. and Weissmann, G. (1968) Phospholipid spherules (liposomes) as a model forbiological membranes. J. Lipid Res. 9:310-318.Shahrokh, Z. and Nichols, A. V. (1982) Particle size interconversion of human lowdensity lipoproteins during incubation of plasma with phosphatidyicholine vesicles.Biochem. Biophys. Res. Commun. 108:888-895.Shapiro, S. S. and Thiagarajan, P. (1982) Lupus anticoagulants. Frog. Hemostas.Thromb. 6:263-285.Sharma, P., Tyrrell, D. A. and Ryman, B. E. (1977) Some properties of liposomes ofdifferent sizes. Biochem. Soc. Trans. 5:1146-1149.Shin, M. L., Paznekas, W. A. and Mayer, M. M. (1978) On the mechanism ofmembrane damage by complement: the effect of length and unsaturation of the acylchains in liposomal bilayers and the effect of cholesterol concentration in sheeperythrocyte and liposomal membranes. J. Immunol. 120:1996-2002.154Shin, M. L., Paznekas, W. A., Abramovitz, A. S. and Mayer, M. M. (1977) On themechanism of membrane damage by C: exposure of hydrophobic sites on activated Cproteins. J. Immunol. 119:1358-1364.Siegel, J., Osmand, A. P., Wilson, M. F. and Gewurz, H. (1975) Interactions of C-reactive protein with the complement system. II. C-reactive protein-mediatedconsumption of complement by poly-L-lysine polymers and other polycations. J. Exp.Med. 142:709-721.Siegel, J., Rent, R. and Gewurz, G. (1974) Interactions of C-reactive protein with thecomplement system. I. Protamine-induced consumption of complement in acute phasesera. J. Exp. Med. 140:631-647.Silversmith, R. E. and Nelsestuen, G. L. (1986) Interaction of complement proteinsC5b-6 and C5b-7 with phospholipid vesicles: effects of phospholipid structural features.Biochemistry 25:7717-7725.Six, H. R., Uemura, K. and Kinsky, S. C. (1973) Effect of immunoglobulin class andaffinity on the initiation of complement-dependent damage to liposomal modelmembranes sensitized with dinitrophenylated phospholipids. Biochemistry 12:4003-4011.Sommerman, E. F. (1986) Factors influencing the biodistribution of liposomal systems.Ph. D. Thesis, The University of British Columbia.Spanjer, H. H., van Galen, M., Roerdink, F. H., Regts, J. and Scherphof, G. L. (1986)Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipidcomposition. Biochim. Biophys. Acta 863:224-230.Steger, L. D. and Desnick, R. J. (1977) Enzyme therapy. VI. Comparative in vivo fatesand effects on lysosomal integrity of enzyme entrapped in negatively and positivelycharged liposomes. Biochim. Biophys. Acta 464:530-546.Stein, Y., Halperin, G. and Stein, 0. (1980) Biological stability of[3Hjcholesteryl oleylether in culture fibroblasts and intact rat. FEBS Lett. 111:104-106.Szebeni, J., Wahl, S. M., Betageri, G. V., Wahi, L. M., Gartner, S., Popovic, M., Parker,R. J., Black, C. D. and Weinstein, J. N. (1990) Inhibtion of HIV-1 in155monocyte/macrophage cultures by 2’, 3’-dideoxycytidine-S’triphosphate, free and inliposomes. Aids Res. Hum. Retroviruses 6:691-702.Szoka, F. and Papahadjopoulos, D. (1978) Procedure for preparation of liposomes withlarge internal aqueous space and high capture by reverse-phase evaporation. Proc. Nat!.Acad. Sci. USA 75:4194-4198.Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L. and Prahi, J. W. (1980)Evidence for the presence of an internal thioester bond in the third component of humancomplement. Proc. Nat!. Acad. Sci. USA 77:5764-5768.Tack, B. F. and Prahl, J. W.. (1976) Third component of human complement:purification from plasma and physicochemical characterization. Biochemistry 15:45 13-4521.Tadakuma, T., Ikewaki, N., Yasuda, T., Tsutsumi, M., Saito, S. and Saito, K. (1985)Treatment of experimental salmonellosis in mice with streptomycin entrapped inliposomes. Antimicrob. Agents Chemother. 28:28-32.Tall, A. (1986) Plasma lipid transfer proteins. J. Lipid Res. 27:36 1-367.Tall, A. R. (1980) Studies on the transfer of phosphatidyicholine from unilamellarvesicles into plasma high density lipoproteins in the rat. J. Lipid Res. 21:354-363.Tall, A. R. and Green, P. H. R. (1981) Incorporation of phosphatidyicholine intospherical and discoidal lipoproteins during incubation of egg phosphatidylcholinevesicles with isolated high density lipoproteins or with plasma. J. Biol. Chem.256:2035-2044.Tall, A. R., Abreu, E. and Shuman, J. (1983a) Separation of a plasma phospholipidtransfer protein from cholesterol ester/phospholipid exchange protein. J. BioL Chem.258:2174-2180.Tall, A. R., Forester, L. and Bongiovanni, G. (1983b) Facilitation ofphosphatidylcholine transfer into high density lipoproteins by an apolipoprotein in thedensity 1.20-1.26 g/ml fraction of plasma. J. LipidRes. 24:277-289.156Thielens, N. M. and Colomb, M. G. (1986) A model for the study of the assembly andregulation of human complement C3 convertase (classical pathway). Eur. J. Immunol.16:617-622.Tschopp, J. (1984) Ultrastructure of the membrane attack complex of complement:heterogeneity of the complex caused by different degree of C9 polymerization. J. Biol.Chem. 259:7857-7863.Uemura, K. and Kinsky, S. C. (1972) Active vs. passive sensitization of liposomestoward antibody and complement by dinitrophenylated derivatives ofphosphatidylethanolamine. Biochemistry 11:4085-4094.Vidal-Naquet, A., Gossage, J. L., Sullivan, T. P., Haynes, J. W., Gilruth, B. H. andBessinger, R. L. (1989) Liposome-encapsulated hemoglobin as an artificial red bloodcell: characterization and scale-up. Biomat. Art. Cells Art. Org. 17:531-552.Volanakis, J. E. and Kaplan, M. H.. (1974) Interactions of C-reactive protein with thecomplement system. II. Consumption of guinea pig complement by CRP complexes:reguirement for human Clq. J. Immunol. 113:9-17.Volanakis, J. E. and Narkates, A. J. (1981) Interaction of C-reactive protein withartificial phosphatidylcholine bilayers and complement. J. Immunol. 126:1820-1825.Weinstein, J. N., Ralston, E., Leserman, L. D., Klausner, R. D., Dragsten, P., Henkart, P.and Blumenthal, R. (1984) Self-quenching of carboxyfluorescein fluorescence: uses instudying liposome stability and liposome-cell interaction. In Liposome Technology, VoLIII. Gregoriadis, G., ed. CRC Press, Boca Raton, Fla., pg. 183-204.Weissmann, G., Bloomgarden, D., Kaplan, R., Cohen, C., Hoffstein, S., Collins, T.,Gottlieb, A. and Nagle, D. (1975) A general method for the introduction of enzymes, bymeans of immunoglobulin-coated liposomes, into lysosomes of deficient cells. Proc.Natl. A cad. Sci. USA 72:88-92.Wessel, D. and Flugge, U. J. (1984) A method for the quantitative recovery of protein indilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141-143.Whaley, K. (1985) Measurement of complement. In Methods in Complement forClinical Immunologists. Whaley, K., ed. Churchill Livingstone, pg.77-139.157Wilkins, D. J. and Myers, P. A. (1966) Studies on the relationship between theelectrophoretic properties of colloids and their blood clearance and organ distribution inthe rat. Br. J. Exp. Pathol. 47:568-576.Williams, K. J. and Scanu, A. M. (1986) Uptake of endogenous cholesterol by asynthetic lipoprotein. Biochim. Biophys. Acta 875:183-194.Williams, K. J., Tall, A. R., Bisgaier, C. and Brocia, R. (1987) Phospholipid liposomesacquire apolipoprotein E in atherogenic plasma and block cholesterol loading of culturedmacrophages. J. Clin. Invest. 79:1466-1472.Williams, K. J., Werth, V. P. and Wolff, J. A. (1984) Intravenously administeredlecithin liposomes: a synthetic antiatherogenic lipid particle. Perspect. BioL Med.27:417-431.Wong, M., Anthony, F. H., Tillack, T. W. and Thompson, T. E. (1982) Fusion ofdipalmitoylphosphatidylcholine vesicles at 4°C. Biochemistry 21:4126-4132.Wright, A. E. and Douglas, S. R. (1903) An experimental investigation of the role of thebody fluids in connection with phagocytosis. Proc. R. Soc. Lond. 72:357-370.Yagawa, K., Onoue, K. and Aida, Y. (1979) Structural studies of Fe receptors. I.Binding properties, solubilization, and partial characterization of Fc receptors ofmacrophages. J. Immunoh 122:366-373.Yoshioka, A., Peake, I. R., Furlong, B. L., Furlong, A., Giddings, J. C. and Bloom, A. L.(1983) The interaction between factor VIII clotting antigen (VIIICAg) and phospholipid.Br. J. Haematol. 55:27-36.158


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