Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Circulation lifetimes and tumor accumulation of liposomal drug delivery systems Parr, Michael J. 1995

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_1995-060373.pdf [ 3.32MB ]
JSON: 831-1.0088357.json
JSON-LD: 831-1.0088357-ld.json
RDF/XML (Pretty): 831-1.0088357-rdf.xml
RDF/JSON: 831-1.0088357-rdf.json
Turtle: 831-1.0088357-turtle.txt
N-Triples: 831-1.0088357-rdf-ntriples.txt
Original Record: 831-1.0088357-source.json
Full Text

Full Text

CIRCULATION LIFETIMES AND TUMOR ACCUMULATION OFLIPOSOMAL DRUG DELWERY SYSTEMSbyMICHAEL J. PARRB.Sc., University of British Columbia, 1990A THESIS SUBMITE’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENT FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BIOCHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAOctober, 1995© Michael J. Parr, 1995In 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.(Signature)Department of 1*’D /&4at.M &ozzyThe University of British ColumbiaVancouver, CanadaDate (/2 /DE-6 (2/88)ABSTRACTOne of the greatest benefits of liposomal encapsulation of therapeutic drugs is the tendency forthese carriers to accumulate in sites of disease. In the case of a solid tumor model employedhere, it was the goal of this thesis to maximize the drug delivery to this site. This requires anunderstanding of the major factors governing tumor delivery. Of central importance to liposomaldelivery to solid tumors is the circulation lifetime of the carrier. In the first section ofexperiments, factors influencing the use of poly(ethylene glycol) (PEG)-lipids to increase thecirculation lifetime of liposomes was examined. Second, the observation that drug loadedliposomes last significantly longer in the circulation was more fully examined. And finally, thedelivery of drug to a murine solid tumor was assessed and the influence of PEG-lipids in thedrug carrier and the effect of entrapped drug has on its delivery determined.PEG-lipid anchor conjugates can prolong the circulation lifetimes of liposomes followingintravenous injection, but this can depend upon the nature of the lipid anchor and the chemicallink between the PEG and lipid moieties. Incorporation of various PEG-lipids into largeunilamellar vesicles (LUVs) composed of distearoylphosphatidylcholine (DSPC) and cholesterol(chol) (DSPC/chol/PEG-lipid, 50:45:5 mol/mol) results in differing liposomal circulationlifetimes in mice. This is shown to be due to differential removal of the hydrophilic coating invivo that arises from exchange of the entire PEG-lipid conjugate from the liposomal membrane,although chemical breakdown of the PEG-lipid conjugate is also possible. This work establishesthat DSPE is a considerably more effective anchor for PEG2000 than POPE and that the chemicalstability of PEG-PE conjugates is sensitive to the nature of the linkage and exchangeability of thePEG-PE complex. It is suggested here that retention of the PEG coating is of paramountimportance for prolonged circulation lifetimes.11The influence of entrapped drug on the circulation lifetimes of liposomal carriers wasinvestigated next. Pre-doses of liposomally entrapped doxorubicin blocked the accumulation ofsubsequently injected liposomes in the reticuloendothelial system (RES). This effect is tennedRES blockade. Liposomal drug doses as low as 2 mg/kg can induce maximum RES blockadewithin 24 h after administration, and this effect lasts as long as 8 days. Full recovery is onlyachieved by 14 days. Another commonly employed liposomal anti-cancer drug, vincristine, haseffects that are similar in magnitude, but more transient, allowing recovery of the RES within 2to 4 days. Liposomes incorporating PEG-lipids or ganglioside GM1 are proposed to avoid theRES, however it is shown that when loaded with doxorubicin these liposomes also induce RESblockade and do not avoid uptake by the RES. Rather, these lipids engender a decrease in the rateof uptake by cells of the RES.The fmal set of experiments consisted of a comparison of tumor accumulation and efficacyproperties of doxorubicin entrapped in liposomes incorporating PEG-lipids versus conventionalliposomes by monitoring drug pharmacokinetics and tumor accumulation at the maximumtolerated dose (MTD)(60 mg/kg liposomal doxorubicin). The tumor model consisted of micebearing Lewis Lung carcinoma solid tumors. In contrast to expected behavior, the efficiency ofdoxorubicin accumulation at the tumor site, evaluated with an area under the curve analysis, washigher for conventional liposomes than for the sterically stabilized liposomes. Both formulations,however, exhibited profound increases of over 500-fold in tumor accumulation of drug ascompared to free drug injected at the MTh (20 mg/kg doxorubicin). These studies suggest thatoptimization of factors nominally leading to longer blood circulation times do not providetherapeutic advantages for liposomal formulation of doxorubicin administered at the MTh. TheII’dominant factor influencing the circulation lifetime for both liposomal carrier systems appears tobe that of entrapped drug, consistent with RES blockade described in this thesis. Improvement inother parameters, such as drug leakage rates, hold greater promise for improving therapeuticproperties of liposomal drug carriers.ivTABLE OF CONTENTSABSTRACTTABLE OF CONTENTS vLIST OF FIGURES viiiLIST OF TABLES xiABBREVIATIONS xiiACKNOWLEDGMENTS xiiiDEDICATION xivCHAPTER 1: INTRODUCTION 11.1 Project overview: liposomes as drug carriers 11.2 Liposomes 51.2.1 Chemistry and physics of lipids Phospholipids Cholesterol 121.2.2 Liposome preparation Multilamellar vesicles (MLVs) Small unilamellar vesicles (SUVs) large unilamellar vesicles (LUV) 161.2.3 Drug encapsulation Passive entrapment Active (ApH) entrapment 191.3 In vivo behavior of intravenously injected liposomes 241.3.1 Interactions of liposomes with plasma proteins 241.3.2 Interactions with the reticuloendothelial system 251.4 Properties of liposomes influencing circulation lifetimes 281.4.1 Poly(ethylene glycol)-lipids (PEG-lipids) 301.4.2 RES blockade: the effect of entrapped drug 341.5 Targeting to sites of tumor growth 351.5.1 Passive versus active targeting 351.5.2.1 Normal vasculature 361.5.2.2 Tumor vasculature 381.5.2.3 Other solid tumor properties 391.6 Objectives 40CHAPTER 2: THE USE OF PEG-LIPIDS TO IMPROVE THE CIRCULATIONLIFETIMES OF LIPOSOMES 422.1 Introduction 42V2.2 Materials and methods.432.2.1 Monomethoxypoly(ethylene glycol)-lipid (MePEG-lipid) synthesis 432.2.2 Preparation of large unilamellar vesicles (LUVs) 482.2.3 Exchange studies 492.2.4 Chemical stability studies 502.2.5 Biodistribution and circulation longevity studies 512.3 Results 512.3.1 PEGcjij-S-POPE is lost from the liposome surface both in vivo and in vitro 512.3.2 Chemical stability of various linker groups in the MePEG-PE conjugate 572.3.3 MePEG-DSPE is retained in DSPC/cholesterol LUVs and exhibits enhancedchemical stability 592.3.4 Biodistributions of DSPC/cholesterol LUVs containing different species ofMePEG2000-P 612.4 Discussion 65CHAPTER 3: CHARACTERIZATION OF RES BLOCKADE WITH DOXORUBICINAND VINCRISTINE 693.1 Introduction 693.2 Materials and methods 713.2.1 Liposome preparation 713.2.2 Drug loading 713.2.3 Animal biodistribution studies 723.2.4 Liver histology 733.3 Results 743.3.1 The presence of GM1 in liposomes with entrapped doxorubicin does not preventRES blockade 743.3.2 Characterization of RES blockade with entrapped doxorubicin and vincristine 793.3.3 Liver histology after RES blockade 833.4 Discussion 83CHAPTER 4: TUMOR ACCUMULATION OF CONVENTIONAL AND STERICALLYSTABILIZED LIPOSOMAL DOXORUBICIN 904.1 Introduction 904.2 Materials and methods 924.2.1 Preparation of liposomes and doxorubicin loading 924.2.2 Animal and tumor models 934.2.3 Assays for liposomal lipid and doxorubicin 944.2.4 Acute toxicity evaluation 954.2.5 Plasma elimination and tumor accumulation 954.2.6 Tumor histology 964.2.7 Tumor growth inhibition 974.2.8 Statistical analysis 974.3 Results 984.3.1 Estimation of Maximum Tolerated Doses 984.3.2 Influence of dose escalation on plasma liposomal lipid levels 98vi4.3.3 Drug elimination from plasma and tumor accumulation in BDF- I mice bearingLewis Lung tumors 1014.3.4 Tumor histology 1094.3.5 Inhibition of tumor growth by liposomal doxorubicin 1124.4 Discussion 114CHAPTERS: SUMMARIZING DISCUSSION 1195.1 Summary of results 1195.2 Discussion 121REFERENCES 126vi’LIST OF FIGURESFigure 1.1Amphipathic lipids in bilayer configuration 6Figure 1.2Structures of common phospholipids 8Figure 1.3Lipid polymorphism 11Figure 1.4The structure of cholesterol and its effect on the structure of lipid bilayers 13Figure 1.5Liposome morphology 15Figure 1.6Active drug entrapment in liposomes 21Figure 1.7Structure of doxorubicin and vincristine 23Figure 1.8Liver cross section 27Figure 1.9Sterically stabilized liposomes (SSLs) 32Figure 1.10Capillary endothelial vascular structure and modes of extravascular transport 37Figure 2.1Summary of PEG-lipid conjugate chemical structures 44Figure 2.2Circulation lifetime ofDSPC/cholesterol/MePEG20- -POP liposomes 52Figure 2.3Loss of PEG coating from the surface of the LUV 54Figure 2.4Thin layer chromatography of the results following incubation of micellar PEG-PEin serum at 37°C 58Figure 2.5In vitro incubation in normal mouse serum at 37°C of DSPC/cholesterol largeunilamellar vesicles incorporating 5 mol % MePEG2000-S-DSPE 60viiiFigure 2.6Circulation lifetime of DSPC/cholesterolllvlePEG2000- -DSP liposomes andin vivo exchange ofMePEG2000-S-DSPE from injected liposomes 62Figure 2.7Models for PEG-PE exchange and breakdown 67Figure 3.1Biodistribution of the pre-dose containing liposomal doxorubicin 75Figure 3.2Biodistribution of the subsequent injection of empty liposomes 76Figure 3.3Dose titration of entrapped doxorubicin in the pre-dose: biodistribution ofthe subsequent injection 78Figure 3.4Dose titration of entrapped doxorubicin or vincristine in the pre-dose:biodistribution of the subsequent injection 80Figure 3.5Time course of recovery of RES blockade achieved following i.v. administrationof liposome entrapped doxorubicin or vincristine 82Figure 3.6Cryostat sections of livers obtained from normal, liposomal doxorubicin treated,and liposomal vincristine treated animals 84Figure 3.7Cryostat sections of normal liver, liposomal DOX treated and liposomal VINCtreated animals after injection of colloidal carbon 85Figure 4.1Dose titration of the liposomal carrier 100Figure 4.2Pharmacokinetic analysis of liposome clearance in tumor bearing mice 102Figure 4.3Pharmacokinetic analysis of drug clearance in tumor bearing mice 105Figure 4.4Tumor loading of liposome and drug loading in the murine Lewis Lung solid tumor model .... 107ixFigure 4.5Lewis Lung solid tumor histology after administration of either free orliposomal doxorubicin 110Figure 4.6Doxorubicin mediated Lewis Lung solid tumor growth inhibition 113Figure 5.1Potential uses for exchangeable PEG-lipids 123xLIST OF TABLESTable 1.1Comparison of Different Drug Carriers Used for Systemic Delivery 2Table 1.2Liposome Drug Carrier Technology 4Table 1.3Transition temperatures (Ta) of various combinations of acyl chain length,degree of saturation, and headgroup moiety 9Table 2.1Biodistribution of DSPC/cholesterol large unilamellar vesicles incorporatingGM1 or PEG-PE one day after i.v. injection 64Table 4.1Toxicity/weight loss in response to the maximum tolerated dose for free andliposomal doxorubicin 99Table 4.2.Comparison of liposomal biodistribution in Lewis Lung solid tumor bearingversus tumor free BDF- 1 mice after i.v. administration of equivalent dosesof lipid (10 jimol per mouse) 104xiABBREVIATIONSApoA- 1 apolipoprotein A-iAUC area under the curvechol cholesterolCHE cholesteryl hexadecyl etherCTm peak tumor drug concentration levelsDCC N,N’-dicyclohexylcarbodiimideDOX doxorubicmDSPC distearoyl phosphatidyicholineEDTA ethylenediaminetetra-acetic acidEPC egg phosphatidylcholineFATMLV frozen and thawed LUVsGM1 monosialoganglioside GMIGM-CSF granulocyte-macrophage colony stimulating factorHAMA human anti-mouse antibodyHBS HEPES buffered salineHDL high density lipoproteinHEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acidIL-2 interleukin-2i.p. intraperitoneali.v. intravenousIgG immunoglobulin GLLC Lewis Lung carcinomaLUV large unilamellar vesiciesLUVET LUVs prepared by extrusionMePEG monomethoxypoly(ethylene glycol)MLV multilamellar vesiclesMPS mononuclear phagocyte systemMU) maximum tolerated doseNTIS N-hydroxysuccinimidePA phosphatidic acidPC phosphatidyicholinePE phosphatidylethanolaminePEG poly(ethylene glycol)PEG-PE poly(ethylene glycol)-modified phosphatidylethanolaminePG phosphatidyiglycerolPT phosphatidylinositolQELS quasielastic light scatteringRES reticulo-endothelial systemSUV small unilamellar vesiciesTAM tumor associated macrophageTe targeting efficiencytB tumor bearingtF tumor freeVEGF vascular endothelial growth factorV1NC vincristineVLS vascular leak syndromeVPF vascular permeability factorVVO vesiculo-vacuolar organellesxliACKNOWLEDGMENTSSo many people have helped me in getting to this point in so many ways that I cannot possiblymention everyone I would like to thank. I would, however, like to acknowledge Dana Masin forher assistance with the animal work, Guoyang Zhang for the tissue sectioning, Steven Ansell forthe chemical syntheses, and Kim Wong and Diane Tanguay for keeping the lab running sosmoothly.Special thanks also go to my best friends in the lab for making my time here immenselyenjoyable: Nancy, for keeping me constantly entertained in her attempt to drive me insane, andTroy and Austin for trying their best to undue the damage either she does or I do to myself.I must especially thank two people: Marcel Bally, for all his encouragement, advice and ideasover the years; and of course Pieter Cullis, for providing such an outstanding and enjoyableenviromnent in which to work in addition to his encouragement and advice.xliiTo my wife,KristixivCHAPTER 1INTRODUCTION1.1 Project overview: liposomes as drug carriersThe concept of a “magic bullet” or targeted drug delivery system for the treatment of humandisease was first proposed long ago (Ehrlich, 1906), but it is only recently that significantadvances toward this goal have been made. In particular, three basic approaches for the deliveryof drugs to disease sites hold promise. These include antibody-drug conjugates, and two carrierbased approaches, polymers and liposomes. Advantages and disadvantages of these systems areoutlined in Table 1.1. While some of the initial problems with antibody-drug conjugates such asaltered antibody and drug properties (Sung et al., 1992: Thomas et a!., 1989) and immuneresponses (Khazaeli et al., 1994) can be overcome in particular cases (Trail et al., 1993; Hale etal., 1988), basic limitations remain. These include the amount of drug that can be attached to theantibody (Ghose and Blair, 1987)(and thus the amount of drug which can be delivered in highconcentration to the intended target), the need to re-engineer the antibody-drug complex for eachdrug or antibody employed (Trail et al., 1983), and an inability to protect the active agent fromdegradative enzymes in the physiological milieu (Weinstein and van Osdol, 1992).As a result there has been interest in carrier based technologies. These systems include polymerbased carriers and lipid based carriers, both of which can overcome the limitations mentionedabove. Polymer based carriers, including nanoparticles and microparticles (reviewed in BrannonPeppas, 1995), are small particulate polymer constructs capable of carrying largeTable 1.1Comparison of Different Drug Carriers Used for Systemic DeliveryDrug Carrier System Advantages DisadvantagesAntibodies specific targeting; low drug-carrying capacity;relatively low MW increases immunogenicity;access to extravascular sitePolymer-based drug stability; low RES uptake; limitedimmunogenicity; controlled extravascular access; difficultrelease; high drug carrying pharmaceutical manufacture;capacity chemical drug modification;potential toxicityLiposomes drug stability; low RES uptake; limited access toimmunogenicity; controlled extravascular siterelease; high drug carryingcapacityamounts of a phannaceutical agent. Lipid based systems, including liposomes and lipoproteins,have the ability to entrap biologically active agents in an interior compartment. Liposomes, morespecifically, have undergone considerable development since their initial characterization in1965 (Bangham et al., 1965). Liposomes can offer flexibility with regard to choice of entrappeddrug(s), and have the potential for sophisticated modifications, including specific attachment oftargeting components, controlled drug release properties, and fusogenic capacity as would berequired for intracellular delivery.One of the first benefits noted for liposomal drug systems was the reduction in toxicity over thefree drug for very simple “non-targeted” systems consisting only of the drug encapsulated insidelipid vesicles (Rahman, 1982; Olson et al., 1982). This resulted in liposomal formulations of2anticancer drugs (Cowens et al., 1993) and antifungal agents (Madden et at., 1990a) that are nowcompleting advanced clinical trials (Cowens et al., 1993; Conley et al., 1993; Gabizon et al.,1994a; Harrison et a!., 1995). The reduction in toxicity has been attributed to the elimination ofpeak plasma concentrations of free drug because most of the drug is sequestered within thecarrier and leaks out slowly. Chemotherapeutic agents often exhibit steep dose response curves,but are limited by the toxicity associated with high free drug doses (Minow et al., 1975;Livingston, 1994). Liposomal encapsulation maintains and often increases anti-tumor potencyand efficacy (Gabizon et at., 1982; Mayer et at., 1990b;). Furthermore, the reduced toxicityallows higher doses of drug to be administered in the liposomally encapsulated form (Gabizon eta!., 1986; Batty et at., 1990b).The second benefit of liposomal drug carriers results from the increased circulation lifetimes ofliposomally encapsulated drug as compared to the free drug. It has been suggested that liposomescan serve as circulating reservoirs for slow release of drug in the blood compartment (Mayer etal., 1 990a; Allen et al., 1992). However, one of the biggest benefits of liposomes is the tendencyof this carrier to accumulate in sites of disease and act as local reservoirs for release of drugdirectly into the affected tissue (Huang et al., 1992, Gabizon, 1992, Mayer et al., 1990a). Such“passive targeting” is strongly correlated with longer circulation times of the carrier (Gabizonand Papahadjopoulos, 1988; Gabizon et al. 1990; Gabizon, 1992).Attempts to further extend the circulation lifetimes and thus the passive targeting of liposomaldrug carriers have thus far focused on size and surface properties of the liposomes. Importantrefmements included (i) incorporation of long-chain saturated lipids for better drug retention andphysical and chemical stability, and (ii) reduced liposome size and neutral surface charge for3Table 1.2Liposome Drug Carrier TechnologyTechnology Description Utility Stages of Developmentfirst generation: base natural and/or synthetic reduced toxicity Phase I, II clinical trialscarrier: phospholipids with enhanced efficacy completeencapsulated drug passive targeting within Phase III initiateddisease site0second generation: sterically stabilized improved circulation approved by FDA in US“stealth” liposomes (SSL): time:incorporation PEG- improved passivelipids targeting to disease sitethird generation: sterically stabilized specific target cell experimentalliposome deliverysurface associated improved therapeutictargeting information index(antibodies)thermosensitive?triggered release?improved circulation times. These first generation systems are in advanced clinical trials inhumans for several anti-cancer drugs, including doxorubicin which is now being tested in PhaseIII trials (Cowens et al., 1993; Conley et al., 1993; Gabizon et al., 1994a; Harrison et al., 1995).Some of these formulations include the so called “sterically stabilized” liposomes, which4incorporate surface modif’ing agents such as the ganglioside GMI or PEG-lipid conjugates wherethe polymer poly(ethylene glycol) is covalently attached to the head group of a lipid anchor.Liposomes incorporating these components can have increased circulation lifetimes as comparedto formulations which do not incorporate these lipids (reviewed in Allen, 1994). The nextgeneration of liposomal drug carriers include “actively” targeted systems which contain targetingligands such as antibodies coupled to the liposome surface (Loughrey et al., 1993; Longman etal., 1995).It is proposed here that actively targeted systems must first achieve maximal levels of passivetargeting to sites of disease before the benefits of active targeting to particular diseased cells canbe realized. Part of this thesis characterizes passive targeting of liposomes in a rodent tumormodel, and included the use of sterically stabilized systems and entrapped drug to influencepassive drug targeting to a disease site.1.2 LiposomesDispersion of bilayer forming lipids in aqueous media results in the spontaneous formation ofliposomes. The multilamellar structure was first described by Bangham et al., (1965) as an onionskin arrangement of concentric lamellae. These lamellae consist of lipids in the bilayerconfiguration, a structure that arises as a result of the “amphipathic” nature of lipids. Thecombination of a hydrophilic head group and hydrophobic tails within the same molecule resultsin an orientation of the lipid head group towards the aqueous environment and the acyl tailstoward each other, as depicted in Figure 1.1. Liposomes have been employed as modelmembranes for studies on the structural and functional roles of lipids in membranes and as5Figure 1.1Amphipathic lipids in bilayer configurationhydophilicaqueousbufferhydrophobicbilayerstructurematrices for reconstituted membrane proteins. These systems allow investigation of processessuch as membrane fusion (Bailey and Cullis, 1994; van Meer et al., 1985), factors leading tocomplement activation (Devine et al., 1994), protein-lipid interactions, among many otherapplications. For example, because liposomes form closed spheres with defined interior andexterior aqueous spacesseparated by lipid bilayers, they are excellent tools to study membranepermeability and ion gradient formation (Deamer and Nichols, 1983; 1989; Viero and Cullis,1990), and the distribution of various molecules in response to these gradients, such as6neurotransmitter uptake and storage (Nichols and Deamer 1976; Bally et al., 1988). The utility ofliposomes as drug delivery vehicles will be covered in later sections.1.2.1 Chemistry and physics of lipidsMost of the liposomal drug delivery systems in use today are composed of a combination ofphospholipids and cholesterol. These components are discussed below. Other membranecomponents will be discussed in the context of more recent advances in this carrier technology. PhospholipidsPhospholipids are composed of various combinations of polar (hydrophilic) headgroups coupledto apolar (hydrophobic) tails via a glycerol-phosphate backbone. A summary of phospholipidstructure is shown in Figure 1.2. Each combination of headgroup and acyl chain compositiondictates the physical properties of the lipid bilayer. For example, at physiological pH liposomescontaining phosphatidyl serine (PS), phosphatidylglycerol (PG), phosphatidylinositol (P1) andphosphatidic acid (PA) will have a negative surface charge. This charge is an important factordetermining serum protein binding (Moghimi and Patel, 1989), which leads to the clearance ofliposomes from the circulation as will be discussed later.Another important parameter for characterizing phospholipids is the temperature of the gel toliquid-crystalline phase transition for bilayers that is principally dependent upon the length andsaturation of the acyl chains. In general, longer, more saturated acyl chains give rise to higherphase transition temperatures (Ta). Acyl chain motion is often characterized by an order7Figure 1.2Structures of common phospholipidsNeutral phospholipids—HeadgroupCholine 4-CH2CHN(3)O=P—O Phosphatidyicholine (PC)EthanolamineCH2H—H Glycerol backbonePhosphatidylethanolamine (PE)4H2CH3Negative phaspholipidsO==O4-HC!HZ (!H2 Phosphatidic acid (PA)t!H2 H2 Serine 4—CH2CH—N3H2 H2 Phosphatidylserine (PS)H2 H2 i—CH2CH(OH)CHOGlycerolH2 Phosphatidyiglycerol (PG) OH OHH2 c!H2H2 H2 Inositol HH2 H Phosphatidyilnosltol (P1)H2 HHH2 H2!H2 tH2 Saturated fatty acids(!H2 I!H2 Lauric (12:0) CH3(C2)100 H!H2 iH2Acyl chainMyristic (14:0) CH(C12OOHH2 cH2 Palmitjc (16:0) CH3(C2)14OOHc!H3 H2 Stearic (18:0) CH(C16OOHH2Unsaturated fatty acidsCH3 I Palmitoleic (16:1 A9) CH3(C2)5H=CH(CHCOOHOleIc (18:1 A9) (CHCH=CH(CHCOOHLinoleic (18:2A9,i2)CH(C )4H=CHCHH=CH(C COOH8Table 1.3Transition temperatures (To) of various combinations of acyl chain length, degree ofsaturation, and headgroup moietylipid species transition temperature T (°C)dilauroyl PC (12:0, 12:0)-1dimyristoyl PC (14:0, 14:0) 24dipalmitoyl PC (16:0, 16:0) 41distearoyl PC (18:0, 18:0) 55stearoyl, oleoyl PC (18:0, 18:1) 6stearoyl, linoleoyl PC (18:0, 18:2) -13dipalmitoyl PA (16:0, 16:0) 67dipalmitoyl PE (16:0, 16:0) 63dipalmitoyl PS (16:0, 16:0) 55dipalmitoyl PG (16:0, 16:0) 41parameter “s” where s = 1 for no motion, whereas for rapid isotropic motion s = 0. Below the T,the acyl chains have a high “order” (s 1) in that their motion is greatly restricted and the chainspack together in a frozen or “gel” phase. Above the T, the acyl chains are less ordered or more“fluid” in nature in what is termed the “liquid-crystalline” phase. Longer acyl chains haveincreased order whereas unsaturated acyl chains disrupt packing and reduce the acyl chain orderof the membrane. In addition, the headgroup can also influence the T. These properties aresummarized in Table 1.3. In general, membranes are more permeable to a variety of solvents andsolutes at or above the T than below (Bittman and Blau, 1972). Increased membranepermeability has also been correlated with increased unsaturation or shorter acyl chains(Papahadjopoulos et al., 1973).9Hydrated phospholipids can adopt a wide variety of structures, the bilayer being only one ofthem (Figure 1.3). The ability of lipids to adopt structures other than the bilayer is described aslipid polymorphism (Cullis et al., 1986). Some lipids have head groups which occupy largevolumes in comparison to their tails, and can be thought of as adopting a cone shape and thushave a tendency to form micelles (see Figure 1.3). These lipids include detergents. Unsaturatedspecies of PE, on the other hand, in general will not form bilayers due to the relatively smallneutral headgroup and more flexible acyl chains that are conceptually thought to resembleinverted cones. These lipids tend to form inverted hexagonal or H11 phase structures (Cullis andde Kruijff, 1979). Most of the lipids employed for liposomal drug carriers fall into the bilayerforming class, and can be thought of as exhibiting a cylindrical shape. These lipids includephosphatidyicholines, commonly employed as the base phospholipid in liposome compositionsfor drug delivery.Other factors can also modulate lipid polymorphism, including the effects that temperature hasacyl chain fluidity, and the influence of pH and ionic strength on head group charge (Cullis et al.,1986). As discussed above, more mobile acyl chains increase the relative volume of thehydrophobic portion, whereas charged headgroups effectively increase the volume occupied bythe hydrophilic portions. The particular combination of tail and head group relative sizedetermines the relative shape of the molecule as described in Figure 1.3. For example,unsaturated PS has a net negative headgroup charge at physiological pH, however upon loweringthe pH, protonation of its carboxyl group results in net neutralization. The neutral headgroupoccupies a smaller area due to the reduction in bound water thereby promoting formation of H11phase structures.10Figure 1.3Lipid polymorphismSome examples of common shapes and resulting lipid shapes are shown belowshape structureinverted conemicellarcylinderconeinverted micellarlamellar111.2.1.2 CholesterolCholesterol is the major neutral component of almost all eukaryotic biological membranes.Cholesterol is an amphipathic molecule due to the poiar 3--hydroxyl group that is orientedtoward the lipid/water interface but sits buried next to the carbonyl groups of the fatty acylchains near the head groups, and the rigid steroid ring lying associated with the acyl chains. Themore flexible aliphatic tail lies deepest in the membrane. Incorporation of cholesterol decreasesthe membrane order for phospholipids in the gel phase (i.e. below their T) and increases theorder of the membrane for lipids in the liquid-crystalline phase (above their T; Demel and deKruijff, 1976). Increasing the cholesterol content above 7 mol % reduces the enthalpy of the gelto liquid-crystalline phase transition, until at 33 mol % and higher, the phase transition can nolonger be detected (Hubbell and McConnel, 1971). The addition of cholesterol to bothunsaturated and saturated PC membranes above their gel to liquid-crystalline transitiontemperature decreases membrane permeability, while increasing permeability for membranescomposed of saturated PC below the T (Bittman and Blau, 1972). In addition, the inclusion ofcholesterol helps to stabilize liposomes used for systemic delivery of drugs (Papahadjopoulos etal., 1973), primarily as a result of reduced lipid exchange with lipoproteins (Kirby et al., 1980;Hunt, 1982). This latter role of cholesterol is discussed in detail in Section Liposome preparationCategories of liposomes include multilamellar vesicles (MLVs), large unilamellar vesicles(LUVs), and small unilamellar vesicles (SUVs) as depicted in Figure 1.5 and depend uponliposome size and the number of bilayers (lamellarity) contained within each vesicle.12Figure 1.4The structure of cholesterol (A) and its effect on the structure of lipid bilayers (B)ALT<T“gel”B+cholestero I“liquifying”III endothermjc4r transitionInLaT>T“liquidCrystalline”+cholesterol“condensing” Multilamellar vesicles (MLVs)Dispersion of a lipid powder or a dry lipid film by mechanical agitation (e.g. vortex mixing) inaqueous solution results in the spontaneous formation of MLVs composed of concentric bilayers.The resulting vesicles are heterogeneous in size, typically ranging in size from 1-10 m, and canbe multilamellar to the extent that less than 10% of the total lipid is present in the outermostbilayer (Mayer et a!., 1985).These MLVs typically have low aqueous trapped volumes (of the order of 0.5 .tlJLtmo1 lipid) dueto the close packing of adjacent lamellae. Trapped volumes can be increased by incorporation ofcharged lipids that lead to charge repulsion between bilayers and increased interbilayer spacing(Hope et al., 1986). Alternatively, repeated cycles of freezing and thawing can also increase theinterbilayer spacing allowing trapped volumes of approximately 7 .tI4tmol lipid to be achieved.These MLVs are often referred to as frozen and thawed MLVs (FATMLVs; Mayer et al., 1985).Reverse phase evaporation (Szoka and Papahadjopoulos, 1978; 1980) is an important alternativemethod for the production of MLVs with large trapped volumes and increased intralamellarspacing. Briefly, the lipid is first dissolved in organic solvent. Then, as the solvent is diluted orevaporated away in the presence of aqueous buffer, the lipids move from the organic to aqueousphase forming vesicles in the process. While MLVs prepared by this method can have hightrapped volumes (up to 10,imol), this procedure can be limited by low lipid solubility ofcertain lipids in the organic solvent as well as difficulties in removing residual solvent from thefinal aqueous preparation.14CDiCDCDtCD— CDCDC)=- CC)C)CD -t tCDCDC/)ct.—-CD‘—CD-t—CDCD CDC)O2CDCl)o—Cl)- CDC)‘‘CD0 CDqSCD-t—0 0Cl) CI c1.2.2.2 Small unilamellar vesicles (SUVs) large unilamellar vesicles (LUV)Unilamellar vesicles (SUVs and LUVs) are grouped by size, usually 25-50 nm, and 50-200 nm,respectively. SUVs can be produced directly from MLVs by several methods, includingsonication (Huang, 1969) and French press (Barenholz et al., 1979). The limiting factor in termsof size appears to be the maximum curvature that the bilayer can assume as a result of packingconstraints generated by the small radius. SUVs can have a outer to inner monolayer lipid ratioas high as 2:1, and typically have low trapped volumes (—0.2 .tl/imol; Huang, 1969; Barenholzet al., 1979). SUVs are often unstable because of the thermodynamically unfavorable stressesresulting from their small radii and can spontaneously fuse into larger structures over time withthe loss of their entrapped contents (Wong et al., 1982). These properties tend to make SUVspoor choices for drug carriers.LUVs are more useful as drug carriers because the higher trapped volumes result in the potentialfor greater drug encapsulation and the greater membrane stability results in longer circulationlifetime and stability of the carrier within the circulation. There are several means for producingLUVs. Detergent dialysis (Mimms et al., 1981) involves solubilization of either dried lipid orpre-formed vesicles in buffer containing detergent. The resulting mixed micelles are thendialyzed against buffer to remove the detergent resulting in the formation of LUVs. The size andtrapped volumes depend upon the detergent, the rate and method of detergent removal, and thelipid composition (Madden, 1986). It is a relatively gentle method compatible with proteinintegrity and thus is often used in studies involving the reconstitution of membrane proteins(Helenius et aL, 1977). A problem often associated with this method, however, is the presence of16residual detergent. Other methods include ethanol injection (Chen and Schullery, 1979), etherinfusion (Deamer and Bangham, 1976), and reverse phase evaporation (Szoka andPapahadjopoulos, 1978) which are similar to that described for MLVs in that lipid dissolved inan organic solvent are hydrated in aqueous buffer and then the organic solvent is slowlyremoved. In these methods more precise control of organic solvent removal can result in largeunilamellar vesicles. Problems regarding residual solvents or detergents limit applications ofthese procedures for production of liposomal drug carriers.One of the most general methods for the production of LUVs involves extrusion. This typicallyrelies on forcing either MLVs or FATh’ILVs through polycarbonate filters of a defined pore size(Szoka and Papahadjopoulos, 1980; Hope et al., 1985; Mayer et al., 1986). The resulting vesicleshave well defined sizes (close to the pore size) and are largely unilamellar if the filter pore size isless than 200 nm. The technique requires that the extrusion step be carried out repeatedly(typically 1 Ox) at a temperature above the transition temperature (Ta) of the phospholipidcomponent. Some advantages of this method are that it allows high lipid concentrations to beused, a wide variety of bilayer fonning lipid compositions can be prepared, and it is simple,rapid, and reproducible. LUV sizes around 100 nm prepared by this method have trappedvolumes of 1-3 I4tmol. These LUVs are sometimes referred to as LUVETs (LUVs by extrusiontechnique). They are useful as drug carriers since the procedure is amenable to pharmaceuticalproduction, allowing control of quality and sterility and does not require residual solvent ordetergent.171.2.3 Drug encapsulationThere are two basic strategies for encapsulating drugs within liposomes. In passive trappingtechniques, the drug is combined with the lipid during liposome formation. For aqueous agents,encapsulation depends upon the trapped aqueous volume of the liposome, whereas for lipophiliccompounds, encapsulation efficiency usually depends on the capacity of the bilayer to solubilizethe agent while maintaining vesicle structure. A second basic trapping method is termed activetrapping and involves the encapsulation of the drug in pre-formed vesicles exhibitingtransmembrane ion gradients. Many anti-cancer drugs are now encapsulated using proceduresbased on active loading. Passive entrapmentHydrophobic drugs can be passively trapped by including the agent in the original lipid mixtureprior to liposome generation. Amphotericin B is one example of a drug which is entrapped in thismaimer (Madden et a!., 1990a). Depending on the packing constraints and the lipidcharacteristics determining compatibility with drug incorporation into the membrane, thistechnique can have a high efficiency of incorporation. However, drugs of this class often exhibitappreciable exchange rates into other membranes and thus in vivo the drug can often rapidlyleave the carrier (Madden et a!., 1990a).The passive entrapment of water soluble drugs into liposomes is similar to hydrophobic drugs inthat both drug and lipid are combined in the original preparation prior to vesicle formation butdepends on the aqueous trapped volume of the vesicle preparation. For SUVs with low trapped18volumes (0.2 jtl4tmoI), efficiency of entrapment is typically around 1% (Szoka andPapahadjopoulos, 1980). For FATMLVs and LUVs with trapped volumes ranging from 1-10jil4tmol combined with high lipid concentrations of the order of 400 mg lipid/mi, efficiencies ofup to 80% can be achieved (Mayer et a!., 1985). The retention of water soluble drugs depends onboth the membrane and drug. For relatively membrane impermeable drugs such as methotrexateand cytosine, the half-lives for retention in EPC/chol vesicles are 50 and 18 hours, respectively(Madden et al., 1990b). On the other hand, passively entrapped doxorubicin, which has lipophilicqualities, has a retention half-life of less than 1 hour in identical vesicles (Juliano and Stamp,1975). As discussed earlier, the incorporation of longer chain saturated acyl chains in the lipidspecies combined with cholesterol can dramatically improve the retention of these drugs. Forboth lipid and aqueous entrapment procedures, the use of MLVs also usually results in improvedretention because of increased numbers of lamellar barriers to pass to reach the external medium. Active (ipH) entrapmentActive loading procedures are designed to load drug into the interior of pre-formed liposomes.Drugs that can be entrapped by this method are typically lipophilic cations or anions with anionizable amino or carboxyl function, respectively. For example, drugs that are weak bases, suchas doxorubicin or vincristine, will accumulate inside liposomes in response to a proton gradient,ApH, where the liposomes have an acidic interior. The mechanism for this accumulation isshown in Figure 1.6. In the external environment a proportion of the weak base exists as theneutral form along with the charged (protonated) form of the drug. In the neutral form the drug ismore membrane permeable (Addanki et al., 1968; Rottenberg, 1979) and able to cross the bilayer19readily. However, on arrival in the acidic interior, the weak base becomes protonated. Thecharged species is not significantly membrane permeable, and is trapped within the interior.The equilibrium transbilayer distribution of this weak base can be described by a simplederivation. The equilibrium constant (Ia) of a weak base B is:Ka = H.B/BHwhere H is the proton activity, B is the activity of the neutral base, and BH is the activity of theprotonated base. To a first approximation activity can be replaced by concentration. The relativeconcentration of the neutral and protonated weak base at a given pH are therefore described bythe Henderson-Hasselbach equation:pH = PKa + log {[B]/[BH1}If the dissociation constants for a weak base are the same on both the inside1 and outside0 of thevesicle membrane, then:Ka= [Hj[B]1I[BHj =[Hj0B]/[ H]As noted previously, in general the uncharged species of an ionizable compound tends to bemore membrane permeable than the corresponding charged species. As a result, at equilibriumthe concentration of the neutral species will be the same on both sides of the membrane. The20Figure 1.6Active drug entrapment in liposomesThe equilibrium redistribution of a lipophilic amine (weak base) in response to a pH gradient(ApH) across the liposome membrane. Only the neutral form of the molecule is significantlymembrane permeable.OUTSIDE INSIDEpH 7.5 pH 4.0BHAl,&B )÷ +H + H +[B]0Hj [B]1 Hj1Ka= Ka=[BHJ0 [BH]At equilibrium, if:[B]0 = [B]Then:[BH].— [Hj.[BH]Q— [H]021above equation can therefore be simplified and the equilibrium distribution will then reflect thep11 gradient according to:[BH]/[BHj0= [H]/[Hj0This indicates that for a zpH of 3 units, for example, a weak base may be accumulated to aninterior concentration 1000 fold higher than in the exterior medium. For 100 nm diameter LUVscomposed of EPC or EPC and cholesterol, doxorubicin active trapping efficiencies can approach100% at drug to lipid mol ratios of 0.2 or lower (Madden et al., 1 990b). In an analogous fashion,weak acids can also be accumulated into liposomes with oppositely directed pFT gradients(interior basic; Eastman et al., 1991).The generation of a pH gradient across the LUV membrane is straightforward and simplyinvolves the exchange of the external buffer for a buffer of a different pH. Methods include sizeexclusion chromatography or simple titration of the external buffer to a new pH. In some casesdialysis of the external buffer may also be useful. pH gradients for several PC membrane speciesappear to be stable for hours or days even at elevated temperatures (Harrigan et al., 1992), whilethe addition of cholesterol further retards the decay of the imposed zpH (Madden et al., 1 990b).The maximum zpH that can be stably maintained in EPC/cholesterol LUVs is 3.7 units(corresponding to a membrane potential of approximately 220 mV; Harrigan et al., 1992).Many other drugs are also lipophilic weak base amines and thus the mechanism of ApHentrapment is of general utility. A survey of drugs (Madden et al., 1990b) illustrates the22Figure 1.7Structure of doxorubIcin and vincristine.DOXORUBICINVINCRISTINE0H3CH NH223generality of pH dependent entrapment, but while most redistribute in response to atransmembrane proton gradient, the level and stability of accumulation varies considerably. Pooruptake or subsequent release of most drugs was shown to be primarily due to loss of the pHgradient. For example, vincristine leakage appears to be directly related to eventual dissipationof the Apil (Boman et al., 1993). For other drugs, however, leakage was correlated withsignificant membrane permeability of the ionized species (Madden et al., 1990b). Fordoxorubicin, the initial accumulation is higher than that predicted by the Henderson-Hasselbachequation and drug retention is longer than the decay of the zpH. This is likely due to a reducedsoluble aqueous fraction (Mayer et al., 1990b; Harrigan et al., 1993). A variation of the zpHloading method involves the use of entrapped ammonium sulfate (Haran et a!., 1993) to generatea zpH gradient that subsequently mediates doxorubicin encapsulation.1.3 In vivo behavior of intravenously injected liposomesThe use of liposomes as intravenous drug carriers requires a detailed understanding of the twomajor factors controlling their pharmacokinetic and pharmacodynamic behavior in vivo. First,interaction of liposomes in vivo involves the binding of plasma proteins and lipoproteins.Second, this then determines the subsequent interaction with the major clearance mechanism forliposomes, the reticuloendothelial system (RES).1.3.1 Interactions of liposomes with plasma proteinsThe interaction of liposomes with lipoproteins can cause the release of encapsulated agents dueto lipid exchange, resulting in dissolution of the carrier (Kirby et al. 1980). The apolipoprotein24ApoA-1, found predominantly in the high density lipoprotein (HDL) fraction, plays a major rolein this process which involves insertion of the protein into the lipid bilayer (Klausner et a!.,1985). Another source of leakage involves interactions with complement proteins that can lead toactivation of the complement cascade (Devine et a!., 1994). Activation of the complementpathway can result in the formation of a membrane attack complex, a pore forming channel 10urn in diameter, and resulting loss of interior contents or lysis of the membrane (Silverman et al.,1984).Complement protein binding also represents one form of opsonization, a particularly importantprocess regulating liposome elimination. Opsonization is the binding of plasma proteins thatpromote the uptake of foreign particulate by the fixed and free macrophages of the RES, alsoknown as the mononuclear phagocytic system (MPS)(Coleman, 1986; Moghimi and Patel,1989). Opsonins include IgG, fibronectin, and certain complement proteins (C3, C3bi). Whilesome opsonins are thought to be only surface bound, such as C3 or IgG, others may havehydrophobic interactions with the liposome membrane. Subsequent RES uptake depends onrecognition of membrane associated opsonins by specific receptors on the macrophage surface(Coleman, 1986), and it has been shown that the total amount of bound protein on the liposomesurface is directly related to the clearance rate (Chonn et al., 1991; 1992).1.3.2 Interactions with the reticuloendothelial systemThe removal of foreign particulate matter such as opsonized liposomes from the circulation iscarried out by the reticuloendothelial system (RES). The RES is primarily composed of theresident macrophages of the liver (Kupffer cells), spleen, lungs, and bone marrow. It has been25more recently recognized that circulating monocytes also play a role in this clearance mechanism(Senior, 1987), hence the total system is also sometimes referred to as the mononuclearphagocyte system (MPS).The vast majority of liposomes cleared from the circulation are found in the liver, and to a lesserextent in the spleen. In the liver, the specialized Kupffer cells lie within the sinusoids (Figure1.8), and phagocytose a wide variety of particles from bacteria and other foreign pathogens aswell as defined particulates such as latex beads (Pratten and Lloyd, 1986), liposomes (Roerdinket al, 1981), carbon particles (Kampschmidt et al., 1966), and other macromolecules. Theprimary role of this specialized macrophage is to capture and then degrade or detoxify foreignagents in the blood.In some cases, liposome uptake by the RES is useful, such as when this cell population is theintended biologic target. Liposomal delivery of macrophage activators, for example, may be ofbenefit for increasing the tumoricidal activity of macrophages (Fidler et al., 1982). Manyintracellular parasites and bacterial infections use phagocytic cells as hosts, and liposomaldelivery of specific drugs to eliminate these intracellular parasites have also shown promise(Alving, 1988). Finally, liposomes have proven to be particularly potent immune adjuvants forcertain antigens, as these macrophages are also involved in antigen processing and presentation(Alving, 1987). However, the RES presents a major obstacle if alternate target sites are beingconsidered. Strategies to avoid the RES, primarily involving the modification of liposomes toreduce opsonization, show promise in terms of reducing RES accumulation.26Figure 1.8Liver cross sectionLiver section taken from a normal CD-i mouse. Identified on the figure are (V), central vein;(H), hepatocyte; (b), red blood cell; (E), endothelial cell; (S), sinusoid; (K), Kupifer cell(hematoxylin and eosin, 5 tm thick paraffin)271.4 Properties of liposomes influencing circulation lifetimesThe primary properties governing circulation lifetimes of liposomes are size, composition,charge, and dose. Large liposomes (>1 tm diameter) are cleared more rapidly than smallliposomes (<200 mn). For example, large MLVs have typical half-lives of less than 15 minutes,whereas LUVs (30-80 nm) of identical lipid composition and dose exhibit half-lives over 4 foldlonger (Juliano and Stamp, 1975). In general large liposomes are taken up by the Kupifer,however the spleen phagocytes and sinusoidal structure also play roles in the uptake of largeliposomes (Litzinger and Huang, 1992). Liposomes that are very small (SUVs), however, areoften also cleared quickly from the circulation. It is thought that SUVs have membrane defectsarising because of the high radius of curvature that may act as sites for opsonin binding. There isalso evidence that many small pores exist in the liver sinusoids that allow small liposomes toextravasate and thus facilitate binding and uptake by liver hepatocytes (Roerdink et al., 1981).The administered lipid dose also plays an important role in determining the circulation lifetimeof injected liposomes. It is often observed that, in tenns of percent of the injected dose, higherdoses result in higher circulation levels and lower liver uptake at a given time point (Poste et al.,1984). This is thought to be due to saturation of the liver macrophages that then results inspillover into the spleen, followed by further spillover into the lung and bone marrow. In studieswith saturating pre-doses of MLVs (1100 mg/kg in mice), Abra et al. (1980) for example wereable to demonstrate over 5-fold depression in liver uptake and a 3 to 4-fold increase in spleenuptake of a second identical injection 1 hour after the pre-dose. More recent work, however, hasfocused on the possibility that large doses of liposomes may actually deplete certain opsonins(Oja et al., 1995).28The clearance behavior of liposomes is also strongly influenced by lipid composition. Asmentioned earlier, surface charge plays a large role in the clearance of liposomes by influencingthe extent of opsonization. While neutral surfaces usually exhibit low levels of opsonization andslow RES uptake, negative liposome surfaces accumulate significantly more opsonins and arecleared faster from the circulation (Chonn et al., 1991, 1992). In vitro studies measuringmacrophage uptake levels of MLVs with 30 mol % PS, PG, or P1 indicated that 25, 18, and 15-fold greater levels of lipid uptake occur compared to similar vesicles composed of thecorresponding neutral PC (Shroit et al., 1986). Available evidence suggests that positivelycharged liposomes are also cleared considerably faster than neutral liposomes (Senior et al.,1991a).The role of lipid composition effects on serum protein binding and RES uptake extends to acylchain composition. In general unsaturated acyl chains result in more fluid lipid bilayers and may,therefore, facilitate association of some opsonins. Long chain saturated acyl chains result inmore rigid lipid bilayers and thus resist protein insertion. However, this rigidity can bedetrimental. In the case of liposomes composed only of highly saturated long acyl chainphospholipids, circulation lifetimes can actually be reduced if the lipid gel-to-crystalline phasetransition temperature is higher than physiological temperature. The inclusion of cholesterol inthis case greatly increases circulation lifetimes, primarily as a result of removal of packingdefects that occur in gel state systems (Kirby et al., 1980; Hunt, 1982).29The process of opsonization appears to be the first critical step in liposome clearance. Attemptsto reduce opsonization and maximize drug retention, therefore, focus on using L1.JVs composedof long chain saturated neutral lipids such as DSPC in combination with cholesterol.1.4.1 Poly(ethylene glycol)-lipids (PEG-lipids)The realization that plasma proteins interact with liposome surfaces has led to furthermodification of surface properties. The incorporation of the ganglioside GM! into the lipidmixture at 10 mol % (Allen and Chonn, 1987) was found to greatly increase the circulationlifetimes of the liposomal carrier. There was a concomitant reduction in RES uptake resulting ina higher blood-to-RES ratio at a given time point. Part of the original rationale for theincorporation of this naturally occurring lipid component was that its sugar residues wouldprovide a surface resembling that of cells. It has since been shown that the reduced clearance isprimarily the result of reduced opsonin binding (Chonn et al., 1991; 1992) The failure of similargangliosides to effectively prolong circulation lifetimes, however, has led to some debate as tothe function of GM! and other gangliosides in the liposome membrane (Allen et al., 1994). Onesuggestion is that GM1 may promote the binding of specific dysopsonins, plasma binding proteinswhich would either label the liposome surface as not foreign, or simply prevent the binding ofclearance-promoting opsonins (Park and Huang, 1993). Unfortunately one of the majordrawbacks to the use of GM1 in drug delivery applications is its cost. The results obtained withGM1 did spark renewed interest in the development of liposomes exhibiting enhanced circulationlifetimes and has led to other means of altering the liposome surface characteristics.30It has been known for some time that polymer coated nanoparticles and other polymeric coatedspheres circulate longer than uncoated particles (reviewed in Brannon-Peppas, 1995). There is awealth of physical chemistry data which describes the steric stabilization effect and reducedprotein binding and this information has recently been applied to drug carriers. This technologyhas also led to the use of polymers directly coupled to proteins and drugs for intravenousinjection, most often using polyethylene glycol (PEG; Abuchowski et al., 1977; Delgado et al.,1992,). First reports on the use of PEG-phosphatidylethanolamine conjugates to modify thesurface of conventional liposomes revealed that circulation lifetimes of these drug carriers couldalso be substantially improved (Blume and Ceve, 1990, Klibanov et al., 1990; Papahadjopoulosetal., 1991).PEG is a flexible hydrophilic polymer of repeating ethylene glycol units (-[O-CH2-CH]-)and isusually coupled to the headgroup of PE via well established chemistries involving reactions withthe primary amine of PE. Although there are methods to attach PEG to the surface of pre-formedliposomes resulting in a polymeric coating only on the outside surface (Senior et al., 1991 b),these tend to be difficult and the preferred method of incorporation is the inclusion of PEG-PE inthe original mixture. Thus, after liposome formation, the polymer coats both the inner and outermonolayer.A PEG coating on liposomes increases the circulation lifetimes. This is thought to be due toreduced recognition by cells of the RES. More detailed studies on the surface characteristics ofliposomal PEG-PE have yielded a refined view of the polymeric coating as a steric barrier,inhibiting the close approach of other surfaces or plasma proteins (Lasic, 1994). These liposomesare now commonly referred to as sterically stabilized liposomes (SSL).31Figure 1.9Sterically stabilized liposomes (SSLs)Shown are the structures of A, ganglioside GMI, B, PEG-PE. GMI (MW 1544) is composed of alipid anchor attached to a hydrophilic branched sugar residue. PEG-PE is typically composed ofa phosphatidylethanolamine lipid anchor to which is attached (via a short linker group -X-) to therepeating (-OCH2CH) hydrophilic polymer poly(ethylene glycol)(PEG). For an averagemolecular weight of the PEG polymer of 2000, n is approximately 45. C depicts the binding andinhibition of opsonin binding to the liposome surface in the absence or presence of a stericallystabilized liposome surface.BGal GalNac Gal Gic CeramideC.S.ANan.S.32Other polymers, thought to have the same relative hydrophilicity and steric stabilization as PEG,fail to perform as well as PEG in terms of extending circulation lifetimes (Lasic, 1994). It is nowhypothesized that PEG is sufficiently flexible when coupled to the liposome surface thatinhibition of protein binding stems from a combination of both steric and dynamic properties ofthe polymer (Lasic, 1994). PEG is envisioned to provide not only an initial barrier to closeapproach but also a sweeping action to inhibit protein binding near the liposome surface. Lowerlevels of PEG provide a “mushroom” conformation that can adopt an extended “brush”conformation at higher PEG contents. In either case, the inhibition of protein binding is largelydue to the increased unfavorable surface free energy provided by the PEG coating (Arakawa andTimasheff, 1985). The optimal PEG concentrations in terms of circulation lifetimes appears to beapproximately 5 mol % PEG-PE for PEG with an average molecular weight for the polymer of2000 (Allen et al., 1994).In summary, the principal effect of PEG on the liposome surface is a major reduction in theinteraction of liposomes with plasma proteins. This leads to a stabilization effect (avoidingdestabilizing apolipoproteins) and a reduction in the total amount of opsonin binding (Senior etal., 1991b; Blume and Cevc, 1990). There may also be a reduction in the binding of somespecific opsonins (Allen et al., 1994). It is speculated that the PEG coating may also reducecellular uptake directly such that even if there is protein bound, the coating may still inhibitreceptor mediated binding at the level of the macrophage (Allen et al., 1994).331.4.2 RES blockade: the effect of entrapped drugAnother possible strategy for increasing the circulation lifetime of injected liposomes is to alterthe cells of the RES itself. As mentioned previously, there have been attempts to reduce theactivity of the RES by administering saturating pre-doses of liposomes (Abra and Hunt, 1981) orimmunoglobulin (Coleman, 1986). In this regard, it has been noted that liposomal drug carrierscontaining doxorubicin exhibit significantly longer circulation lifetimes over the identical emptycarrier (Bally et al., 1 990a). This is almost entirely due to reduced liver uptake. It was recognizedmany years ago that there was the potential for harmful effects with liposomally entrappedcytotoxic drugs because of accumulation in the RES or other non-target tissue (Patel, 1984).Both mouse and human studies, however, have suggested that there are no specific livertoxicities as measured by histology or biochemical functions following i.v. administration ofliposomal doxorubicin (Cowens et al., 1993). The question remains, however, whether REStoxicity is responsible for the altered pattern of clearance seen with liposomal doxorubicin.Drugs such as liposomal dichloromethylene diphosphonate eliminate macrophages by deliveringdrugs designed to be toxic to phagocytic cells directly to the macrophage population (Classenand Van Rooijen, 1986; Van Rooijen, 1989). Doxorubicin has a number of modes of actionwhich might be expected to affect Kupffer cells, including DNA intercalation and topoisomeraseII interference, free radical formation, covalent binding to DNA, and various cell binding effects(Cummings et al., 1991). Non dividing cells including Kupifer cells might be killed bydoxorubicin as a result of destructive free radical formation or interference with the normal DNAto RNA to protein pathway. Vincristine on the other hand binds to microtubules (Owellen et al.,341972; 1976). Microtubules are important for normal cellular structure and rearrangement and formembrane trafficing which is important in phagocytic proceses.1.5 Targeting to sites of tumor growth1.5.1 Passive versus active targetingLiposomes with long circulation lifetimes result in increased accumulation in extravascular sitesof disease including sites of infection, inflammation, and tumors (Gabizon and Papahadjopoulos,1988; Gabizon et al. 1990; Gabizon, 1992). This targeting ability is termed passive targeting, andconstitutes targeting because up to 10-100 time more liposomal drug can be directed to thedisease site as compared to injection of the same amount of free drug. Active targeting, on theother hand, involves the attachment of specific ligands such as antigen specific antibodies ontothe liposome surface and thus target to specific cell populations. It should be noted that activetargeting also requires long circulation times and excellent passive targeting capabilities to allowpenetration to the disease site.It is usually observed however that antibody coated liposomes are cleared more rapidly than theirnon-targeted counter parts. Even when immune reactions to the antibody are minimized via hostcompatibility or use in immunocompromised animals such as SCID mice, clearance is usuallyfaster than non-targeted systems, but with the addition of a PEG coating, circulation times canapproach that of carriers which are not coated with antibodies (Loughrey et al., 1993). It wouldbe expected that a surface bound antibody would have target binding problems. The problem oftarget binding can also be overcome by coupling of the antibody to the end of the PEG chain. Amajor effect that the PEG coating has on circulation lifetimes of the targeted systems is that it35reduces the size of the liposome aggregates which often form during the antibody couplingprocedure (Harasym et al., 1995). As mentioned earlier, size plays a critical role in determiningliposome circulation lifetimes.Thus for both non-targeted and targeted systems, the accumulation at a disease site depends uponthe circulation lifetime. However the primary barrier to drug delivery remains access to that site.Even for targeted systems, this remains a two step process, the first being the passiveaccumulation within the site. Once there, “targeting” can occur by altering the distribution of theliposomes within that site (Longman et al., 1995). The process by which the specific barriers arecrossed during movement from the blood to a defined interstitial space is referred to asextravasation. Normal vasculatureIn the normal vasculature, blood capillaries act as the primary sites of nutrient exchange betweenthe blood and tissue. The movement of material from the blood to extravascular sites isdependent on the capillary blood vessel structure. In general there are three classifications (Posteet al., 1984; Jam, 1987; see Figure 1.10) defined by the endothelium and basement membranestructure. Continuous capillaries are found in tissues such as muscle, connective tissue, and skin.They are composed of a continuous lining of endothelial cells and have an uninterruptedsubendothelial layer of basement membrane. Fenestrated capillaries, found in many glands, thegastrointestinal tract, and renal glomerous has an endothelial layer interrupted by fenestrae 30 to80 nm in diameter sealed by a membranous diaphragm. The basement membrane is usuallycontinuous. The last class of capillary structure is called discontinuous or sinusoidal because of36Figure 1.10Capillary endothelial vascular structure and modes of extravascular transportThe three major classes of capillary endothelial vascular structure (continuous, fenestrated, anddiscontinuous) are shown with the various modes of transport possible across them (adaptedfrom Jam, 1987; Poste 1984) (1), direct diffusion across endothelial cell; (2) lateral membranediffusion; (3), interendothelial transport - (a) narrow, (b) wide; (4) endothelial fenestrae - (a)closed, (b) wide; (5), vesicular transport - (a) transcytosis, (b) vacuolar-vesicular organelles(VVOs)(channels).Transport pathways: lipophilic solutes: (1), (2), (3), (4); hydrophilic solutes, macromolecules (3),(4), (5).continuouscapillaryfenestratedcapillarydiscontinuouscapillary123a 4a 4b 5a 5b1 2 3b 5b37its prevalence in the sinusoids of the liver, spleen, and bone marrow. The endothelial layer haslarge openings (>100 mn) between or even within cells. The basement membrane is either absent(liver) or interrupted and fragmented (spleen, bone marrow).The permeability of normal vascular structure is tightly controlled. Whereas small moleculescan, in general, pass freely through normal capillaries and other vessels with intactinterendothelial junctions, macromolecules are retained in the circulation. Extravasation ofcirculating macromolecules can occur via vesicular transport or related mechanisms (Kohn et al.,1992). During inflammation, macromolecular leakage is increased. This may involve contractionof the postcapillary endothelial cells, creating gaps that macromolecules such as liposomes canpenetrate (Kohn et al., 1992).The various modes of transport across these classes of vessels are also illustrated in Figure 1.10.These include the direct diffusion of some molecules across the endothelial cell, lateralmembrane diffusion, movement through the interendothelial junctions (both tight and wide),across the endothelial fenestrae, and via vesicular mechanisms (Jam, 1987; Kohn et al., 1992). Tumor vasculatureIn general, the vascular lining of blood vessels within a tumor are hyperpermeable to circulatingmacromolecules. This is due, in large part, to larger defects in the endothelial layer such asfenestrations, widened interendothelial junctions, and in some cases blood channels that havelittle or no endothelial lining at all (Dvorak et al., 1988; Kohn et al., 1992). Most of the leakageis thought to occur across post-capillary venules and even large veins. Interestingly enough,38these vessels often have continuous endothelium with closed junctions (Dvorak et al., 1988;Kohn et a!., 1992). The mechanism of transport in these cases is increased vesicular structurescalled vesiculo-vacuolar organelles (VVOs) that may actually be more stable and long livedstructures than previously assumed.One of the mechanisms responsible for increased vascular permeability in tumors is due tosecretion by tumor cells of vascular permeability factor (VPF; Dvorak et al., 1991), also knownas vascular endothelial growth factor (VEGF). VPF is important in the development of growingtumors by allowing the leakage of various macromolecules and nutrients into the tumor tissue.The leakage of plasma clotting proteins into a newly formed tumor region due to increasedpermeability of nearby host blood vessels allows the formation of provisional stroma. This theninitiates the process of angiogenesis, the ingrowth of blood vessels necessary for continued solidtumor growth and development via perfusion and paracrine support (Blood and Zetter, 1990). Other solid tumor propertiesMost solid tumors appear to be more highly vascularized than the surrounding host tissue.However, despite the abundance of relatively large blood vessels, tumor blood flow is slowerthan normal tissues. This is due to the tortuous blood vessel pathways, including dead ends andcut off loops (Jam 1987; 1988). The general organization also consists of poorly definedartery/vein subsystems. Therefore, the amount of blood and associated circulating liposomesflowing through the tumor per unit time is very low.39A second barrier to macromolecular delivery is the high interstitial pressure (Jam, 1988). Innormal tissues, the pressure is 0 mm Hg due to the easy drainage of extravascular fluid via thelymphatic system. In solid tumor tissue, however, the lymphatic system is usually absent anddrainage is therefore very poor except at the periphery/host tissue interface. This results in veryhigh interstitial pressures, found in some mouse solid tumor models to be upwards of 10-30 mmHg.The actual physical basis for transport across the vascular barrier falls under two basicmechanisms (Jam, 1987). The transport of large macromolecules is governed by convection, theflow of fluid from vessel to interstitium, while small molecules are governed by diffusion,dependent upon concentration differences for that molecule for the blood versus interstitium.Interstitial pressures are often as high as the post-capillary venous pressure, where most of theavailable sites are for extravasation of large macromolecules. The minimal pressure differencesgive rise to minimal convection and therefore limited extravasation of macromolecules.1.6 ObjectivesOf central importance to liposomes as drug carriers is their circulation lifetime. This thesis isdivided into three areas of investigation. First, it is well established that the incorporation ofPEG-lipids into liposomes can significantly prolong their circulation lifetime. However, severalkey questions remain regarding important factors influencing their retention and stability in theliposomal carrier. Second, the effect that entrapped drug can have on clearance characteristicshave not been adequately addressed. This is investigated using sensitive assays to monitor thefunction of the RES. And third, the delivery of liposomal drug to a murine tumor model is40examined comparing a conventional liposomal drug carrier to one which has incorporated PEGlipid to optimize its circulation time.41CHAPTER 2THE USE OF PEG-LIPIDS TO IMPROVE TIlE CIRCULATION LIFETIMES OFLIPOSOMES2.1 IntroductionAs outlined in Chapter 1, the use of liposomes as systemic drug delivery vehicles requires longcirculation lifetimes. The incorporation of monomethoxy poly(ethylene glycol)phosphatidylethanolamine (MePEG-PE) conjugates into liposomal systems has been shown tosignificantly extend the circulation lifetimes of intravenously injected liposomes (Klibanov et al.,1990; Blume and Cevc, 1990; Senior et al., 1991b; Allen et al., 1991a; Allen and Hansen, 1991).However, the extent to which the PEG coating remains associated with the liposome afterinjection into the circulation has not yet been adequately addressed in the literature. Therefore,the studies outlined in this chapter investigate factors influencing the retention and chemicalstability of poly(ethylene glycol)-lipid conjugates incorporated into large unilamellar vesicles.The lipid moiety of the molecule must obviously be sufficiently lipophilic to firmly anchor thehydrophilic coat to the surface. In this regard, liposomally incorporated PEG-cholesterol or PEGmonostearate are relatively ineffective at improving the circulation lifetimes of intravenouslyinjected liposomes (Allen et al., 1991a). Presumably, the hydrophobic moiety in thesecompounds is an ineffective anchor and thus the hydrophilic coat is rapidly lost from injectedLUVs. Reports on the anchoring properties of diacyl phosphatidylethanolamine anchors havebeen conflicting. It has been suggested that the lipid moiety has little effect on the circulationcharacteristics of LUVs incorporating PEG-lipids (Woodle et al. 1992), while others indicate that42the lipid anchor is a important factor (Allen et al., 1991 a). In addition, the chemical stability ofMePEG-PEs in vivo has not received detailed attention.This chapter examines the influence of the lipid anchor and linkage chemistry on the ability ofMePEG-PE to improve circulation lifetimes of LUV systems. It is shown that the anchoringcapacity of PE anchors is extremely sensitive to the acyl chain composition, where distearoyl PEspecies are considerably more effective anchors than palmitoyl oleoyl species. Second,depending on the type of linkage between the PEG and the PE, breakdown can occur either onthe LUV surface or after release of PEG-PE from the LUV. These factors should be consideredwhen discussing the usefulness or mechanisms of PEG-PEs incorporated into liposomes.2.2 Materials and methods2.2.1 Monomethoxypoly(ethylene glycol)-lipid (MePEG-lipid) synthesisThe overall chemical structures of the of the various MePEG-lipids synthesized are shown inFigure 2.1, which include MePEG linked to phosphatidylethanolamine via succinate (MePEG-SPE), carbamate (MePEG-C-PE) and amide (MePEG-A-PE) linkages, and directly tophosphatidic acid (MePEG-PA). All PEG-lipids were isolated as a single component on TLC,with similar Rf values, and showed 1H NMR resonances characteristic of the MePEG and lipidgroups. MePEG2000-S-POPE and MePEG2000-S-DSPE were synthesized as follows.Monomethoxypoly(ethylene glycol) (MePEG2000-OH) (10 g) was treated in pyridine with tenequivalents of succinic anhydride (0.5 g) at room temperature for two days. The solution wasdiluted with water, acidified, extracted with methylene chloride, and the organic extracts weredried over magnesium sulfate, filtered, and the solvent removed. The resulting residue was43Figure 2.1Summary of PEG-lipid conjugate chemical structures(a), succinate linkage: MePEG-S-PE; (b), carbamate linkage: MePEG-C-PE; (c), amide linkage:MePEG-A-PE; (d), direct linkage: MePEG-PA. (see Materials and Methods for detaileddescriptions of the compounds synthesized)abcdsubjected to silica gel column chromatography in methylene chloride/methanol (96/4 v/v) andMePEG2000-succinate isolated. The dry MePEG2000-S(1.5 equivalents), DCC (1.2 equivalents),and NHS (1.6 equivalents) were dissolved in chloroform, stirred for an hour and filtered. Dry 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) or 1 ,2-distearoylphosphatidylethanolamine (DSPE) (1.0 equivalents) was dissolved in warm chloroform andadded to the filtrate, then 5 equivalents triethylamine was added and the reaction mixture stirredfor half an hour. Combined organic extracts were dried over anhydrous magnesium sulphate,filtered and the solvent removed under reduced pressure. The residue was subjected to silica gelcolumn chromatography. Fractions collected were analyzed by TLC using methanol/chloroform44as the solvent (15/85 v/v) and visualized by exposure to iodine. Fractions containing pureMePEG2000-S-POPE or MePEG2000-S-DSPE were combined, taken up into distilled water,centrifuged at 1500 x g for 30 mm. and the supernatants dialyzed against distilled waterovernight. The resultant solutions were lyophilized to yield a white powder.The synthesis ofMePEG2-[’4C]S- OPE was carried out as described for MePEG2000-S-POPEwith the exception that succinic anhydride- 1,4-[’4C1 was reacted first before addition of excesssuccinic anhydride (non-radioactive). The product was isolated by preparative TEC using twosuccessive plates run in methanollchloroform (15/85 v/v). The MePEG2-S-POPE componentwas extracted from the appropriate scraped bands with methanol and then methanol/water (1:1)to yield MePEG2o-[14CjS- OPE with specific activity 1.74 mCilmmol. The extract wasdispersed in water, centrifuged at 1500 x g for 30 minutes, frozen and lyophilized.The synthesis ofMePEG2000-S-[3HIDSPE first required making MePEG2000-S-DOPE similarly tothe procedure described for MePEG2000-S-POPE. The MePEG2000-S-DOPE and Pd-C were thenadded with methanol to a vial which was sealed and flushed with nitrogen. Sodium borotritide(490 mCi/mmol) was injected and the mixture stirred for an hour. Sodium borohydride wasadded and the solution stirred for three hours. After carefully opening in a fume hood, thesuspension was acidified with a drop of hydrochloric acid (10%), more methanol added, and thencentrifuged. The supernatant was filtered through celite, diluted with water and extracted withmethylene chloride. The organic fractions were dried over magnesium sulfate, filtered, and theMePEG2000-S-[3H]DSPE was purified by preparative TLC as described above (99% of theremaining radioactivity was located in the MePEG2-S-DSPE component, specific activity 140mCilmmol).45To synthesize[3H]MePEG20-S-DSPE, a sample of[3H]MePEG20-OH (Amersham, customsynthesis) was used as the starting material with the remainder of the synthetic procedurefollowed that of MePEG23-S-DSPE. Purification was carried out using preparative TLC platesas described above to yield[HjMePEG2000-S-DSPE (specific activity 44 mCilmmol).[3HjMePEG5000-S-POPE (specific activity 156 mCilmmol) was synthesized similarly toH]MePEG2000-S-DSPE.MePEG2000-S-(1 -palmitoyl-2-(4-pyrenyl)-butyroyl)PE was made by first synthesizingMePEG2000-S-(1 -palmitoyl-2-hydroxy)-phosphatidylethanolamine as the starting species usingthe procedure described for MePEG-S-POPE. A solution of 3 equiv. pyrenebutyric acid andDCC (1.5 equiv.) in alcohol free chloroform was allowed to stir at room temperature for 1 h,filtered and then 1.0 equiv. (100 mg) of the lyso-PEG-PE added to the reaction mixture with 10mg 4-dimethylaminopyridine. After work up, the product was isolated by columnchromatography and preparative thin layer chromatography as before to yield a pure fluorescentcompound, MePEG2000-S-(1 -palmitoyl-2-(4-pyrenyl)-butyroyl)PE (MePEG2000-S-PPBPE).MePEG2000-C-POPE was made according to the following procedure. Dry MePEG2000-OH (10 g)was dissolved in chloroform/toluene (50/2 v/v), reacted with 3 equiv. triphosgene and theproduct precipitated by addition of ether. The precipitate, MePEG20-ch1orofonnate(MePEG20-C-Cl), was filtered and dried under vacuum. The MePEG2000-C-Cl (1.0 g) and dryPOPE (1 equiv.) were dissolved in chloroform and treated with 5 equiv. triethylamine. Thesolvent was removed under vacuum and the residue dissolved in water. The aqueous solutionwas acidified and extracted with methylene chloride. The combined organic fractions were dried46over magnesium sulphate, filtered, solvent removed and the residue subjected to columnchromatography using silica gel and methanol/methylene chloride to yield pure MePEG2000-carbamate-POPE (MePEG2000-C-POPE). The product was dispersed in water, centrifuged at1500 x g for 30 mm. and the supernatant lyophilized to yield a white powder.MePEG2000-A-POPE and MePEG2000-A-DSPE were synthesized as follows. MePEG2000-aceticacid was synthesized by an adaption of the procedure in Sessler et al. (1992). Briefly,MePEG2-OH (10 g) was added to a solution of sodium dichromate (1.5 equiv.) in dilutesulfuric acid (10%) and the solution stirred at room temperature overnight. The solution wasextracted with methylene chloride and the combined organic extracts washed with sodiumhydroxide solution (1 M). The organic fraction was dried over magnesium sulphate, filtered andthe solvent removed under reduced pressure. The residue was dissolved in a minimum ofchloroform and precipitated with ether. The precipitate was filtered and dried, yieldingMePEG2000-acetic acid as a pale blue powder (colour due to complexed chromium). MePEG2000-A-POPE and MePEG2-A-DSPE were then prepared using the same procedure as the succinateanalogs, substituting MePEG2000-acetic acid for MePEG2000-succinate.To synthesize MePEG2000-POPA, a mixture of 1 -palmitoyl-2-oleoyl-phosphatidic acid (POPA)( 1equiv.), MePEG20-OH (1.0 g, 1.1 equiv.) and 2,6,6-triisopropylbenzenesulphorylchloride(TIPBSC)(3 equiv.) was suspended in dry pyridine. The reaction mixture was protected fromlight and allowed to stir overnight. Water was added and the mixture allowed to stir for a furtherthree hours. The solution was diluted with water, acidified and extracted with methylenechloride. After removal of the solvent, the residue was dispersed in water, filtered, centrifugedand the supernatant lyophilized. The resultant powder was subjected to column chromatography47using silica gel and methanollchlorofonn. Pure fractions were combined, taken up in water,centrifuged and lyophilized to yield a white powder.All lipids utilized in the above synthetic procedures were from Avanti Polar Lipids. Unlessindicated otherwise, all other materials were from Sigma. Column chromatography was carriedout using silica gel 60 (70-230 Mesh ASTM) (Merck). Analytical TLC employed aluminumbacked silica gel 60 - F254, 0.2 mm thick (Merck) and preparative TLC employed glass backedsilica gel 60, 0.5 mm thick (Merck).2.2.2 Preparation of large unilamellar vesicles (LUVs)The production of LUVs was carried out as previously described (Hope et al., 1985). Briefly,lipid mixtures composed of distearoylphosphatidylcholine (DSPC) and cholesterol (55:45mollmol), DSPC, cholesterol and MePEG-PE (50:45:5), or DSPC, cholesterol and GM1(45:45:10), each with trace amounts of[14C] or[3Hlcholesteryl hexadecylether (CHE) as a nonmetabolizable and non-exchangeable liposome marker (Derksen et al., 1987) were freeze-driedfrom benzene/methanol solution and hydrated in physiological sterile HEPES buffered saline(HBS) (20 mM HEPES, 150 mM NaC1, pH 7.4). The sample was freeze-thawed five times andthen extruded at 65°C ten times through two stacked 100 nm pore size polycarbonate filters(Costar/Nuclepore, Canada) employing an Extruder (Lipex Biomembranes, Canada). Liposomesize ranged from 95 to 115 nm as determined by quasi-elastic light scattering on a NICOMPModel 270 submicron particle sizer. The resultant LUVs were loaded onto a conventional BioGel A-15m (200-400 mesh)(Bio-Rad, Canada) 10 x 1 cm column equilibrated with HBS toremove unincorporated MePEG-PE, and the pooled liposome peak diluted with HBS to an48appropriate concentration. All initial liposome preparations were checked for concentration bydetermination of phosphorous (Fiske and Subbarow, 1925) using a Shimadzu UV-visiblerecording spectrophotometer at 815 nm, and thereafter by scintillation counting using a BeckmanLS3801 with Pico-Fluor 40 scintillation fluid (Packard). [‘4C] and [3H]CHE was fromNEN/DuPont, DSPC from Avanti Polar Lipids, and cholesterol and other chemicals were fromSigma.2.2.3 Exchange studiesThe MePEG-PE to liposome radiolabel ratios for 5 mM (total lipid) preparations incorporatingeither MePEG2000-[‘4C]S-POPE,[3HJMePEG2000-S-DSPE, or MePEG2000-S-[3HIDSPE weretaken to determine the starting ratio for the exchange studies. Then 500 jil of the liposomepreparation was diluted in 500 jil of either FiBS or normal mouse serum (Cedar Lane, Canada).For MePEG2000-S-POPE, an additional incubation involving 500 iil of liposomes with 200 .tl ofmouse serum and 300 il of BBS was carried out. These mixtures were incubated at 37°C and atvarious times, two 50 aliquots were removed and passed down 1 ml Bio-Gel A-15m spincolumns to separate liposomes in the void volume from serum and unincorporated MePEG-PEradiolabel. (The use of these spin columns to separate liposomes from serum components hasbeen previously described (Chomi et al., 1991)). The peak two liposome fractions from bothcolumns were counted and the MePEG-PE to liposome radiolabel ratios were determined by acorrected DPM dual label determination.492.2.4 Chemical stability studiesFive mg of the MePEG-lipid indicated was dissolved first in 200 of water, and then incubatedin 1000 jil normal mouse serum at 3 7°C. Additional incubations involving 500 tl of liposomes(20 mM total lipid) composed of DSPC/cholesterollMePEG-lipid in 500 serum at 37°C werealso carried out. At various times, aliquots from the micellar or liposomal incubations wereremoved and lipid components extracted by the following procedure. 80 jil of sample was addedto 920 jil of water. To this was added 2.1 ml of methanol and 1.0 ml of chloroform. Aftermixing, 1.0 ml of water was added, vortexed, and then an additional 2.0 ml of chloroform wasadded. After thorough vortexing, the sample was allowed to sit for 10 mm. before centrifuging at1500 x g for 1 hour. The organic layer was isolated, concentrated, and then spotted on 0.25 mmthick, silica gel 60, 5 x 10 cm TLC glass plates (Merck). The solvent system used to develop theplates was chloroform/methanol (85:15 vollvol), and were visualized with iodine vapor. Where[3H]MePEG5000-S-POPE was used, 0.5 cm sections of each running lane were scraped andextracted with 3 x 1.0 ml chloroform/methanollwater (50:40:10 vol/vol). The extract was placedin scintillation vials, solvent removed, 5.0 ml scintillation fluid added, and left overnight beforecounting. Where MePEG2000-S-P BPE was used, fluorescence associated with scraped sectionswas extracted as above, solvent removed, and resuspended in 4.0 ml of 0.5% (w/vol) sodiumcholate detergent. Fluorescence was read on a Perkin Elmer LS5O luminescence spectrometeroperating at 600 V using an excitation wavelength of 339 nm (2.5 mm slit width), emissionwavelength of 377 urn (2.5 mm slit width), and filter set at 350 nm. TLC standards used to aididentification included the appropriate free MePEG-Olls, extracted serum, free 4-(l-pyrenyl)butyric acid, and the appropriate MePEG-lipids.502.2.5 Biodistribution and circulation longevity studiesThe LUV preparations employing trace [H1 or[‘4C]CHE as liposome markers were injected vialateral tail vein in a volume of 200 .tl (1 ilmol total lipid) into 25 g CD-I mice (Charles River,Canada). At various times, the mice were sacrificed and blood withdrawn by cardiac punctureand collected in microtainer tubes with EDTA (Becton-Dickinson, Canada). After centrifuging at1500 x g for ten minutes, the plasma was isolated and showed no hemolysis. Two 100 .tlsamples from each mouse were counted directly in 5.0 ml scintillation fluid. The percentagerecovery of liposomes remaining in circulation was based on a plasma volume of 4.55% ofindividual mouse body weight. Liver and spleen tissue were homogenized by Polytron to 20%and 10% in saline, respectively. 200 jil of tissue homogenate was solubilized with 500 jilSolvable (NEN/DuPont) for 2 hours at 60°C, after which the samples were cooled and treatedovernight with 200 jil hydrogen peroxide. Five ml scintillation fluid was then added beforecounting. Liver and spleen associated liposomes are expressed as percent injected dose per tissue(total organ weight). Where in vivo exchange studies were carried out, two 50 il aliquots ofplasma were passed down spin columns as described above and the MePEG-lipid to liposomeratio determined.2.3 Results2.3.1 PEG20-S-POPE is lost from the liposome surface both in vivo and in vitroThe first series of experiments were designed to ascertain the influence of MePEG2000-S-POPEon the circulation lifetimes of 100 nm diameter DSPC/cholesterol (55:45 mol/mol) LUVs inmice. As shown in Figure 2.2, the incorporation of 5 mol % MePEG2-S-POPE results in a51Figure 2.2U)Cl)w50c1)Circulation lifetime of DSPC/cholesterollMePEG2000- -POP liposomesLarge unilamellar vesicles composed of DSPC/cholesterol (55:45) (0),DSPC/cholesteroIJMePEG2000- -POP (50:45:5) (•), (or DSPC/cholesteroJJMePEG2000- -DSP(50:45:5) (B), dashed line, taken from Figure 2.6A) were injected via the lateral tail vein into 25g CD-i mice (5 mM total lipid in 200 iil, 1 i.Lmol total lipid/mouse). At various times, the micewere sacrificed and plasma isolated. The marker[3HJCHE was used to determine liposomerecovery ([‘4CJCHE for (B)). Results shown represent the mean of four animals ± S.E. of themean.1008060402000 4 8 12 16 20 24time (hours)52relatively modest increase in LUV circulation lifetimes. At 24 h, the LUV preparationincorporating PEG-POPE is almost completely cleared. This may be contrasted with previousreports (Allen et a!., 1991a) that incorporation of 5 mol % PEG-PE can result in up to 30% ofDSPC/cholesterol LUVs remaining in the circulation at 24 h, and the third curve whereliposomes incorporating MePEG2000-S-DSPE have greater circulation levels (over 20% at 24 h).In order to determine whether this could be due to the loss of the PEG coating arising frominteractions with serum protein factors, the DSPC/cholesterol LUVs (labeled with[3F1]CFIE as anon-exchangeable liposome marker) incorporating MePEG2000-[14C]S- OPE were incubatedwith normal mouse serum at 37°C and the retention of radiolabel monitored. As shown in Figure2.3A, this incubation results in rapid loss of the MePEG-PE radiolabel. While the MePEG-PEcontent of the LUVs is relatively unaffected when incubated in BBS alone, the MePEG-PEradiolabel rapidly drops when incubated in the presence of either 20% or 50% serum. After 24 hin 50% serum, the MePEG-PE to liposome marker ratio has dropped to nearly 50% of its initialvalue, suggesting that practically all of the MePEG-PE radiolabel in the outer monolayer hasbeen removed. This is supported by the results of Figure 2.3B, which shows the spin columnprofile for the 24 h time point. There is an exact overlap of the MePEG-PE and liposomal peaks,and good separation from the MePEG-PE no longer associated with the liposomes. Integration ofthe two peaks yields a 52:48 ratio for liposome associated to free MePEG-PE. After an additional6 days at room temperature in the 20% incubation, this ratio was also nearly 50%. Given ourdata, it is not unreasonable to assume that little more than 50% of the label is available forexchange. In addition, studies with MePEG1900-carbamate-DSPE at 4 mol % indicate that thePEG extends outward from the surface in a brush formation approximately 5 nm (Needham eta!., 1992). Although direct evidence is lacking, it has been suggested that the distribution should53Figure 2.3 (following page)Loss of PEG coating from the surface of the LUVA, in vitro incubation of DSPC/cholesteroIJMePEG2oo-[14C]S-POP (50:45:5 mol/mol) largeunilamellar vesicles in normal mouse serum at 37°C. 500 il of the LUV preparation (5 mM totallipid) was incubated in the presence of 500 il HBS (•), 200 serum plus 300 Itl HBS (A), or500 .tl serum (s), representing 0%, 20%, and 50% serum concentrations, respectively. Atvarious times, two 50 aliquots of the incubation mixture were removed and liposomesseparated from the free PEG-PB and serum components by loading onto a 1 ml Bio-Gel A-15mspin column. The {‘4Cj (PEG-PB) to [RH] (CHE, liposome marker) ratios were obtained from thepeak two liposome fractions from both columns and expressed as a percentage (± S.D.) of theinitial ratio before incubation.B, Bio-Gel A-i Sm (200-400 mesh) spin column profile of DSPC/cholesterollMePEG2000-[’4]SPOPE LUVs in 50% serum at 37°C for 24 hours. (0), the [RH] DPM (liposome) label, and (D),the [‘4C] DPM (PEG-PE) label measured for 30 gl of each collected fraction.C, the in vivo loss of the PEG-PB coating from injected liposomes. The [‘4CJ (PEG-PE) to [3HJ(CHE) ratio was determined for liposomes recovered from mice injected with DSPC/chol/PEGPE liposomes both before (A) and after (y) separation of liposomes from plasma componentsvia spin column. Results represent the means of[14C] (PEG-PB) to [3H] (CUE) ratios obtainedfrom the four mice before, or after the peak two liposome fractions from two columns for eachmouse plasma sample, and expressed as a percentage (± S.D.) of the initial ratio before injection.54Figure 2.3Loss of PEG coating from the surface of the LTJV__________//—100 15B200908O8100E 0o i’-’ 0 5 10 15 20 25Cl) frction#0- 60-0Lu 50A 40I I I I I IT(-‘3Li-i- 100c-: 90080-70-60-50-0 4 8 12 16 20 24 168time (hours)55be approximately equal between the two leaflets of the bilayer as long as the radius of the vesicleis large (greater than a factor of 10) relative to the length of the polymer (Woodle and Lasic,1992) as is the case here.The in vivo exchange results presented in Figure 2.3C confirm that the MePEG-PE is lost fromthe surface of the injected LUVs. Here, plasma samples were taken at various times from micewhich had received[3H}CHE labeled liposomes incorporating MePEG2000-[’4C]S- OPE andwere counted to compare the MePEG-PE to liposome ratios both before and after separation ofthe liposomes from the plasma. In agreement with the in vitro data, this ratio drops significantlywith time indicating rapid loss of the MePEG-PE. The differences in the ratios for unseparatedand separated liposomes can be attributed to MePEG-PE which is no longer associated with theliposomes but which continues to circulate for a short period of time and to the short timerequired to isolate plasma and elute the spin columns. The level of MePEG-PE still associatedwith the liposome approaches 50% by 8 h, and by 24 h has completely leveled off. The fasterrate of loss of this component from the outer monolayer in vivo versus in vitro is expected giventhe greater plasma/serum to liposome ratio in vivo. It would also appear that the rate of loss ofMePEG-PE is faster than the clearance rate of the LUVs initially containing this component. Ifonly approximately 50% of the label is available for exchange, label loss leads clearance by asignificant amount. For example, at 1 hour post injection 76% of the vesicles remain in the blood(from Figure 2.2), whereas after serum removal, 75% of the total label remains. This indicatesthat 50% of the available exterior label has been lost and 24% of the vesicles have been cleared.At 4 hours, by the same argument, 75% of the available outer label has been removed while 45%of liposomes have been cleared. Thus, at these early time points outer label removal leadsclearance by 25 to 30%. It is also of interest to compare the clearance rate to the control. One can56see that when at least some proportion of the PEG coating remains, these liposomes have aslower clearance rate than the control. However, beyond some critical value of PEG loss between4 and 8 hours after which there is very little PEG remaining on the surface, the clearance of theseliposomes quickly approaches that of the control.2.3.2 Chemical stability of various linker groups in the MePEG-PE conjugateThere are two possible mechanisms for the loss of the MePEG-PE radiolabel from the LUVs invitro and in vivo. These are cleavage of the MePEG moiety from the lipid anchor or exchange ofthe entire MePEG-PE out of the LUV. Chemical breakdown was monitored by thin layerchromatography (TLC) after incubation of MePEG-PE micelles in normal mouse serum. Thedata of Figure 2.4 shows the effect of different chemical linkages between MePEG and the lipidanchor on the chemical stability of MePEG-PE. All four versions degrade significantly afterexposure to serum at 3 7°C. The succinate linked version rapidly generates a compound whichcorresponds to free MePEG, while the other three (carbamate, amide and direct linked) versionsshow very little of this product. The breakdown of the succinate ester linkage is perhaps notunexpected given a similar phenomenon observed in the corresponding MePEG-proteinconjugates (Zalipsky et al., 1992), however, this has not been previously reported for PEG-lipidconjugates. In addition, all four versions show the appearance of a product which is slightly morepolar than the starting MePEG-lipid, which in turn is broken down to yield a product with furtherincreased polarity by 24 h. When these four compounds were incorporated into liposomes,incubation in serum yielded the same patterns of breakdown products, but the rates at whichthese products are formed was significantly slower (results not shown).57Figure 2.4Thin layer chromatography of the results following incubation of micellar PEG-PE inserum at 37°C(a), MePEG20-S-POPE; (b), MePEG20-C-POPE; (c), MePEG2000-A-POPE; (d), MePEG2000-PUPA. 5.0 mg of the PEG-PE indicated was first dissolved in 200 jii of water, then incubatedwith an additional 1000 of normal mouse serum at 37°C. At various times, 80 jtl aliquots werewithdrawn, extracted, and concentrated. The samples were run on 0.25 mm silica platesdeveloped with chloroform/methanol (85:15 vol/vol) and spots visualized with iodine vapor.Lanes were: (1), before incubation; (2), 5 mm. incubation; (3), 1 h; (4), 4 h; (5), 8 h; (6), 24 h. (e)shows TLC standards normal mouse serum extract (7) and free MePEG2000-OH (8).11101 II•Oe ObOe 0I 4 oa!!,. ... . ..•. I....123456 123456 123456 123456 78a b c d e58Additional experiments were performed to characterize the breakdown products, utilizing[3HIMePEG5000-S-POPE and MePEG2000-S-(1 -palmitoyl-2-(4-pyrenyl)-butyroyl)PE(MePEG2000-S-PPBPE). These experiments confimed that free MePEG-OH was being generatedin addition to lyso-MePEG-lipid compounds. Furthermore, heat inactivation of serum (65°C for10 mm) or addition of 5 mM EGTA was found to reduce the rate of breakdown by approximately80%. All versions of the MePEG-lipid conjugates remain stable in HBS (pH 7.4) over 24 h,although the succinate version did show some slow breakdown (approximately 10 % over 24 h).Lower pH values (pH 2) resulted in the appearance of iyso compounds within several hours dueto acid catalyzed hydrolysis at the sn-i and sn-2 positions (Derksen et al., 1987).2.3.3 MePEG2000-DSPE is retained in DSPC/cholesterol LUVs and exhibits enhancedchemical stabilityThe chemical breakdown of MePEG-lipids can occur either on the surface of the liposome orafter exchange of the whole molecule out of the LUV. In order to reduce the possibility ofexchange, MePEG2-PEs were synthesized with a DSPE anchor which may be expected toresult in improved retention. The exchange of either[3HJMePEG2000-S-DSPE or MePEG2000-S-{3H]DSPE from [‘4C]CHE labeled LUVs in serum or HBS at 37°C is illustrated in Figure 2.5.The acyl chain label remains associated with the liposome in both HBS and in serum. However,the MePEG leaves the liposome to a measurable extent, resulting in approximately 7% loss inHBS over 24 h. In serum, the loss of this label is somewhat greater, up to approximately 12%loss over 24 h, although this rate is reduced compared to MePEG2000-S-POPE. Thus, the resultsof Figure 2.5 demonstrate that a primary factor for retention of the MePEG coating is the lipidanchor and that these compounds are relatively chemically stable if they remain associated with59Figure 2.5In vitro incubation in normal mouse serum at 37°C of DSPC/cholesterol large unilamellarvesicles incorporating 5 mol % MePEG2000-S-DSPE500 jil of the LUV preparations (5 mM total lipid) incorporating[3H]MePEG2000-S-DSPE(circles) or MePEG2000-S-[3H]DSPE (squares) was incubated in the presence of 500 il HBS(open symbols), or 500 il serum (closed symbols). At various times, two 50 jil aliquots of theincubation mixture were removed and liposomes separated from free components by loadingonto a 1 ml Bio-Gel A-15m spin column. The [I-Ij (PEG-PE) to [14CJ (CHE) ratios wereobtained from the peak two liposome fractions from both columns and expressed as a percentage(± S.D.) of the initial ratio before incubation.80-c0U)700060-50-_____________________I I I I I I0 4 8 12 16 20 24time (hours)60the liposome. However, it also appears that slow hydrolysis of the succinate bond can occur onthe liposome surface leaving the lipid anchor behind.The ability of MePEG20-S-DSPE, when incorporated into DSPC/cholesterol LUVs, to prolongthe circulation lifetime is significantly improved over that observed for MePEG2000-S-POPE.Using [‘4C]CHE labeled liposomes incorporating[3HJMePEG2000-S-DSPE or MePEG2000-S-[3H]DSPE, approximately 20% of the injected dose remains in the circulation at 24 h as shown inFigure 2.6, with the two preparations exhibiting very similar clearance behavior. When the [3H]to [14C) ratio was checked at various times both before and after separation of liposomes fromplasma components (Figure 2.6B and 2.6C), both the MePEG and acyl moiety labels of theMePEG2-S- DSPE are shown to remain associated with the liposome. It may, however, bemore accurate to say that the liposomes recovered, which are representative of those still incirculation, have retained most of their PEG coating. It is probable that liposomes which havelost their protective coating would have been rapidly cleared.2.3.4 Biodistributions of DSPC/cholesterol LUVs containing different species of MePEG2000-PEThe final series of experiments were performed to characterize the biodistribution at 24 h forDSPC/cholesterol LUVs incorporating various species of MePEG2000-P varying in acyl chaincomposition or PEG-PE chemistry at a liposome dose level of 1 .imol total lipid per mouse. Theresults are shown in Table 2.1. In the absence of a PEG-PE coating, DSPC/cholesterol LUVs arealmost completely removed from the circulation at 24 h (less than 1% of the injected doseremains), with high levels accumulated in the liver and spleen. The incorporation of 10 mol %GM1 significantly increases the circulation levels to approximately 11% of the injected dose61Figure 2.6 (following page)Circulation lifetime of DSPC/cholesteroUMePEG- -DSP liposomes and in vivoexchange ofMePEG2000-S-DSPE from injected liposomesA, circulation lifetime. Large unilamellar vesicles composed ofDSPC/cholesteroIJ[3HJMePEG20- -DSPE (50:45:5) (D) or DSPC/cholesterolIMePEG2-S-[H]DSPE (50:45:5) (•) were injected via lateral tail vein into 25 g CD-i mice (5 mM total lipidin 200 ti, 1 jimol total lipid/mouse). At various times, the mice were sacrificed and plasmaisolated. The marker[14H]CHE was used to determine liposome recovery. Results shownrepresent the mean of four animals ± S.E. of the mean.B, the [RH] to [14CJ ratio for plasma isolated from A before separation of liposomes from plasmacomponents via spin column. (D),[3HJMePEG2000-S-DSPE, and (s), MePEG2000-S-[3H]DSPEpreparations. Results represent the means of ratios obtained from the four mice and expressed asa percentage (± S.D.) of the initial ratio before injection.C, the ll] to [‘4C] ratio for plasma isolated from (a) after separation of liposomes from plasmacomponents via spin column. (D),[3HIMePEG2000-S-DSPE, and (•), MePEG2000-S-{3HjDSPEpreparations. Results represent the means of ratios of the peak two liposome fractions from twospin columns from four mice, and expressed as a percentage (± S.D.) of the initial ratio beforeinjection.62Figure 2.6Circulation lifetime of DSPC/cholesterolJMePEG2000- -DSP liposomes and in vivoexchange ofMePEG-S-DSPE from injected liposomes100 -80-a)0VVa) 60-c)r 40-20-100 -190 -080-a)E 70-0U)o 60-.9-Bo 50-LU0..0LU___________________a- 100H90-.91o 80-70 -60 -C50 -0 4 8 12 16 20 24time (hours)63Table 2.1Biodistribution of DSPC/cholesterol large unilamellar vesicles incorporating GMI or PEG.PE one day after i.v. injectionThe 5 mM 100 mn LUV preparations were injected via lateral tail vein in a volume of 200 tl (1.tmol total lipid) into 25 g CD-i mice. At 24 h, the mice were sacrificed and plasma, liver, andspleen isolated. The percentage recovery of liposomes remaining in circulation was based on aplasma volume of 4.5 5% of individual mouse body weight. Liposomes associated with liver andsleen tissues were determined based on total organ weight. Each preparation employed trace[H]CHE as a liposome marker, and the results represent the mean of four animals ± S.E. of themean.Liposome composition (molar ratio) % of injected dose recovered per total tissueblood liver spleenDSPC/chol (55:45) 0.20 ± 0.10 56.4 ± 3.3 7.76 ± 1.50DSPC/choIIGMl (45:45:10) 11.3 ± 0.5 25.4 ± 0.4 1.90 ± 0.09DSPC/choVMePEG2000- -POP (50:45:5) 3.31 ± 1.15 42.2 ± 2.8 3.85 ± 0.45DSPC/chol!MePEG2000-A-POP (50:45:5) 2.57 ± 0.92 42.6 ± 2.8 3.49 ± 0.49DSPC/choL’MePEG2000- -DSP (50:45:5) 16.3 ± 1.1 24.9 ± 2.6 1.60 ± 0.10DSPC/chol/MePEG2000-A-DSP (50:45:5) 18.0 ± 0.9 18.8 ± 1.4 1.41 * 0.07remaining while decreasing the amount found in the liver and spleen at 24 h by factors of 2 and 4respectively. Incorporation of 5 mol % MePEG2000-S-POPE and MePEG2000-A-POPE havesmaller effects than GMI in altering the LUV biodistribution. However, the presence of eitherMePEG2-S-DSPE or MePEG2-A-DSPE greatly increased the circulation levels present at24 h to almost 20%, higher than achieved with GMI, while the accumulation by the liver andspleen is reduced to an equal or better extent as GM1. While there is little difference inbiodistribution behavior between the succinate and amide versions for MePEG-POPE, the datafor DSPE anchored species suggest that the amide linkage may be slightly superior in bothimproved circulation lifetimes and reduced liver and spleen uptake.642.4 DiscussionThe use of liposomes as systemic drug delivery vehicles depends upon their ability to remain incirculation for extended periods of time. The incorporation of PEG-lipids clearly allowsextended circulation lifetimes to be achieved. However, the results presented here emphasize twomajor points. First, relatively subtle changes in the acyl chain composition of the PE anchor cansignificantly influence retention of the PEG-PE in the outer monolayer of the liposome. Second,significant chemical breakdown of PEG-PE conjugates may occur, particularly after the PEG-PEis lost from the LUV surface.The influence of acyl chain composition on PEG-PE retention and related clearance behavior isparticularly profound. As shown here, when MePEG2000-S-POPE is incorporated intoDSPC/cholesterol LUVs, the circulation lifetime is only modestly increased. This increase is lessthan that reported for MePEG2-DSPE (Allen et al., 1991a; Woodle et al., 1992), butcomparable to other studies using MePEG2000-DOPE (Mon et al., 1991). The poor performanceof PEG-POPE is due to rapid removal of the exterior PEG coating, with a half-time ofapproximately two hours in vitro (50% mouse serum at 3 7°C) and approximately one hour invivo. This may be compared with the rate of clearance of the injected LUVs, which exhibit ahalf-life in the circulation of approximately five hours. The fact that loss of the hydrophiliccoating precedes liposome clearance suggests that loss of the PEG coating hastens clearance. Asshown here, the loss of the PEG-POPE coating is primarily due to exchange of the entire PEGPOPE molecule out of the external monolayer.65The use of DSPE as the lipid anchor in place of POPE results in a dramatic improvement on theretention of the PEG coating. When MePEG2-S-DSPE is incorporated into LUVs andincubated in 50% mouse serum, approximately 90% of the PEG-PE remains associated with theLUVs after 24 h. The DSPE anchor also exhibits markedly superior properties in vivo. Thecirculation half-life of LUVs incorporating MePEG2000-S-DSPE is approximately 10 h, with over20% of the injected dose remaining in circulation at 24 h. In addition, the LUVs recovered fromthe circulation even up to 24 h show no exchange or breakdown of the MePEG20-S-DSPE,although any liposomes which have lost their PEG coating would likely have been cleared fromthe circulation.Previous work (Silvius and Zuckermann, 1993) examining the intervesicular exchangeability ofseveral PEG-lipids in vitro showed that transfer of saturated diacyl conjugates of MePEG2000decreased exponentially with increasing chain length. In addition, transfer of POPE derivativesof MePEG20 and MePEG5000 was found to be 30 to 40 fold slower than the correspondingDMPE derivatives. Thus, the increase in LUV retention in vitro and in vivo between POPE andDSPE anchors observed here is not unreasonable.The chemical stability studies on pure (micellar) PEG-lipids indicate that the succinate linkage islabile in mouse serum, generating free MePEG-OH by one hour with complete hydrolysis of thislinkage within 24 hours. A variety of other linkages proved to be more stable in this respect,including carbamate, amide, and direct linkages. The results presented here also indicate that thesuccinate bond is protected by retention of the PEG-lipid in the LUV. For MePEG2000-S-DSPE, aslow loss of the PEG headgroup as compared to the lipid anchor (which is completely retained)indicates that it is possible to remove the PEG from the LUV surface and leave the lipid anchor66Figure 2.7Models for PEG-PE exchange and breakdownA, intact PEG-PE remaining on liposome surface providing steric stabilization; B, exchange ofthe entire molecule from the membrane as in PEG-POPE; C, breakdown of chemical linkage(succinate) after exchange, or; D possible breakdown of chemical linkage (succinate) on surfaceof liposome membrane when PEG-PE is well anchored.A B C Dbehind, however, this effect is small compared to loss of the POPE anchored version which isalmost completely removed from the outer monolayer within several hours.The rationale for the use of PEG-lipids is their ability to significantly reduce the rate of clearanceof liposomes from the circulation. At a practical level, the results presented here demonstrate that67chemical stability and lipid anchoring ability are determining factors for the ability of PEG-lipidsto provide improved circulation lifetimes for LUVs in vivo. While it has been reported that thenature of the anchor of PEG-lipids influences the circulation lifetimes of injected liposomes(Allen et al., 1991 a) and that the LUV lipid composition can also affect the circulation lifetimesof liposomes incorporating PEG-PEs (Maruyama et al., 1992; Litzinger and Huang, 1992), otherssuggest that different PEG anchors are equivalent and that the lipid composition may be variedwith little effect if PEG-PEs are incorporated (Woodle et al., 1992). Our results conclusivelydemonstrate the importance of the PEG-lipid anchor.In summary, the lipid anchor is a primary factor in the retention of a PEG polymeric coating forLUVs. Chemical breakdown of the conjugate largely occurs after exchange out of the LUV butcan occur on the LUV surface depending upon the PEG-lipid linkage. The use of a strongmembrane anchor (DSPE) and chemically stable conjugate bond (amide) results in retention ofthe polymeric coating and greatly enhanced circulation lifetimes. It is concluded that since themajor rationalization for the use of PEG-lipids in liposomes is the prolonged circulation lifetimesand hence the greater chance of accumulation in targets other than the RES, by whatever detailedmechanisms PEG-PE is proposed to work, this requires that the PEG coating is retained andshould be a consideration in any practical discussion of the use of PEG-lipids.68CHAPTER 3CHARACTERIZATION OF RES BLOCKADE WITH DOXORUBICIN ANDVINCRISTINE3.1 IntroductionThe studies in this chapter are based on previous work from this laboratory concerning thecharacterization and biodistribution of doxorubicin-loaded liposomes (Bally et al., 1990a). It wasshown that the RES function was strongly influenced by liposome containing doxorubicin assuch liposome were cleared from the circulation at a slower rate than empty liposomes. Further,it was found that pre-dosing with liposomes containing doxorubicin resulted in greatly extendedcirculation lifetimes of a subsequent injection of empty liposomes. These results were attributedto an ability of doxorubicin-loaded liposomes to impair or “blockade” RES function. RES“blockade” provides an alternative means to achieve long circulation lifetimes and does notnecessarily require the use of lipids such as those described in Chapter 2.The experiments performed here are divided into two sections. It has been suggested that stealthor sterically stabilized liposomes exhibit an ability to avoid uptake by the phagocytic cells of thereticuloeridothelial system (RES) found predominantly in the liver and spleen (Allen and Chonn,1987; Klibanov et al., 1990; Lasic et al., 1991; Oku et al., 1992). In the first part of this study thevalidity of this hypothesis is examined. Specifically, the influence of GM1 on the ability of a predose of liposomes containing doxorubicin to blockade RES function, as expressed by theextended circulation lifetimes exhibited by a subsequent injection of empty liposomes, isexamined. One of two possible results would be expected. If GM1-containing liposomes with69entrapped doxorubicin do in fact avoid the RES, the liver and spleen function should not beaffected and the subsequent injection of empty liposome should be cleared normally. On theother hand, if GM1-containing liposomes do not avoid the RES, one would expect to see animpaired ability of the RES to clear liposomes and thus extended circulation lifetimes forsubsequent injections.The second component to the studies described in this chapter concern potential adverse sideeffects due to the use of procedures involving RES blockade. Because the RES is the primarysite for foreign particulate clearance, it has long been recognized that many potential harmfuleffects may result following the delivery of cytotoxic liposomal agents. The liver macrophagesin particular play a key role in host defense mechanisms (Phillips, 1989, Toth and Thomas,1992). The effect that entrapped doxorubicin has on the RES is further examined here.Preliminary studies indicated that liposomally delivered doxorubicin had a maximum effect at 24h after injection, and that very little drug was needed to achieve optimal blockade. These studieswere extended to more fully characterize the RES blockade technique, including the amount ofliposomally delivered doxorubicin necessary to achieve blockade as well as the duration of theeffect. In addition a different anticancer agent, vincristine, was also studied in terms of inducingRES blockade. This agent was selected as an appropriate alternative since, unlike doxorubicinwhich is a cytotoxic drug, vincristine is a cytostatic agent known to act by inhibiting formationof microtubules required for formation of the spindle apparatus in dividing cells (Owellen et al.,1972, 1976). It is anticipated that this cell-cycle specific agent will not directly affect theviability of mature macrophages such as Kupffer cells. Vincristine, will, however, affect thefunction of these cells by inhibition of cellular filament formation required for membranetrafficking and cellular rearrangement as seen in phagocytic processes. It is shown that either70liposomal drug can induce RES blockade even at very low doses, although the duration isconsiderably shorter for vincristine. These findings are discussed in terms of the differentpotential modes of action of these two drugs.3.2 Materials and methods3.2.1 Liposome preparationLiposomes were prepared as described in Chapter 2. Briefly, lipid mixtures in chloroform weredried to a film under a stream of nitrogen gas, then further dried under high vacuum for aminimum of 4 h. For the pre-dose composition, the lipid was hydrated with 300 mM citric acid(pH 4.0), frozen and thawed five times, and then extruded at 65°C ten times through threestacked polycarbonate filters (Nuclepore, Canada) of 100 nm pore size employing and extrusiondevice (Lipex Biomembranes, Canada). An approximate. 100 nm mean diameter for the resultantLUVs was determined employing a NICOMP 370 particle sizer. A transmembrane pH gradientwas established by passing the LUV preparations down a Sephadex G-50 column equilibratedwith 150 mM sodium carbonate buffer (pH 7.5) and collecting the LUVs in the void volume.For the empty LUVs employed for the subsequent injection, the lipid film was hydrated in FIBS(20 mM HEPES, 150 mM NaC1, pH 7.4) and extruded as described above.3.2.2 Drug loadingDoxorubicin (Adria Laboratories) and vincristine (Lyphomed Canada) were loaded into LUVs asdescribed previously (Mayer et al., 1986; Mayer et al., 1 990a; 1 990b). Aliquots of preheated71drug in saline were added to pre-heated liposomes at 65°C to achieve the indicated drug/lipidratios (mol/mol) and incubated at this temperature for a further 10 mm. Entrapment efficiencieswere in excess of 95%.3.2.3 Animal biodistribution studiesFemale BDF- 1 (for the first series of experiments) and CD-i (for the second) mice (20-23 g,Charles River, Canada) were injected with the specified dose of empty and drug-loadedliposomes via the lateral tail vein. Pre-doses consisted of 100 nm diameter LUVs composed ofDSPC/cholesterol (55:45, mol/mol), DSPC/cholesterollGMl (45:45:10), orDSPC/cholesterolIMePEG2-A-DSP (50:45:5) with or without entrapped doxorubicin orvincristine. These LUVs were injected at a dose of 0.33 iimol lipid per mouse (10 mg/kg lipiddose for DSPC/cholesterol) delivered in a volume of 200 jil. A trace amount of[3H]cholesterylhexadecylether (NEN, Canada) was used as a non-exchangeable lipid marker (Derksen et a!.,1987) for detennining the biodistribution of this pre-dose at 24 h. Blood was collected by heartpuncture and placed in EDTA-treated microtainers (Becton-Dickinson, Canada). Plasma wasprepared by centrifuging (200 x g) the blood samples for 10 mm. followed by liquid scintillationcounting of 200 tl samples to determine radioactivity. Liver and spleen were removed wholefrom the animal carcass and weighed. 20% or 10% homogenates in water were prepared for liverand spleen, respectively. 200 jil of this was then digested with 500 il Solvable (NEN/DuPont)for 1 h at 60°C, cooled, bleached with 200 tl H20 (30%), and tissue-associated radioactivitydetermined by liquid scintillation counting. For the biodistributions of the subsequent injection,the pre-doses described above (but not containing any radiolabeled lipid marker) were given, andthen a subsequent injection of empty liposomes was administered 24 h later. This later injection72was composed of 100 nm diameter DSPC/cholesterol (55:45) LUVs with a trace amount of[3lljcholesteryl hexadecylether, injected at a dose of 3.3 jimol lipid per mouse (100 mg/kg lipiddose) delivered in 200 .tl. One control group of mice received no pre-dose. The mice weresacrificed 24 h later and the liposome biodistribution in blood, liver, and spleen was determined.Biodistribution results were analyzed using a two-tailed Student’s t-test.DSPC was obtained from Avanti Polar Lipids, and cholesterol, GMI, and all other chemicalswere obtained from Sigma. MePEG2-A-DSPE was synthesized as described in Chapter Liver histologyGroups of 4 CD-i mice were injected with pre-doses (0.33 imol lipid per mouse) consisting ofDSPC/cholesterol LUVs with entrapped doxorubicin (0.2 mol:mol drug:lipid ratio) or vincristine(0.05). Four days later the mice were sacrificed and liver removed. Cryostat sections wereprepared by washing the excised liver lobes in PBS at 4°C and then fixed in 3%parafomaldehyde in PBS. They were then washed passed through graded concentrations ofsucrose, and embedded in OCT and frozen in liquid nitrogen. Sectioning (5 urn thick) wascarried out at -20°C using a Frigocut 2800N Reichert-Jung Leica microtome. These sectionswere then washed in PBS, stained with Carazzis hematoxylin, washed with water, 1.5%NaHCO3,and water again, and then mounted for viewing. Sections were scored for Kupffer cellsover 15 randomly selected fields (40x, Leitz Dialux microscope) over a range of liver sectionsfor each treatment group. Photomicrographs were obtained using a Orthomat microscopecamera. All images were recorded on Fuji color ASA400 negative film.73Liver uptake of colloidal carbon (du Souich et a!., 1981) was also carried out. A commerciallyavailable India Ink (Koh-I-Noor)(80 mg/mi) was diluted 20 fold and 200 il injected i.v. 24 hafter the injection of pre-doses described for the histology experiment above. Four hours after theinjection of colloidal carbon the mice were sacrificed and liver cryostat sections were preparedas described above.3.3 Results3.3.1 The presence of GM1 in liposomes with entrapped doxorubicin does not prevent RESblockadeThe biodistribution of the pre-dose at 24 h after injection is shown in Figure 3.1. In the blood, theliposomes which did not contain GMI are present in very low levels (<1% of injected dose)whereas those containing GM1 are present at levels corresponding to approximately 25% of theinjected dose at 24 h. The difference is largely accounted for by reduced liver and (to a smallerextent) spleen uptake for the GM1 containing formulation. Both DSPC/choiesterol liposomes andGM1-containing liposomes exhibit significantly reduced liver uptake when the liposomes containentrapped doxonibicin (p<O.O5 for both groups). This results in greatly increased uptake in thespleen for the DSPC/cholesterol LUVs which did not contain GMI. However this effect is notseen for Gl-containing liposomes where the two spleen panels are not significantly different (p> 0.05). The biodistribution 24 h after the subsequent injection of empty DSPC/cholesterolLUVs is shown in Figure 3.2. The biodistribution observed in a group of mice which received nopre-dose is shown in the left bars. This pattern of blood clearance and liver and spleen uptake istaken as a primary control. Pre-doses of liposomes which did not contain drug results in nosignificant difference in the uptake into the liver and spleen from their respective controls (pvalues all> 0.05) and indicates that pre-injection of a low dose of lipid alone does not alter the74Figure 3.1Biodistribution of the pre-dose containing liposomal doxorubicinLarge unilamellar vesicles of various compositions were injected via lateral tail vein at a dose of0.33 tmol lipid/mouse. At 24 h, the mice were sacrificed and tissue samples indicated weremeasured for liposomal lipid levels. Where employed, doxorubicin was entrapped at a drug:lipidratio of 0.2 (mol:mol). Values shown represent the mean of results from 8 animals ± S.E. of themean.+n(I fr00 o oQ-DQ_ a.U) U)- plasma. ±-+--HE- liver100806040200120Cu2 1008060402006050403020100-- HE— spleen-n075Figure 3.2Biodistribution of the subsequent injection of empty liposomesAt 24 h after the injection of the pre-doses indicated at bottom (as in Figure 3.1), LUVscomposed of empty DSPC/cholesterol were injected at a dose of 3.3 tmol lipid per mouse.Twenty four hours after this the mice were sacrificed and lipid levels of this subsequent injectionin the tissues determined. Values represent the mean of 8 animals ± S.E. of the mean.14001200-100080060040020001400120001000800600400020005004003002001000- ÷ plasma+n----- spleen. +flI). .C.C .C0 c.CO tLCl, Cl)0cC50 0C)3Z50Cl) Cl)0076pattern of uptake into these tissues. A slight increase in blood levels of the subsequent injectionfor the pre-dose containing empty GM1 is liposomes is observed. However, pre-injection of drug-loaded liposomes which did not contain GM1 substantially blocks liver uptake of the laterinjection of empty liposomes, resulting in elevated blood levels and spleen uptake. Pre-doses ofdoxorubicin-loadedG1-containing LUVs also results in dramatic blockade of liver uptake aswell as elevated spleen uptake. The reduction of liver uptake is slightly less than that observed inthe absence of GM1. Interestingly, there is no significant difference for blood levels of GM1-containing LUVs. It is important to note that these results represent the effect of liposomallyentrapped doxorubicin; the administration of free doxorubicin prior to injection of emptyliposomes has been shown not to alter the liposome clearance (Bally et al., 1990a).These studies were extended to determine the minimum dose of doxorubicin required in bothcontrol and GM1-containing liposome to induce significant RES blockade. These studies wereperformed by injection of the same lipid pre-doses which contained varying amounts ofdoxorubicin, and then determining the biodistribution of a subsequent injection of emptyliposomes. The drug/lipid ratios were 0.00, 0.02, 0.05, 0.10, 0.20, and 0.30 (mollmol). The meanvalues obtained for mice which received no pre-dose are indicated by the dashed lines. In boththe blood and liver, the effect of entrapped doxorubicin in liposomes without GM1 on thebiodistribution of the subsequent injection are readily apparent even at very low drug levels(drug/lipid ratios <0.02 mol/mol), whereas for theG1-containing pre-dose, higher drug doses(drug/lipid ratios of 0.10 mol/mol) are required to induce the same effect. It should be noted thatthese dose levels are very small in comparison to the dose required to result in therapeuticbenefit. A drug/lipid ratio of 20 would be required to achieve the maximum tolerateddoxorubicin dose of 20 mg/kg (Mayer et al., 1990b; Bally et al., 1990b). for example. Thus at77Figure 3.3Dose titration of entrapped doxorubicin in the pre-dose: biodistribution of the subsequentinjectionPre-doses with entrapped doxorubicin at drug:lipid ratios indicated were injected at a dose of0.33 imol lipid per mouse and then at 24 h, a 3.3 j.tmol lipid per mouse injection of DSPC/cholliposomes was given. 24 h after this lipid levels of the subsequent injection were determined.Pre-dose compositions were (D), DSPC/chol, and (B)DSPC/cholJGMl, with entrappeddoxorubicin as indicated by the drug:lipid ratio. Dashed line indicates results from mice whichreceived no pre-dose. Values shown represent the mean of results from 4 animals ± S.E. of themean.I_______________200- plasma1400 liver1200Ei000-’800-600-400-200-0- I I0.00 0.05 0.10 0.15 0.20 0.25 0.30drug to lipid ratio (mol:mol) of pre-dose78any reasonable dose of doxorubicin in GMI-containing liposomes, strong RES blockade would beexpected. In addition to the ability of GMI-containing LUVs with entrapped doxorubicin toblockade liver uptake, a further point of interest concerns the different uptake behavior of theliver and spleen. An ability to dramatically block liver uptake by a small pre-dose of liposomallyentrapped doxorubicin is consistent with specific uptake of liposomes by Kupifer cells (Roerdinket a!., 1981; Senior, 1987) and argues against any non-specific mechanism. Similarly, theincreases in spleen uptake seen as a result of liver blockade imply that liposome uptake in thespleen by fixed macrophages plays a relatively minor role in liposome clearance. Rather, theseresults suggest a non-specific filter model (Liu et al., 1991; 1992) where the spleen accumulatesliposomes not cleared by the liver.3.3.2 Characterization of RES blockade with entrapped doxorubicin and vincristineFigure 3.4 shows a dose titration of liposomally entrapped drug in the pre-dose and the clearanceof the subsequent test injection. The results for doxorubicin are similar to that from the previoussection (Figure 3.3) where a maximum liver depression is observed at drug to lipid ratios ofapproximately 0.05 (nominally 0.5 mg/kg DOX). As indicated in Section 3.3.1, there is analmost 5 fold depression in liposomal lipid uptake in livers of these pre-treated animals. Theabsolute levels, however, differ between the BDF- 1 mice used previously and the CD-i miceused here.Dose titration of entrapped vincristine at this time point (24 h) shows similar dose titrationeffects as entrapped doxorubicin. Following administration of a second dose of liposomes (100mg/kg) one observes increases in plasma liposomal lipid levels and decreases in liver levels.79Figure 3.4Dose titration of entrapped doxorubicin or vincristine in the pre-dose: biodistribution ofthe subsequent injectionPre-doses consisted of 0.33 j.tmol lipid per mouse (10 mg/kg) DSPC/chol with entrapped drug atvarious drug:Iipid ratios indicated at bottom. 24 h later the subsequent injection (emptyDSPC/chol, 3.3 tmol lipid per mouse; 100 mg/kg) was given, and then the biodistribution of thesubsequent injection 24 h after this was then determined. (top plasma, bottom liver). The dashedline represents results from mice that received no pre-dose. Pre-dose compositions containedliposomally entrapped (•), VINC or () DOX. Values shown represent the mean of results from4 animals ± S.E. of the mean.a)Cl)Cl)I1. 0.05 0.10 0.15 0.20 0.25 0.30drug:Iipid ratio80Maximum effects are achieved when the drug to lipid ratio is greater than 0.025. The depressionin liver uptake levels off past the 0.02 drug:lipid ratio and only represents a 2-3 fold decrease.Interestingly, a higher elevation of plasma levels at lower drug doses is seen with liposomalvincristine.A time course for RES recovery is shown in Figure 3.5, in which the timing between the predoses and subsequent injection is varied. For this experiment, the drug to lipid ratiocorresponding to the maximum effect from the dose titration data was used (0.2 mol:mol fordoxorubicin and 0.05 mol:mol for vincristine). When the pre-dose and subsequent injection weregiven simultaneously (day 0), this dose of doxorubicin had little effect on the clearance of thesubsequent injection. However for vincristine, there was an immediate liver depression andgreatly increased plasma levels. By one day, the doxorubicin pre-dose produced maximumdepression of liposomal lipid uptake in liver (now under the same conditions as that for Figure3.4). Liposomal vincristine pre-injection also resulted in a suppression that was comparable tothat shown in Figure 3.4 on day 1. For vincristine treated animals, normal liver uptake wasrestored by day 2, whereas doxorubicin treated animals did not recover until approximately day 8to 14.Additional data shown in this figure for the pre-dose compositions includes a pre-dose consistingof DSPC/cholesterolIMePEG2000-A-DSP liposomes with entrapped doxorubicin (0.2 drug:lipidratio), analogous to the GM1 liposomes used as a pre-dose composition in the previous section.The timing of recovery of clearance behavior closely parallels that for the conventionalliposomal doxorubicin treated animals, although the effect is attenuated overall. There is similarplasma elevation throughout the time course (p> 0.05), and the liver is also similar, although81Figure 3.5Time course of recovery of RES blockade achieved following i.v. administration ofliposome entrapped doxorubicin or vincristinePre-doses consisted of 0.33 tmo1 lipid per mouse (10 mg/kg) DSPC/chol with entrapped drug ata drug:lipid ratio of 0.05 for VJNC and 0.2 for DOX. At various times later, as indicated by thebottom scale, the subsequent injection was given (empty DSPC/chol, 3.3 j.tmol lipid per mouse;100 mg/kg), and then the biodistribution of this subsequent injection 24 h after this was thendetermined. (top plasma, bottom liver). The dashed line represents results from mice thatreceived no pre-dose. Pre-doses contained liposomally entrapped (•), V1NC or () DOX asdescribed above. The results for doxorubicin entrapped within a DSPC/cholfPEG pre-dose (0.2mol:mol drug to lipid ratio are also shown (A). Values shown represent the mean of results from4 animals ± S.E. of the mean.C)Cl)C’,ctsI1. 2 4 6 8 10 12 14time (days)82significantly reduced in magnitude (p <0.05 at 1, 4 and 14 days compared to the conventionalliposomal DOX liver blockade) and possibly recovery period.3.3.3 Liver histology after RES blockadeLiver cryostat sections taken 4 days after administration of liposomal doxorubicin or vincristine(Figure 3.6) were scored for Kupffer cells and, surprisingly, revealed no differences in thenumber of Kupffer cells identified via morphological and staining characteristics betweencontrol and drug treated animals. A second approach to assess RES status involved carbonclearance. No quantitative differences in the number of cells shown to accumulate colloidalcarbon one day after the pre-dose treatments were found (Figure 3.7). Qualitative differencesmay, however, exist. Control liver sections, for example, revealed a close association betweenthe colloidal carbon and Kupffer cells (panel A), whereas in the liposomal doxorubicin treatedanimals the carbon appeared to be somewhat more associated with hepatocytes (panel B). For thevincristine treated animals, carbon distribution appeared both qualitatively and quantitativelysimilar to the controls (panel C).3.4 DiscussionLonger circulation lifetimes are observed for doxorubicin loaded liposomes in general (Bally etal., 1 990a). In order to better elucidate the mechanism behind this phenomenon, experimentswere performed here in which pre-doses of various amount of liposomal doxorubicin could begiven and their effect on the RES determined by testing with a subsequent test injection of emptyliposomes. In the first part of this chapter it was established that pre-doses without entrapped83Figure 3.6Cryostat sections of livers obtained from normal, liposomal doxorubicin treated, andliposomal vincristine treated animalsFour days after a pre-dose of liposomal drug, the mice were sacrificed and liver sections fixed,frozen and stained with hematoxylin. (A) normal liver, (B) liposomal DOX treated and (C)liposomal VINC treated animal livers sections. Only those sinusoidal cells which were large,irregular (length to width ratio of 2 or greater), and darkly staining were scored as Kupffer cells.84Figure 3.7Cryostat sections of normal liver, liposomal BOX treated and liposomal VINC treatedanimals after injection of colloidal carbon24 h after a pre-dose of liposomal drug, 40 mg/kg colloidal carbon was injected and then 4 hafter this the mice were sacrificed and liver sections fixed, frozen and stained with hematoxylin.Colloidal carbon appears as sharply defined, aggregated black particles. Liver sections werefrom (A) normal, (B) liposomal DOX treated, and (C) liposomal VINC treated animals:485drug had no effect on RES uptake of a subsequent injection and thus allowed us to isolate theeffect of entrapped drug. Pre-doses containing doxorubicin were found to greatly impair theRES, and even when the pre-dose contained GM! RES blockade was readily apparent. In thesecond part of the chapter, additional data suggested that vincristine, another liposomal drugunder current development, could also induce RES blockade, although a time course suggestedthat the effect was more transient. Animals pre-dosed with liposomal vincristine recoveredwithin several days whereas those treated with liposomal doxorubicin took over 8 days torecover to normal liver liposome clearance behavior. Additional experiments with PEG-PEcontaining liposomes confirmed that such longer circulation lifetime formulations do not strictlyavoid the RES as evidenced by the similar albeit attenuated RES blockade.Based on the time course data it was originally assumed that even at the extremely low levels ofliposomal doxorubicin being employed, the liver macrophages were being killed. The 8-14 dayrecovery period is consistent with the turnover times for the Kupifer cell population (Crofton etal., 1975). Modes of action for doxorubicin include DNA intercalation and topoisomerase IIinterference, free radical formation, covalent binding to DNA, and various cell binding effects(Cummings et al., 1991). While most effective against rapidly dividing cells such as tumor cells,non dividing cells including Kupffer cells are also killed by doxorubicin as a result of some or allof the above modes of action (Barranco, 1984).Vincristine on the other hand is more specific in its mode of action in that it binds to the growingend of microtubules and prevents their assembly (Owellen et al., 1972, 1976). Given thatrecovery of the capacity of the liver to accumulate liposomes was rapid (within 1-2 days), itseems reasonable that vincristine inhibits phagocytosis only for the time that the drug is present86and does not kill Kupffer cells. While phagocytosis of larger (> 1 jim) particles is dependent onactin filaments, microtubules appear involved in the uptake of smaller (< 0.9 jim) particles, witha gradual continuum of different filament involvement between the two extremes (Pratten andLloyd, 1986; Toyohara and Inaba, 1989).When the livers obtained from animals pre-treated with liposomal doxorubicin or vincristinewere examined, no reduction in the number of Kupifer cells was observed. The 4 day time pointwas selected for these studies because depression of liposome uptake in the liver afterdoxorubicin pre-treatment was maximal. This also eliminated potential problems due to cells thatwere killed but not yet eliminated from the tissue at earlier time points. The conclusion from thisdata is that liposomal doxorubicin, although functionally shutting down mechanisms responsiblefor liposome uptake in liver, is not eliminating Kupffer cells. Daemen et al. (1995) have donevery similar work in rats and shown that a single injection of 5 mg/kg liposomal doxorubicin canimpair foreign particle clearance mechanisms of the liver but requires multiple injections toshow actual elimination of Kupifer cells.The results summarized above were confirmed using data derived following colloidal carboninjection 24 hours after the liposomal drug pre-doses were administered. Even though bothtreatment groups showed maximum liver depression of liposome uptake at the time pointselected, there was little quantitative difference between the control and treatment groups forcolloidal carbon uptake. This raises the intriguing possibility that the “RES” blockade approachdescribed here targeted a specific uptake mechanism or, alternatively, a particular sub-populationof cells. The liver macrophage population can be divided into several sub-populations based onsize and maturity and thus a variety of functions (Daemen et al., 1989; Hoedemakers et al.,871993). In general, large liver macrophages are more active for phagocytosis than smaller cells(Daemen et al., 1989), although elimination or inhibition of large macrophages can induceactivation and thus a shift to the smaller population (Lazar et al., 1989).Commercial colloidal carbon suspensions are reported to have particle sizes of the order of 60-70nm (Miyata et al., 1994), although sizes exceeding several hundred nanometers were measuredfor the carbon suspensions used here, possibly due to aggregation. Particles that are 60-70 nmwould have access to the liver parenchyma and can be taken up by the endothelial cells lining theliver sinusoids (Roerdink et al., 1981). It is possible that Kupffer cell activity has been shutdown, resulting in a shift in the distribution of particulates to these other areas of the liver. Inaddition, the differences between the duration of P.ES blockade for doxorubicin versusvincristine may be related to the retention properties of the carriers within the Kupffer cells.Doxorubicin is known to be very stable and leak slowly from engulfed liposomes and thus theinterior of macrophages may act as a slow release reservoir (Storm et al., 1988). Vincristine isexpected to leak out rapidly from both the liposome and the cell (Mayer et al., 1 990a; Boman etal., 1994). This is likely why co-injection of the pre-dose and subsequent test injection togetherresulted in immediate liver depression in the case of vincristine but not for doxorubicin.The results presented in this chapter show that very low doses of liposomal doxorubicin orliposomal vincristine can have large effects on the liposome clearance mechanisms of RES, evenwhen the carriers are composed of “RES avoiding” liposomes. This effect becomes particularlyimportant in terms of understanding the behavior of liposomal anticancer drugs when used attherapeutically active levels as described in the following chapter. While the experiments heredid not demonstrate Kupifer cell elimination when using low (< 10 mg/kg) pre-doses of88liposomal drugs, higher or multiple doses certainly will (Daemen et al., 1995). At 30 times thedose employed here, the effect that entrapped doxorubicin has on the circulation lifetime of theliposomal carrier is shown in Chapter 4 to be even more dominant than that of the lipidcomposition.89CHAPTER 4TUMOR ACCUMULATION OF CONVENTIONAL AN]) STERICALLY STABILIZEDLIPOSOMAL DOXORUBICIN4.1 IntroductionTherapeutic responses obtained following administration of anti-cancer drugs are dependent ontumor physiology and tumor cell heterogeneity. These drugs must access the target cellpopulations at levels sufficient to cause cytotoxic effects and should be effective in all thediffering microenvironments present with tumors. In humans, strategies designed to maximizethe anti-tumor activity of chemotherapeutic agents must also contend with a heterogeneouscombination of proliferating cells that are in various phases of the cell cycle, proliferating atwidely different rates, growing in different tissues and capable of adapting rapidly to thechemotherapeutic stresses exerted on them. In practical terms this means that chemotherapytypically involves the use of multiple drugs that exert antitumor activity via differentmechanisms. It also means that the maximum dose intensity of antineoplastic agents should beemployed (Livingston, 1994). Tumor cells must be exposed to the highest levels of drug for thelongest time periods if maximum therapeutic effects are to be realized (Lin, 1994).Efforts to maximize the dose intensity of chemotherapeutics are limited by the non-specific toxicside effects exhibited by these drugs. Doxorubicin, one of the most commonly employed anticancer drugs, provides a good example in that it is a potent myelosuppressive agent (Gabizon etal., 1986; Bally et al., 1990b; Bonadonna et al., 1970). Therapeutic doses must be limited toschedules and amounts that do not dangerously compromise regeneration of blood cells or cellsof the immune system. In addition, doxorubicin exhibits a dose limiting cardiotoxicity(ithinehart et al., 1974; Minow et al., 1975) limiting the total drug dose to approximately 450mg/rn2. Myelosuppression can be counteracted using the hematopoietic growth factor90granulocyte-macrophage colony stimulating factor (GM-CSF; Vose and Armitage, 1995).Cardiotoxicity on the other hand can be reduced by administering the drug by infusion or byutilizing this drug in a liposomally encapsulated form (Gabizon et al., 1982; Olson et al., 1982).It has been shown that the therapeutic activity of the liposomal drug is greater than or equal tofree doxorubicin in a variety of pre-clinical and clinical studies (Conley et al., 1993; Cowens etal., 1993).Pharmacokinetic studies to establish the mechanisms whereby liposomes improve the therapeuticprofile of doxorubicin have focused in two areas. First, there is good evidence from pre-clinicalstudies that the reduced cardiotoxicity of liposomal formulations is a consequence of reduceddrug accumulation in cardiac tissue (Gabizon et al., 1982; Herman et al., 1983). Second,therapeutic activity arises from liposome mediated increases in drug circulation lifetimes whichresults in improved drug delivery to tumor sites (Gabizon et al., 1990). Higher lipid doses canalso lead to increased liposome circulation lifetimes (Abra and Hunt, 1981) and may be expectedto facilitate increased accumulation of the carrier at solid tumor sites, provided that the carrierretains encapsulated drug following i.v. administration.The studies summarized here evaluate the accumulation of drug at the tumor site for freedoxorubicin and liposomal doxorubicin when administered at the maximum tolerated dose via asingle bolus intravenous injection in mice. The major aim was to characterize the potentialtherapeutic advantage of liposomes containing PEG polymers, which exhibit longer circulationlifetimes (Blume and Cevc, 1990; Allen et al., 1991 a). It is demonstrated that incorporation ofPEG lipids in the liposomal doxorubicin formulation did not lead to improved tumor delivery orenhanced therapeutic activity under these conditions.914.2 Materials and methods4.2.1 Preparation of liposomes and doxorubicin loadingThe production of 100 mu large unilamellar vesicles (LUVs) was carried out in general aspreviously described (Hope et al., 1985). Dry lipid mixtures composed of DSPC/chol (5 5/45mollmol) or DSPC/cholJPEG-DSPE (55/45/5) each with trace amounts of [3HJCHE(NEN/Dupont) as a non-metabolizable and non-exchangeable liposome marker (Derksen et al.,1987) were dissolved in chloroform. In a warm water bath (50°C) this was reduced to aminimum volume under at stream of nitrogen gas and in order to avoid precipitation ofcholesterol, quickly placed under high vacuum and dried for a further 4 hours. This procedureresults in a homogeneous expanded lipid foam with a significant total surface area whichfacilitates complete lipid hydration. Multilamellar vesicles (MLVs) were produced first,hydrating the dried lipid in 300 mM citrate buffer (pH 4.0) followed by vigorous vortexing,warming, and five freeze-thaw cycles. The MLVs were then extruded ten times through twostacked 100 nm pore size polycarbonate filters (Costar/Nuclepore, Canada) employing anextrusion device (Lipex Biomembranes, Canada) equilibrated at 65°C. The resulting LUVs had amean diameter of 100 ± 15 urn, as determined by QELS on a NICOMP Model 270 submicronparticle sizer operating at a wavelength of 632.8 nm. No differences were observed betweensystems prepared with and without PEG-modified lipids. The lipid concentration of eachliposome preparation was determined by a phosphorous assay (Fiske and Subbarow, 1925),where the colored product was measured spectrophotometrically at 815 nm using a ShimadzuUV-visible recording spectrophotometer. This measurement was used to derive a specificactivity for the radiolabeled liposomes (DPMJjimol total lipid), and thereafter liposomal lipidconcentrations were estimated by scintillation counting using a Beckman LS 3801 instrument.92PicoFluor 40 scintillation fluid (Packard) was used as a high efficiency scintillation cocktail.Distearoylphosphatidylcholine (DSPC) was from Avanti Polar Lipids, cholesterol (chol) andother chemicals were from Sigma, and poly(ethylene glycol)distearoylphosphatidylethanolamine (PEG-A-DSPE)(PEG-PE) was synthesized as previouslydescribed (see Chapter 2).Doxorubicin was encapsulated using the transmembrane pH gradient loading procedure (Mayeret al., 1986). To establish a pH gradient across the LUVs for doxorubicin loading, the resultantLUVs were dialyzed (12-14000 molecular weight cut off, Spectrapor) against 150 mM NaHCO3buffer, pH 7.5 for several hours to remove most of the external citrate and raise the external pHto 7.5. Subsequently, preheated (65°C) aliquots of these LUVs and doxorubicin (doxorubicinHCI, Adria Laboratories dissolved in saline) were combined in a 0.2 mole drug to lipid ratio.These samples were incubated for an additional 10 mm. at 65°C, resulting in over 95% trappingefficiency. “Empty” liposomes were prepared using a parallel procedure, with saline as areplacement for doxorubicin.4.2.2 Animal and tumor modelsAll mice used in this study were 20-22g female BDF-1 mice (Charles River, Canada). The LewisLung carcinoma (LLC) was obtained from the National Cancer Institute Tumor Repository(Bethesda, Maryland) as a frozen tumor fragment from stock number G50132. Tumor cellsuspensions were prepared by mechanical then enzymatic (Dispase/Collagenase/Dnasetreatment) processing of excised tissue and used for experiments on passage number 2 to 5. Ineach passage, 3 x cells in a volume of 50 il were implanted subcutaneously (s.c.) in each of93the mouse flanks (bilateral tumors). Tumors were left to progress to a estimated 0.2 to 0.4 g sizebefore initiation of pharmacology or therapeutic studies. At this time the doubling time of thetumor was approximately 3 days. All drug and liposome injections were delivered intravenously(i.v) through the lateral tail vein in a volume of 200 .tl. At various times after injection the micewere anesthetized by i.p. administration of ketamine/xylazine (160 mg/kg, 20 mg/kg, MTCPharmaceuticals, Canada). Blood was collected via cardiac puncture, placed in microtainer tubeswith EDTA (Becton Dickinson, Canada) and centrifuged at 1500 x g for 10 mm. to isolateplasma. Tissues were carefully removed, washed, blotted to remove attached blood, weighed andhomogenized with a Polytron to a 20% (liver, tumor) and 10% (spleen) homogenate (wt:vol) insaline.4.2.3 Assays for liposomal lipid and doxorubicinTo determine lipid levels, 100 il plasma and 200 ii tissue homogenate were solubilized with500 jil Solvable (NEN/Dupont) for 2 hours at 60°C. Subsequently the samples were cooled andtreated overnight with 200 tl 11202 before addition of 5 ml scintillation fluid. The samples werecounted to determine[3HjCHE. For doxorubicin levels, 100 il plasma and 200 jtl tissuehomogenate were diluted with water up to 800 .tl. Then 100 tl of 10% SDS and 100 jil of 10mM H2S04 were added. After these samples were vortexed well 2 ml ofchloroformlisopropylalcohol (1:1 v/v) was added prior to additional mixing. The resultingsamples were frozen overnight, thawed, and centrifuged for 10 mm. at 1000 x g. The organic(lower) phase was removed and the amount of associated doxorubicin fluorescent equivalentswas measured with a Perkin-Elmer fluorimeter (excitation/emission at 500/550 nm).Doxorubicin standards (0 to 20 nmol) were prepared for each set of assays and these were94prepared after mixing appropriate volumes of the standard with tissue homogenates derived fromorgans isolated from untreated mice. All tissues drug and lipid levels were corrected for drug andlipid in the plasma compartment of these tissues using published plasma volume correctionfactors (Bally et a!., 1993).4.2.4 Acute toxicity evaluationTumor free mice were used to test the doxorubicin mediated acute toxicity and to establish themaximum tolerated dose (MTD) for both free and liposomal drug formulations. Previous workfrom our laboratory indicated that in BDF- 1 mice free doxorubicin has a MTD of between 20and 25 mg/kg while conventional liposomal doxorubicin has a MTD above 60 mg/kg. Therefore0.66 jimol DOX-HC1 dissolved in saline as the free drug and 2.00 j.tmol DOX-HC1 entrappedwithin 10 jimol lipid (LUV) was administered (i.v) per mouse. For an additional comparison,0.66 jimol DOX entrapped within 3.3 jimol lipid was evaluated. Toxicity was measuredqualitatively through evaluations of mean body weight lose and survival up to 40 days aftertreatment. These studies were done in accordance to Canadian Council on Animal Care (CCAC)Guidelines and it should be noted that only one of 25 animals used in this experiment suffered adrug related death and no animals had to be terminated as a result of unacceptable suffering.4.2.5 Plasma elimination and tumor accumulationIn order to demonstrate the influence of lipid dose on elimination of liposomal lipid from theblood compartment a dose titration of liposomal lipid was completed up to the amount of lipidrequired to deliver the MTD of associated drug. For the dose titration, increasing doses of empty95and drug loaded (0.2 drug:lipid ratio) liposomes were administered i.v in tumor free mice. Themice were sacrificed 24 hours after injection and the level of liposomal lipid in plasma andselected organs were determined as described above.Additional plasma elimination and tissue distribution studies were completed in BDF- 1 micebearing Lewis Lung tumors. All mice receiving liposomal doxorubicin were given 2 iimoldoxorubicinll 0 imol total lipid (approximately 60 mg doxorubicin/kg) and sacrificed at 1, 4, 24,48, 96 and 168 hours. Free doxorubicin treated mice were given (i.v.) 0.66 itmol doxorubicin(approximately 20 mg drug/kg) and sacrificed at 15 mm., 1, 4, 24, 48 and 72 hours. Lipid anddrug levels in plasma and tissues were determined as described above.4.2.6 Tumor histologySelected tumors derived from animals treated as described were carefully excised and fixed in3% paraformaldehyde in PBS. After passing through graded concentrations of sucrose (0% to15%), the tissue was imbedded in O.C.T. (Tissue-Tek, Miles Inc., USA), frozen in liquidnitrogen, and then 5 jim cryostat sections were prepared using a Frigocut 2800E microtome fromLeica. Cryostat sections were washed, blocked with BSA and then stained with an anti-MAC-iFITC-antibody conjugate (Pharmingen, CA). A Leitz Dialux fluorescence microscope (at 40magnification) was used to evaluate FITC fluorescence of the sections (430-490 nm cut offfilter) and doxorubicin fluorescence (530-560 nm cut off filter) with fluorescentphotomicrographs obtained using a Orthomat microscope camera. Normal phase contrastphotomicrographs of the sections were also obtained. All images were recorded on Fuji colorASA400 negative film.964.2.7 Tumor growth inhibitionAnimals bearing tumors of between 0.2 and 0.4 g were treated with free and both liposomaldoxorubicin formulations at doses of 0.66 and 2.00 jimol drug per mouse (approximately 20 and60 mg/kg), respectively. Tumor size was determined at various times after a single drug doseusing a caliper to estimate length and width. Tumor mass (g) was calculated using the followingformula (Mayer et al., 1990b):(a) x (b)22where a = length and b = width measurements in cm. Mice were terminated on the nearest wholeday when tumor mass equaled or exceeded 1.5 g. According to the CCAC approved protocolused animals must be terminated when total tumor mass exceeds 20% body weight or whentumors become ulcerated.4.2.8 Statistical analysisDifferences between results obtained after administration of the two liposomal formulations ofdoxorubicin and free drug were determined using an ANOVA analysis. Comparisons were madefor various common time points incorporating all sets of collected data for that time point usingthe Post Hoc Comparison of Means, Scheffé test. Differences were considered significant at ap<O.O5 criterion.974.3 Results4.3.1 Estimation of Maximum Tolerated DosesDose range studies in tumor-free BDF- I mice indicated that the maximum tolerated dose (MTD)for free and liposomal doxorubicin was approximately 20 mg/kg and 60 mg/kg, respectively.Both liposomal formulations of doxorubicin were tolerated up to 60 mg/kg, however there wassignificant loss of body weight at these high doses (Table 4.1). A nadir equivalent to almost a25% decrease in mean body weight was observed between days 8 and 10 after drugadministration. Recovery of normal body weight was achieved by day 18 and all mice survivedthis drug dose with the exception of 1 (out of 5) that died in the group treated with doxorubicinencapsulated in the liposomes containing PEG-PE. At necropsy (day 40), there were no signs ofgross pathological abnormalities. Free drug treated animals (20 mg/kg) exhibited a mean bodyweight loss of 10 to 12% (observed from day 4 through to day 10). Weight loss data observedafter a comparable drug dose (20 mg/kg) given in either liposomal formulations showed reducednadir weight loss and faster recovery to normal body weight (day 7) consistent with the wellestablished liposome mediated reduction in doxorubicin toxicity (Balazsovits et al., 1989;Gabizon et al., 1 994a). Based on weight loss toxicity and long-term (40 day) survival, doses of20 mg/kg for free and 60 mg/kg liposomal doxorubicin were used for the plasma elimination andtumor accumulation studies summarized below.4.3.2 Influence of dose escalation on plasma liposomal lipid levelsIn general, the circulating blood levels of liposomal lipid increase as the dose of liposomesincreases (Mauk and Gamble, 1979; Abra and Hunt, 1981). In the case of PEG-PE containingliposomes dose independent pharmacokinetic characteristics are observed (Allen and Hansen,98Table 4.1Toxicity/weight loss in response to the maximum tolerated dose for free and liposomaldoxorubicinFemale BDF- 1 mice (20-22 g) were administered various doses of free or liposomallyencapsulated doxorubicin delivered i.v. in 200 .t1 volume via the lateral tail vein and weight loss(mean for 4 mice per group) was recorded daily over several weeks.drug dose formulation nadir percent estimated MThjimol per mouse weight loss (day) mg/kg0.66 free drug 12% (5) >202.0 DSPC/chol 23% (9) >602.0 DSPC/chol!PEG-PE 25% (9) >600.66 DSPCIchol 8% (3)0.66 DSPC/chol/PEG-PE 7% (5)1991; Huang et al., 1992), while lower doses of conventional liposomes are cleared from thecirculation more rapidly than higher doses (Mauk and Gamble, 1979). These effects areillustrated in Figure 4.1, which shows the lipid dose remaining in the plasma at 24 h as a functionof the total lipid dose. These results illustrate two important features of drug loaded liposomesand PEG-PE containing liposomes. First, the addition of PEG-modified lipids greatly improvescirculation lifetimes for both the empty and doxorubicin loaded systems. At doses under 1 !Imollipid/mouse, typically 40% of the injected dose is present in the plasma 24 h after administrationof PEG-liposomes, whereas less than 5% of the injected dose is observed in plasma 24 h afteradministration of conventional liposomes (Figure 4. lÀ). As the lipid dose increases thedifferences between the PEG-containing and conventional liposomes are still significant, butthese differences are reduced from a 10-fold (observed below the 1 imol lipid per mouse dose)to less than a 3-fold increase (observed above the 2 jimol lipid per mouse dose). The results99E(I,0a)0ci)Cl)0ci)C.)ci)CFigure 4.1Dose titration of the liposomal carrierVarious doses of empty or drug loaded liposomes (0.2 drug:lipid ratio) were administered in 200jil volume i.v. into tumor free mice. A, plasma recovery at 24 h expressed as a percent of theinjected dose per total plasma; B, the same results expressed as an absolute lipid concentration.(0), DSPC/cholesterol; (•) DSPC/cholesterol, DOX; (D) DSPC/cholesterol/PEG-PE; (•)DSPC/cholesterollPEG-PE, DOX. Results shown represent the mean of four animals ± S.E. pergroup.1008060402001086420CaECl)cci0Eci)00.0E0 2 4 6 8 10total lipid dose injected (tmol)100shown in Figure 4. lB clearly demonstrate a linear relationship between administered lipid doseand the levels of lipid in the circulation at 24 h, regardless of the liposomal formulation used(correlation coefficients (r2) for the conventional formulations were 0.89 and 0.97 for empty andloaded systems, and 0.98 and 0.99 for the PEG liposomes, empty and loaded, respectively). Intotal, these results are consistent with dose independent and dose dependent pharmacokineticbehavior for PEG-containing liposomes and conventional liposomes, respectively. The secondpoint from data in Figure 4.1 is that entrapped doxorubicin significantly increases the plasmablood levels obtained 24 h after i.v. administration of PEG-PE containing liposomes orconventional liposomes. This effect has been noted previously in Chapter Drug elimination from plasma and tumor accumulation in BDF-l mice bearing LewisLung tumorsA comprehensive examination of drug and liposome circulation lifetime following i.vadministration was completed in BDF- 1 mice bearing Lewis Lung tumors (Figures 4.2 and 4.3).Several trends are evident for elimination of the liposomal carriers from the blood compartment(Figure 4.2A) for the tumor bearing mice. First, at 24 h and later, the dominant factor dictatingenhanced circulating blood levels of liposomes is the presence of entrapped doxorubicin. Thedrug loaded liposomes, for both PEG-containing and conventional liposomes, are consistently athigher concentrations in the blood than the respective empty systems, resulting in 3- to 10-foldincreases in the plasma concentrations of liposomal lipid. It should be noted that for bothdoxorubicin loaded liposomal carriers there was an approximately equivalent reduction inliposome uptake by the liver (data not shown). The plasma liposomal lipid levels obtained after24 h are still significantly greater (p<O.O5) for the PEG-containing liposomes. The differences101Figure 4.2ECl)2ciV02Pharmacokinetic analysis of liposome clearance in tumor bearing miceMice were administered 10 j.tmol total lipid per mouse of either conventional or PEG-containingliposomes with or without entrapped doxorubicin (2 i.tmol drug). Mice were sacrificed at 1, 4, 24h, 2, 4, and 7 d, and lipid and drug plasma concentrations determined. A, total time course ofliposomal clearance. B, drug to lipid ratio for the conventional vs. the PEG liposomal systems.(0), DSPC/chol; (•), DSPCIcho1 + DOX; (D) DSPC/choIJPEG-PE; (•), DSPC/cholIPEG-PE +DOX. Results shown represent the mean of four animals ±S.E. per group.10864200.20020. 1 2 3 4time (days)5 6 7102between the two liposomal preparations, however, are reduced substantially as compared to thebehavior in tumor free animals.A second important observation evident from the results in Figure 4.2A is related to tumorinduced increases in liposome elimination from the circulation. For the conventionalformulations, nearly half of the injected dose is eliminated from the circulation within 1 hour.Comparison of the 24 hour data in Figure 4.2A with that from equivalent dose data shown inFigure 4.1 indicates that liposomal blood levels at this time point are approximately two or fourfold lower in tumor bearing mice than in tumor free mice given empty liposomes anddoxorubicin loaded liposomes, respectively. This contrast with previous studies in our laboratoryusing BDF-1 mice with solid tumors derived following s.c. injection of B16/BL6, L1210, P388and FSA cells, which typically show equivalent pharmacokinetic behavior in tumor and non-tumor bearing animals. The differences between the pharmacokinetic behavior for tumor bearingversus tumor free mice in terms of absolute amounts of lipid are shown in Table 4.2. For theconventional liposomes, all of the circulating lipid lost due to the presence of tumor could beaccounted for through increased liposome uptake by the liver, spleen, and solid tumors, whereasfor the PEG-PE containing systems, over 75% could be accounted for by increased uptake inthese three tissues.Drug retention by liposomes in the blood compartment can be measured by assaying lipid anddrug concentrations in plasma over time. This analysis (Figure 4.2B) shows that bothconventional and PEG- liposomes retain encapsulated drug with half-times for drug release offive days or longer. This is consistent with previous reports for doxorubicin retention inDSPC/cholesterol liposomes (Bally et a!., 1 990a). There was no measurable change in the drug103Table 4.2.Comparison of liposomal biodistribution in Lewis Lung solid tumor bearing versus tumorfree BDF-1 mice after i.v. administration of equivalent doses of lipid (10 tmol per mouse)tumor bearing tB versus DSPC/chol DSPC/choIIPEG-PEtumor free tF_(j.tmol)’empty DOX empty DOXplasma (tB-tF) -2.4 -1.6 -4.3 3.8tissues2 (tB-tF) 2.4 1.5 3.2 2.9NETtB-tF [ 0.0 -0.1 -1.1 -0.91 total lipid uptake for tumor bearing minus tumor free mice (jimol)2sum of liver, spleen, tumor tissue total uptakesto lipid ratio measured over the initial 24h period after i.v. injection. After 2 and 4 days incirculation, the drug to lipid ratio is approximately 90% and 70% of the value measured prior toinjection. The doxorubicin leakage rate after 24 h can thus be estimated to be approximately 0.75nmollj.tmol lipid/hour for both liposomal formulations studied.The plasma concentrations of doxorubicin obtained after injection of free and liposomal systemsare shown in Figure 4.3. For the liposomal drugs, drug elimination rates were similar toelimination rates for liposomal lipid, a reflection of the slow drug release rates. Measurementsmade one and two days after injection indicate circulating drug levels of greater than 0.5 .tmol ofdrug per ml plasma (equivalent to 25% of the injected drug). An estimation of doxorubicin areaunder the curve (AUC,) for the blood compartment indicates a 1.5 fold increase in AUC whenthe drug is given encapsulated in PEG-liposomes (AUC = 78 ,imol’mr1.h)versus conventionalliposomes (50 jtmol•mr’.h). Figure 4.3 also includes results obtained following administration104Figure 4.3Pharmacokinetic analysis of drug clearance in tumor bearing miceDrug plasma concentrations from Figure 4.2 were plotted. Also shown is the clearance of 0.66imol free drug per mouse. (A), 0.66 imol free DOX; (I), 2 j.tmol DOX in DSPC/chol; (•), 2j.tmol DOX in DSPC/choIIPEG-PE. Results shown represent the mean of four animals ±S.E. pergroup.1-S(1)I- 0.01-SI I I0 1 2 3 4 5 6 7time (days)105of free drug at the MTD. In the absence of a carrier, plasma doxorubicin levels fall belowdetectable limits within hours. Assuming that the plasma volume of a 20-22 g mouse is 1 ml andan injected drug dose of 0.66 imol doxorubicin per animal, it is estimated that greater than 99%of the injected free drug was eliminated from the circulation within 15 minutes after injection.The AUC for free drug was estimated to be 87 nmol’mr’•h which is approximately 600 and 900fold less than that obtained for doxorubicin given in conventional and PEG-containingliposomes, respectively.In order to ascertain whether the increased blood levels of PEG-PE containing liposomesincreased tumor accumulation the drug and liposomal lipid levels were compared in Lewis Lungtumors for 7 days following i.v. administration for PEG-PB and conventional liposomaldoxorubicin. These data, shown in Figure 4.4, indicate that there is similar tissue uptake for bothdrug loaded and “empty” liposomal carriers during the initial 24 h after i.v. administration. Theempty conventional liposomes reach a peak level in tumor tissue of 0.6 jimol Iipid/g tissue. Thisvalue was achieved 24 h after administration. Following administration of “empty” PEG-containing liposomes the peak lipid concentration is achieved 48 h after administration and avalue of 1.0 jimol lipid/g tissue was obtained. A gradual decline in peak values is primarily aconsequence of continued tumor growth. The amount of liposomal lipid delivered per tumor wasequivalent to approximately 5% and 8% of the injected lipid dose for conventional liposomesand PEG-containing liposomes, respectively. In the case of empty liposomes the level ofliposomal lipid achieved per g tumor was significantly greater (p < 0.05 at time points beyond48h) when using PEG-containing liposomes. This is consistent with the fact that the circulationlifetimes of the PEG-containing liposomes are greater than that of conventional liposomes (seeFigure 4.2A). For the drug loaded systems however, tumor levels of liposomal lipid (jimol106Figure 4.4Tumor loading of liposome and drug loading in the murine Lewis Lung solid tumor modelA, Liposome accumulation in the Lewis Lung solid tumor.(O), DSPC/chol; (•), DSPC/chol +DOX; (D) DSPC/cholIPEG-PE; (•), DSPC/cholfPEG-PE + DOX. B, drug accumulation. (A),free DOX; (•), DOX in DSPC/chol; (s), DOX in DSPC/choIIPEG-PE. Results shown representthe mean of four animals ±S.E. per group.3.5 -A3.0-ED 2.5-E2.0-a)a-toE- 0.5-time (days)107lipid/g) increased over the 7 day time course. This apparent increase in liposome delivery isprimarily a consequence of doxorubicin mediated regression of the solid tumors. Although amaximum lipid concentration cannot be estimated from the data in Figure 4.4, levels ofliposomal lipid achieved in the tumor exceed 2.5 j.tmol lipid/g. There is little difference betweenthe PEG-containing and conventional formulations when the carriers contain encapsulateddoxorubicin.Drug levels achieved within the tumors are shown in Figure 4.4B and results obtained followingadministration of free drug (20 mg/kg) have been included for comparison. For both liposomaldrug formulations, peak solid tumor concentrations of drug (CTmax) are achieved after 48 h. Thecombined effects of tumor regression and drug release from liposomes within the tumor leads tothis plateau. Drug levels delivered up to 48 h via conventional liposomes are slightly higher thanthat delivered via PEG-liposomes, with no significant difference past this time point. Using theAUC (imol doxorubicinlg tissue - time curve) as an estimate of tumor drug exposure,conventional liposomes (AUCT of 38 .tmol.g’.h) expose the tumor to slightly more doxorubicinthan PEG-containing liposomes (AUCT of 31 j.tmol.g’.h). This suggests that more efficient drugdelivery to tumors is obtained for conventional liposomes.The peak level of drug obtained in tumors was reached 4 days after i.v. administration of bothconventional and PEG-containing liposomal formulations, when values of 250 nmol per g weremeasured. This represents approximately 140 jig equivalents of doxorubicin per g tumor. Incontrast, peak drug levels are achieved within 15 mm. after administration of free drug and theselevels (10 nmol per g) were 25-fold lower than those obtained following administration of theliposomal drug. In animals receiving liposomal doxorubicin formulations there was a progressive108decline in the drug to lipid ratios measured within the tumor. For conventional liposomes, drugto lipid ratios (mol:mol) dropped from 0.2 at the earliest time points to 0.13 and 0.10 at 4 and 7days, respectively. Similar results were obtained in tumors from animals given PEG-liposomaldoxorubicin, where ratios of 0.14 and 0.09 were obtained at 4 and 7 days, respectively. Thesetumor drug to lipid ratios are comparable to those measured in the circulation (Figure 4.2B).Based on these changes in drug to lipid ratio, it can be estimated that liposomes within the tumorare releasing drug at 0.60 to 0.65 mnol drug!jimol lipid/h, rates which are comparable to those inthe circulation.4.3.4 Tumor histologyIn order to gain a more precise understanding of the fate of doxorubicin localized within thetumor, histological studies were initiated to qualitatively assess the distribution of doxorubicin.In addition, these studies attempted to correlate drug distribution with the distribution of tumorassociated macrophages (TAMs)(detected using an anti-MAC-i antibody). The results, shown inFigure 4.5, illustrate selected regions within tumors obtained from control and doxorubicintreated animals as a function of time after i.v. administration. Doxorubicin, a fluorescent drug,appears red when viewed using a fluorescent microscope equipped with a rhodamine filter.TAMs were labeled with an FITC conjugated anti-MAC-i antibody and appear green under afluorescene filter. From the phase contrast photomicrographs of control tumors, it is evident thatthe Lewis Lung solid tumor is a loose, poorly formed network of cells. Although not shown inthe photomicrographs incorporated in Figure 4.5, these tumors have a large necrotic center andthis is surrounded by more densely packed cells near the outer surface of the tumor. FITC MAC1 positive cells are interspersed within the outer region of the tumor. It should also be noted that109Figure 4.5 (following page)Lewis Lung solid tumor histology after administration of either free or liposomaldoxorubicinCryostat sections were visualized (40 x) under either phase contrast or fluorescence microscopyand selected slides shown. The top labeling identifies mode of visualization: Phase, phasecontrast; anti-MAC-i -FITC, fluorescence to identify MAC-i positive cells; DOX, doxorubicinfluorescence. The various treatments were: A, control tumor; B, free doxorubicin (0.66j.tmol/mouse) at 24 h; C, 2.00 jimollmouse doxorubicin in DSPC/chol at 48 h; D, 2.00 jimoldoxorubicin in DSPC/choIJPEG-PE at 48 h.110PHASE-— —-I.. r’ a-c’..t’‘c’.’C2tb% ?.a —aAe-C.y A.meq• :.-. a •:c.?r.j ;:et:;c -:t:.•t’.-• qrC, ‘• ‘ I 4Li % IiI;4V%%1e4p_er’ %J;1y• ais 4•-Pt A . -s——;t,t.ç •4 a 44’••‘—. ..‘. i!-i— Sei .. Cr4 IF.• 4 (_is$ .mpY4ft,•._ 4• oilIv.:‘• —•_4’.’_ •1r t• 3e ‘ 4 5..’,. ‘•••e -• ..•.1.t 4a1” IF4 tD ç• . t• .4•,st• A %I ‘IV r SflanU-MAC-1-FITC DOX111the distribution of MAC-I positive cells is variable, where some regions were almost devoid ofTAMs. This is a clear reflection of the microenvironment heterogeneity with this particular solidtumor. Sections derived from tumors treated with free doxorubicin exhibited a weak and diffusedoxorubjcjn fluorescence that was difficult to differentiate from auto fluorescence. Sectionsderived from tumors treated with conventional liposomal doxorubicin exhibited strongdoxorubicin fluorescence over the entire time period evaluated. Many of the MAC-i positivecells also appeared to contain doxorubicin, presumably a consequence of liposome uptake byTAMs. In contrast, sections derived from tumors treated with PEG-liposomal doxorubicinexhibit a poor correlation between drug fluorescence and FITC positive TAMs. These resultssuggest that doxorubicin delivery achieved with conventional liposome and PEG-liposomes,although quantitatively similar, may result in different intratumor drug distributions. It shouldagain be emphasized however that doxorubicin distribution within tumors from animals treatedwith either liposomal drug formulations was highly variable. The photomicrographs presented inFigure 4.5 were selected for the presence of both doxorubicin and TAMs.4.3.5 Inhibition of tumor growth by liposomal doxorubicinThe antitumor activity of free doxorubicin and both liposomal doxorubicin formulations (givenat the MTD) was determined. Tumor size was followed after a single bolus injection of drug,where treatment was initiated when the tumors were 0.2 to 0.4 g. These data are illustrated inFigure 4.6. The control group (receiving no treatment) grew rapidly, reaching more than 1.5 gwithin eight days. The administration of empty liposomes (10 jimol total lipid) for eitherconventional or PEG systems had no significant effect on tumor growth. Treatment with freedrug resulted in a slight delay in tumor growth, where the time required for the tumor to double112Figure 4.60200202AcontrolgroupsDoxorubicin mediated Lewis Lung solid tumor growth inhibitionTumor bearing mice were given various treatments and tumor mass was estimated daily usingcaliper measurements. Control groups: (A), normal control; (0), 10 imol empty DSPC/chol;(D), 10 jimol empty DSPC/choIJPEG-PE. Treatment groups: (A), 0.66 jimol free DOX; (•), 2j.tmol DOX in 10 imol DSPC/chol; (a), 2 jimol DOX in 10 .tmol DSPC/choIfPEG-PE. Resultsshown represent the mean of four animals ± S.E. per group. -Btreatmentgroups1.0 -0.5 -0.0 —— I I0 2 4 6 8 10time (days)12 14 16113in size increased from 3 days in control animals to 4 to 6 days in free drug treated animals. Afterthis time point the tumors progressed rapidly to achieve a tumor mass of 1.5 g by day 9 (similarto control animals). Conventional and PEG liposomal doxorubicin were more effective than freedrug, increasing the tumor doubling time to approximately 7 days in treated groups. The tumorscontinued to grow and within 16 days after treatment the tumor mass approached 1.5 g. Theresults indicate that there is little difference between the two liposomal systems in terms oftherapeutic activity.4.4 DiscussionMaximization of dose intensity is important for effective cancer chemotherapy. Accordingly,studies presented here employ MTD values of 20 mg/kg (0.66 jimol per mouse) for the free drugand 60 mg/kg (2.0 .tmol per mouse) for both the conventional and sterically stabilized liposomaldoxorubicin. For liposomal systems, a consequence of working at high drug levels is a higherlipid dose. At a drug to lipid ratio of 0.2 (mol:mol), for example, a dose of 2 imo1 drug permouse corresponds to a lipid dose of 10 jimol lipid. As can be seen in Figure 4.1, increasing thelipid dose up to this level produces a steady increase in the plasma concentration of theliposomal carrier. This, in turn, may be expected to lead to higher tumor levels, as enhancedcirculation lifetime is positively correlated with accumulation of these carriers in tumors(Gabizon et al., 1990). This result has been confirmed here for empty liposomes where PEG-PEcontaining liposomes accumulate in the tumor to a higher level than conventional liposomes.However, when these systems are loaded with drug, similar levels of liposome delivery to thetumor are observed for both carrier systems.114The observation that plasma clearance of liposomes is faster in tumor bearing mice has beennoted elsewhere for a subcutaneous S180 tumor model (Oku et al., 1992). The differencebetween the tumor free vs. tumor bearing mice plasma levels observed here is largely accountedfor by increased uptake in the liver, spleen, and tumor. Metastatic solid tumors such as the LewisLung carcinoma shed large amounts of cells and other debris (Butler and Gullino, 1975; Glaves,1983), and it has been suggested that the release of this material into the circulation can stimulatethe RES (Thomas et al., 1995). In addition, solid tumors can either directly or indirectlystimulate the release of TNF-a or other lymphokines such as IL-2 (Nagarkatti et al., 1990;Thomas et al., 1995). Such molecules are implicated in vascular leak syndrome (VLS; Fujita etal., 1991; Deehan et al., 1994). Although no evidence for increased tissue plasma volumes wasfound, a slightly increased liver size and a greatly increased spleen size was observed, an effectthat has also been noted for IL-2 induced VLS (Fujita et at., 1991).There are two major ways in which liposomal drug carriers can be used, as a circulating reservoirfor free drug or as a vehicle to deliver drug to the tumor site. For some anti-cancer drugs,frequent or continuous administration can produce the best therapeutic effect, and therefore theprinciple of encapsulating drugs in slow release liposomes has practical significance (Mayer etat., 1990a; Allen et at., 1992; Vaage et al., 1993). However, there is considerable evidence thatthe mechanism of solid tumor delivery involves extravasation of the intact liposome to the tumorsite followed by slow release of drug. Thus, anti-tumor effects may be attributed largely torelease from liposomes that localize in the tumor as opposed to systemic release of drug (Huanget at., 1992). In fact, studies have shown that intact liposomes can be found within the interstitialspace between tumor cells (Gabizon, 1992, Huang et al. 1992). The lipid and drug data presentedhere is consistent with this mode of doxorubicin delivery to tumor cells.115A central question that arises from the concept that intact liposomes access sites outside theblood compartment concerns mechanisms of transfer. Several routes for transendothelial transferof liposomes have been suggested, including both open channels and some forms of transcytosis(Huang et al., 1993). Recent analysis for sterically stabilized liposomes suggests that the mainroute is through gaps in the endothelial layer and not via vesicular transport or leukocytemediated extravasation (Yuan et al., 1994), and the data presented here can be interpretedsimilarly. Doxorubicin delivered via conventional liposomes is positively correlated withmacrophages within the tumor, whereas the correlation between these TAMs and doxorubicin forthe PEG-PE containing liposome mediated delivery is not as strong. This is consistent with areduced affinity of PEG-PE containing liposomes with liver macrophages noted elsewhere(Allen et al., 1991b).The results presented here indicate that at higher drug dose levels the PEG coating may not resultin improved drug delivery to solid tumors, as shown here for the Lewis Lung solid tumor model.This contrasts with previous results which indicate that sterically stabilized liposomes haveincreased microvascular permeabilities compared to conventional liposomes (Wu et al., 1993).These studies (Wu et al., 1993) were compromised, however, by the fact that the plasmaconcentrations of the sterically stabilized systems were considerably higher than theconventional systems. For longer time courses and comparable plasma concentrations, the resultspresented here show little difference in either the kinetics or total amount of drug accumulationin a solid tumor. The movement of drug from the plasma compartment to the tumor site can bedescribed employing a drug targeting efficiency parameter Te relating the AUC in the circulationto the tumor AUC (Te = AUCT/AUCp). The conventional liposomes gave Te values (0.76) which116were almost a factor of 2 higher than that for the PEG-PE containing liposomes (0.40). Incontrast, the free drug exhibited a Te of 3.0. The greater efficiency of tumor targeting for the freedrug is consistent with increased small molecule penetration within solid tumors when comparedto penetration of larger particles such as liposomes (Yuan et al., 1994; Jam, 1987).The peak drug concentrations (CTmx) obtained here for tumor drug loading are extremelyimpressive. For both liposomal formulations, peak drug levels were achieved after 4 days,closely paralleling liposome lipid uptake, consistent with a model of intact liposomeextravasation (Gabizon et al., 1994b). The CTm values obtained in this study wereapproximately 250 nmol doxorubicin per g tumor, or roughly 140 .tg doxorubicin equivalentsper g tumor. This is far higher than achieved in previous studies, and dramatically illustrates theeffect of dosing at the MID rather than lower dose levels. Previous tumor drug loading valueshave been of the order of 20 nmol per g solid tumor (Mayer et al., 1990b; Gabizon, 1992;Maruyama et al., 1993) to 40 nmol per g (Ning et al., 1994). While the levels of drug obtained inthe experiments here do result in a significant reduction in tumor growth in a rapidly growingand aggressive solid tumor model, these results also point to the problem of drug bioavailability.The rate of drug release in the tumor microenvironment (0.60 to 0.65 nmol drug/j.tmol lipid/h),which corresponds to a half-time for release of over 5 days, is likely too slow to be of therapeuticconsequence to the tumor. The development of techniques which result in increased rates of drugrelease, combined with the techniques employed here for delivering maximal levels of liposomaldrug to tumors could potentially be of great therapeutic advantage.In summary, the results of this work establish that by increasing the liposomal carrier dose up tothe MTD for the encapsulated drug, increasingly higher plasma concentrations, longer117circulation lifetimes, and very high levels of tumor associated drug can be obtained. Inclusion ofPEG-PB in the liposomal composition does not improve tumor delivery of drug under theseconditions. Maximum tumor drug levels (CTmX) were similar for both conventional and PEGcontaining formulations, however limited drug release was evident, supported by limitedtherapeutic effects. Given the extremely high levels of drug delivery to the tumor achieved here,it is proposed that techniques leading to the triggered release of liposome contents may lead tomajor improvements in therapeutic outcome.118CHAPTER 5SUMMARIZING DISCUSSION5.1 Summary of resultsThis thesis incorporates a progression of studies designed to more fully understand certainimportant parameters for the use of liposomes as drug delivery vehicles, covering three basicareas of investigation. These included the use of PEG lipids incorporated into the liposomecomposition in order to prolong the circulation lifetime of the carrier in the circulation, the effectthat entrapped drug has on the RES and the circulation lifetime, and fmally the use of liposomalcarriers to deliver drug to a solid tumor site as affected by these two areas of investigation. InChapter 2 it is shown that poly(ethylene glycol)(PEG)-lipid anchor conjugates can prolong thecirculation lifetimes of liposomes following intravenous injection. This chapter investigated theinfluence of the lipid anchor and the nature of the chemical link between the PEG and lipidmoieties on circulation lifetime. It is shown that incorporation of N-(monomethoxypoly(ethylene glycol)2-succiny )-1 -palmitoyl-2-oleoyl-phosphatidylethanolamine(MePEG2000-S-POPE) into large unilamellar vesicles (LUVs) composed ofdistearoylphosphatidylcholine (DSPC) and cholesterol (DSPC/cholesteroIIMePEG200o-S-POPE,50:45:5 mollmol) results in only small increases in the circulation lifetimes as observed in mice.This is shown to be due to rapid removal of the hydrophilic coating in vivo, which likely arisesfrom exchange of the entire PEG-lipid conjugate from the liposomal membrane, althoughchemical breakdown of the PEG-lipid conjugate is also possible. The chemical stability of fourdifferent linkages was tested, including succinate, carbamate and amide linkages betweenMePEG derivatives and the amino head group of PE, as well as a direct link to the phosphatehead group of phosphatidic acid (PA). The succinate linkage was found to be the most labile.119The anchoring capability of DSPE as compared to POPE in PEG-PE conjugates was alsoexamined. It is shown that incorporation of MePEG2-S-DSPE conjugates intoDSPC/cholesterol LUVs results in little loss of the PEG coating in vivo, long circulationlifetimes and reduced chemical breakdown of the PEG-lipid conjugate. This work establishesthat DSPE is a considerably more effective anchor for PEG2000 than POPE and that the chemicalstability of PEG-PE conjugates is sensitive to the nature of the linkage and exchangeability of thePEG-PE complex. It is therefore suggested that retention of the PEG coating is of paramountimportance for prolonged circulation lifetimes.In Chapter 3, it is shown that the incorporation of ganglioside GM1 or phosphatidylethanolaminepoly(ethylene glycol) conjugates into liposomes can result in extended circulation lifetimes invivo. However, pre-doses of LUVs which incorporate either GMI or PEG-PE, with entrappeddoxorubicin, block the accumulation of subsequently injected emptydistearoylphosphatidylcholine/cholesterol liposomes in liver. It is therefore concluded thatliposomes exhibiting extended circulation lifetimes can induce RES blockade and do not strictlyavoid uptake by liver phagocytes. Further characterization of RES blockade revealed thatextremely low doses of doxorubicin could induce a maximum blockade effect, and that theinhibition of liver uptake can last past 8 days, only approaching full recovery by 14 days.Another commonly employed liposomal anticancer drug, vincristine, had effects which weremore transient, although significant RES blockade could also be demonstrated.The final set of experiments presented a comparison of tumor accumulation and efficacyproperties of doxorubicin entrapped in “sterically stabilized” liposomes and conventionalliposomes. The conventional liposomes were composed of distearoyl phosphatidylcholine and120cholesterol, whereas sterically stabilized liposomes contained in addition 5 mol % ofpolyethylene glycol coupled to phosphatidylethanolamine. Drug pharmacokinetics and tumoraccumulation at the maximum tolerated dose (MTD)(60 mg/kg liposomal doxorubicin) weremonitored in mice bearing Lewis Lung carcinoma solid tumors. In contrast to expected behavior,the efficiency of doxorubicin accumulation at the tumor site, measured employing an area underthe curve analysis, was higher for the conventional liposomes than for the sterically stabilizedliposomes. Both formulations exhibited profound increases of over 500-fold in tumoraccumulation of drug as compared to injection of the MTh of free doxorubicin (20 mg/kg).These studies suggest that optimization of factors nominally leading to longer blood circulationtimes do not provide therapeutic advantages for liposomal formulation of doxorubicinadministered at the MTD. Improvement in other parameters, such as drug leakage rates, holdgreater promise for improving therapeutic properties.5.2 DiscussionThe results presented here highlight several important conclusions for drug delivery andintravenously injected carriers in general. First, it is important to establish that the properties ofthe carrier in vivo must be well understood. This is particularly evident for liposomesincorporating PEG-lipids. The studies outlined here were the first to address the possibility thatthe PEG coating could be lost. Second, most liposomal drug carrier work has been carried outusing empty liposomes. It is not necessarily true that results obtained for empty carriers can bedirectly translated or implied for drug loaded systems. As shown in Chapters 3 and 4, entrappeddrug can have a profound impact on the in vivo clearance behavior of liposomes. Furthermore, itis important to assess tumor loading or other goals in terms of a variety of therapeutic conditions.121PEG-liposomes may accumulate to a significantly greater extent in solid tumors compared toconventional liposomes at extremely low dose ranges, however at the maximum tolerated doseof therapeutically interesting liposomal anticancer drugs, there may be little difference in drugdelivery to the tumor. Perhaps of greater general interest, further application of the workcontained in this thesis may be applicable to the generation of a new class of lipid based carriersystems. This is discussed below.The information gained from the studies of various parameters in the construction of PEG-lipidsmay have implications for a variety of applications. For example, it may be possible toincorporate PEG-lipids in liposomes which are designed to exchange out of the membrane aftersome pre-set time. Chapter 4 demonstrated that not only can extremely high levels of liposomaldrug be delivered to a solid tumor, but this delivery largely occurs within the first 2 days afterintravenous injection. Furthermore, the limited therapeutic effects observed were likely a resultof drug release that was too slow to be of toxic consequence to the tumor cells. Whereas the PEGcoating was of limited benefit under conditions maximizing dose intensity, its subsequent lossfrom the membrane after tumor delivery may be used to destabilize the membrane. Theincorporation of lipids such as unsaturated PEs which normally form the inverted hexagonal H11phase can be stabilized in a bilayer configuration by the presence of selected lipids (Cullis et al,1986), such as PEG-PE. However, upon loss of the PEG-lipid component, such a system may beinduced to undergo bilayer destabilization with the rapid loss of entrapped contents. Such ascheme is depicted in Figure 5. 1A. Work by other members of this laboratory stemming directlyfrom the information gained from Chapter 2 has recently shown promise in this regard in vitrofor doxorubicin loaded systems.122Figure 5.1Potential uses for exchangeable PEG-lipidsIn A, the loss of the PEG-lipid component destabilizes the membrane resulting in rapid release ofencapsulated agent. In B, loss of the PEG-lipid component triggers fusion with the targetmembrane.A_____B__Another possible role for such meta-stable liposomes is that of fusion with a target membrane(Figure 5. 1B). Such systems could potentially be very useful for delivering their contents directlyinto a target cell. The control of membrane fusion in vitro is fairly straight forward, however forsystemic delivery such control is exceedingly difficult. With the timed loss of a PEG-Lipidcomponent, such a system would have the benefit of extended circulation lifetime allowingtarget accumulation followed by the subsequent release of fusion inhibition. Chapter 4 suggestedthat a PEG coating may alter the distribution of liposomes within a solid tumor away from theS-I123macrophage population. Combined with targeted system to further enhance the association of theliposomal carrier with the tumor cell, the fusion of such a carrier with the cell should havepotentially huge therapeutic benefit. Preliminary data from our laboratory with erythrocyteghosts again suggests that this approach may be feasible.The potential future application of RES blockade as described here is questionable. Recentexperiments have shown that pre-dosing with low doses of liposomal doxorubicin can not onlysignificantly increase plasma levels of a subsequent injection as shown here, but also that thisresults in increased accumulation in a remote site not normally accessed by liposomes whichwould otherwise have shorter circulation lifetimes (Longman et al., 1995). It was interesting tonote, however, that the addition of PEG-PE to the subsequent injection was not additive in termsof circulation lifetime increases or disease site accumulation as was expected based on the datafrom RES blockade or PEG-PE liposomal strategies separately. This is in general agreement withour findings in Chapter 4 that when plasma levels are already significantly elevated, the additionof PEG to the liposome does not significantly improve tumor targeting.There are several potential problems with RES blockade, most notably the potential for celldeath, although doxorubicin and vincristine at the doses employed in Chapter 3 do not appear tokill a major portion of Kupffer cell population. This is probably not true for higher doses suchwere employed in Chapter 4. Although clinical trials with liposomal doxorubicin have reportedno major poisoning of important liver functions (Cowens et al., 1993), the impairment of Kupifercell phagocytosis is only recently being considered (Daemen et al., 1995). It is possible that otherliposomally delivered agents may be used to temporarily shut down the RES without significantharm to this cell population.124Finally, the last chapter demonstrated that maximization of dose intensity can result in thedelivery of massive amounts of potentially therapeutic drug but results in minimal therapeuticeffect. This illustrates the problem of bioavailability. This kind of approach combined with arelease mechanism (thermosensitive liposomes, or triggered release as discussed above) couldhave huge therapeutic effects.125REFERENCESAbra, R. M. and Hunt, C. A. (1981) Liposome distribution in vivo: III. Dose and vesicle sizeeffects. Biochim. Biophys. Acta, 666: 493-503.Abra, R. M., Bosworth, M. B., and Hunt, C. A. (1980) Liposome disposition in vivo: effects ofpre-dosing with liposomes. Res. Commun. Chem. Pathol. Pharmacol. 29, 349-360.Abuchowski, A., McCoy, J. R., Palezuk, N. C., van Es, T., and Davis, F. F. (1977) Effect ofcovalent attachment of polyethylene glycol on immunogenicity and circulation life of bovineliver catalase. J. Biol. Chem. 252: 3882-3886.Addanki, S., Cahill, F. D., and Sotos, J. F. (1968) Reliability of the quantitation ofintramitochondrial pH and pH gradient of heart mitochondria. Analytical Biochemistry 25, 17-29.Allen, T. M. (1994) A study of phospholipid interactions between high-density lipoproteins andsmall unilamellar vesicles. Biochim. Biophys. Acta 640, 3 85-397.Allen, T. M. and Hansen, C. (1991) Pharmacokinetics of stealth vs. conventional liposomes:effect of dose Biochim. Biophys. Acta, 1068: 133-141.Allen, T. M. and Chonn, A. (1987) Large unilamellar liposomes with low uptake into thereticuloendothelial system. FEBS Lefl. 223, 42-46.Allen, T. M. Mehra, T., Hansen, C., and Chin, Y. C. (1992) Stealth liposomes: an improvedsustained release system for 1-3-D arabinoftiranosylcytosine. Cancer Res., 52: 2431-2439.Allen, T. M., Agrawal, A. K., Ahmad, I, Hansen, C. B., and Zalipsky, S. (1994) The use ofglycolipids and hydrophilic polymers in avoiding rapid uptake of liposomes by the mononuclearphagocyte system. J. Liposome Res. 4, 1-26.Allen, T. M., Hansen, C., Martin, F., Redemann, C., and Yau-Young, A. (1991a) Liposomescontaining synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Aeta 1066, 29-36.Allen, T. M., Austin, G. A., Chonn, A., Lin, L., and Lee, K. C. (1991b) Uptake by culturedmouse bone marrow macrophages: influence of liposome composition and size. Biochim.Biophys. Acta 1061, 56-64.Alving, C. R. (1987) Liposomes as carriers for vaccines. In: Liposomes - from biophysics totherapeutics. Ostro, M. J., ed. Dekker, New York, p 195.Alving, C. R. (1988) Macrophages as targets for delivery of liposome-encapsulated antimicrobialagents. Adv. Drug Delivery Rev. 2, 107-128.Arakawa, T. and Timasheff, S. N. (1985) Mechanism of poly(ethylene glycol) interaction withprotein. Biochemistry 24, 6756-6762.Bailey, A. L. and Cullis, P. R. (1994) Modulation of membrane fusion by asymmetricaltransbilayer distributions of amino lipids. Biochemistry 33, 12573-12580.Balazsovits, J. A. E., Mayer, L. D., Bally, M. B., Cullis, P. R., McDonnell, M., Ginsberg, R. S.,and Falk, R. E. (1989) Analysis of the effect of liposome encapsulation on the vesicantproperties, acute and cardiac toxicities, and anti tumor efficacy of doxorubicin. Cancer.Chemother. Pharmacol., 23: 81-86.126Bally, M. B., Mayer, L. D., Loughrey, H., Redelmeier, T., Madden, T. D., Wong, K., Harrigan,P. R., Hope, M. J., and Cullis, P. R. (1988) Dopamine accumulation in large unilamellar vesiclesystems induced by transmembrane ion gradients. Chem. Phys. Lipids 47, 97-107.Bally, M. B., Nayar, R., Masin, D., Hope, M. J., Cullis, P. R., and Mayer, L. D. (1990a)Liposomes with entrapped doxorubicin exhibit extended blood residence times. Biochim.Biophys. Acta, 1023: 133-139.Bally, M. B., Nayar, R., Masin, D., Cullis, P. R., and Mayer, L. D. (1990b) Studies on themyelosuppressive activity of doxorubicin entrapped in liposomes. Cane. Chemother. Pharm., 27:13-19.Bally, M. B., Mayer, L. D., Hope, M. J., and Nayar, R. (1993) Pharmacodynamics of liposomaldrug carriers: methodological considerations. In Liposome Technology, 2nd ed, Vol III,Gregoriadis, G. (ed.) CRC Press, Boca Raton, FL. pp. 27-41.Bangham, A. D., Standish, M. M., and Watkins, 3. C., (1965) Diffusion of univalent cationsacross the lamellae of swollen phospholipids. 3. Mol. Biol. 13, 238-252.Barenholz, Y., Amselem, S., and Lichtenberg, D. (1979) A new method for preparation ofphospholipid vesicles (liposomes) by French press. FEBS Left. 99, 210-214.Barranco, S. C. (1984) Cellular and molecular effects of adriamycin on dividing and non-dividing cells. Pharmacol. Ther. 24, 303-319.Bittman, R. and Blau, L. (1972) The phospholipid-cholesterol interaction. Kinetics of waterpermeability in liposomes. Biochemistry 11, 483 1-4839.Blood, C. H. and Zetter, B. R. (1990) Tumor interactions with the vasculature: angiogenesis andtumor metastasis. Biochim. Biophys. Acta 1032, 89-118.Blume, G., and Cevc, G. (1990) Liposomes for the sustained drug release in vivo. Biochim.Biophys. Acta 1029, 91-97.Boman, N. L., Masin, D., Mayer, L. D., Cullis, P. R., and Bally, M. B. (1994) Liposomalvincristine which exhibits increased drug retention and increased circulation longevity curesmice bearing P388 tumors. Cancer Res., 54: 2830-2833.Boman, N. L., Mayer, L. D., and Cullis, P. R. (1993) Optimization of the retention properties ofvincristine in lysosomal systems. Biochim. Biophys. Acta 1152, 253-258.Bonadonna, G., Monfardini, S., and De Lena, M. (1970) Phase I and preliminary phase IIevaluation of Adriamycin (NSC 123127). Cancer Res., 30: 2572-2582.Brannon-Peppas, L. (1995) Recent advances on the use of biodegradable microparticles andnanoparticles in controlled drug delivery. mt. J. Pharmaceutics 116, 1-9.Butler, T. P. and Gullino, P. M. (1975) Quantification of cell shedding into efferent blood ofmammary adenocarcinoma. Cancer Res., 35: 512-516.Chen, C. Y. and Schullery, S. E. (1979) Gel filtration of egg phosphatidylcholine vesicles. J.Biochim. Biophys. Methods 1, 189-192.Chonn, A, Semple, S. C. and Cullis, P. R. (1991) Separation of large unilamellar liposomes fromblood components by a spin column procedure: towards identifying plasma proteins whichmediate liposome clearance in vivo. Biochim. Biophys. Acta 1070, 2 15-222.127Chonn, A., Semple, S. C. and Cullis, P. R. (1992) Association of blood proteins with largeunilamellar liposomes in vivo. Relation to circulation times. J. Biol. Chem. 267, 18759-18765.Classen, E. and Van Rooijen, N. (1986) Preparation and characterization of dichloromethylenedisphosphonate containing liposomes. J. Microencapsulation 3, 109-114.Coleman, D. L. (1986) Regulation of macrophage phagocytosis. Eur. J. Clin. Microbiol. 5, 1-5.Conley, B. A., Egorin, M. J., Whitacre, M. Y., Carter, D. C., Zuhowski, E. G., and Van Echo, D.A. (1993) Phase I and pharmacokinetic trial of liposome-encapsulated doxorubicin. CancerChem. Pharm., 33: 107-112.Cowens, J. W., Creaven, P. J., Greco, W. R., Brenner, D. E., Tung, Y., Ostro, M., Pilkiewicz, F.,Ginsberg, R., and Petrelli, N. (1993) Initial clinical (phase I) trial of TLC D-99 (doxorubicinencapsulated in liposomes). Cancer Res., 53: 2796-2802.Crofton, R. W., Diesseihoff den Dulk, M. M. C., and van Furth, R. (1975) The origin, kinetics,and characteristics of the Kupffer cells in the normal steady state. J. Exp. Med. 148, 1-17.Cullis, P. R. and de Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipid inbiological membranes. Biochim. Biophys. Acta 559, 399-420.Cullis, P. R., Hope, M. J., and Tilcock, C. P. S. (1986) Lipid polymorphism and the roles oflipids in membranes. Chem. Phys. Lipids 40, 127-144.Cummings, J. Willmott, N., and Smyth, J. F. (1991) The molecular pharmacology of doxorubicinin vivo. Eur. J. Cancer 27, 532-53 5.Daemen, T., Veniga, A., Roerdink, F. H., and Scherphof, G. L. (1989) Endocytic and tumoricidalheterogeneity of rat liver macrophage populations; implications for drug targeting. Selec. Canc.Ther. 5, 157-168.Daemen, T., Hofstede, G., Ten Kate, M. T., Bakker-Woudenberg, I. A. J. M., and Scherphof, G.L. (1995) Liposomal doxorubicin-induced toxicity: depletion and impairment of phagocyticactivity of liver macrophages. mt. J. Cancer 61, 716-721.Deamer, D. W. and Bangham, A. D. (1976) Large volume liposomes by an ether vaporizationmethod. Biochim. Biophys. Acta 443, 629-634.Deamer, D. W. and Nichols, J. W. (1983) Proton-hydroxide permeability of liposomes. Proc.Natl. Acad. Sci., USA 80, 165-168.Deamer, D. W., and Nichols, J. W. (1989) Proton flux mechanisms in model and biologicalmembranes. J. Memb. Biol. 107, 91-103.Deehan, D. J., Heys, S. D., Simpson, W., Herriot, R., Broom, J., and Eremin, 0. (1994)Correlation of serum cytokine and acute phase reactant levels with alterations in weight andserum albumin in patients receiving immunotherapy with recombinant IL-2. Clin. Exp. Immunol.95, 366- 372.Delgado, C., Francis, G. E., and Fisher, D. (1992) The use and properties of PEG-linked proteins.Crit. Rev. Ther. Drug. Carrier. Syst. 9: 249-304.Demel, R. A. and de Kruijff, B. (1976) The function of sterols in membranes. Biochim. Biophys.Acta 457, 109-132.128Derksen, J. T. P., Morselt, H. W. M., and Scherphof, G. L. (1987) Processing of differentliposomes markers after in vivo uptake of immunoglobulin-coated liposomes by rat livermacrophages. Biochim. Biophys. Acta, 931: 33-40.Devine, D. V., Wong, K., Serrano, K., Chonn, A., and Cullis, P. R. (1994) Liposomecomplement interactions in rat serum: implications for liposome survival studies. Biochim.Biophys. Acta 1191, 43-3 1.du Souich, P., Bemier, J., and Cote, M. G. (1981) Dose-dependent storage capacity of colloidalcarbon as a cause of reticuloendothelial blockade. J. Reticuloendothelial Soc. 29, 91-104.Dvorak, H. F., Nagy, J. A., Dvorak, J. T., and Dvorak, A. M. (1988) Identification andcharacterization of the blood vessels of solid tumors that are leaky to circulatingmacromolecules. Amer. J. Pathol. 133, 95-109.Dvorak, H. F., Sioussat, T. M., Brown, L. F., Berse, B., Nagy, 3. A., Sotrel, A., Manseau, B. J.,Van de Water, L., and Senger, D. R. (1991) Distribution of vascular permeability factor (vascularendothelial growth factor) in tumors: concentration in tumor blood vessels. J. Exp. Med., 174:1275- 1278.Eastman, S. J., Hope, M. J., and Cullis, P. R. (1991) Transbilayer transport of phosphatidic acidin response to transmembrane pH gradients. Biochemistry, 30, 1740-1745.Ehrlich, P. (1906) in Collected Studies on Immunology, Vol. 2, John Wiley, New York. pp 442-447.Fidler, I. 3., Sone, S., Fogler, W. E., Smith, D., Braun, D. G., Tarcsay, L., Gisler, R. J., andSchroit, A. (1982) Efficacy of liposomes containing a lipophilic muramyl dipeptide derivativefor activating the tumoricidal properties of alveolar macrophages in vivo. J. Biol. Response Mod.1, 43-55.Fiske, C. H. and Subbarow, Y. (1925) The colorimetric determination of phosphorous. J. Biol.Chem., 66: 375-400.Fujita, S., Pun, R. K., Yu, Z-X., Travis, W. D., and Ferrans, V. J. (1991) An ultrastructural studyof in vivo interactions between lymphocytes and endothelial cells in the pathogenesis of thevascular leak syndrome induced by interleukin-2. Cancer 68, 2169-2174.Funato, K., Yoda, R. and Kiwada, H. (1992) Contribution of complement system ondestabilization of liposomes composed of hydrogenated egg phosphatidyicholine in rat freshplasma. Biochim. Biophys. Acta 1103, 198-204.Gabizon, A. A. (1992) Selective tumor localization and improved therapeutic index ofanthracyclines encapsulated in long-circulating liposomes. Cancer Res., 52: 89 1-896.Gabizon, A. and Papahadjopoulos, D. (1988) Liposome formulations with prolonged circulationtime in blood and enhanced uptake by tumors. Proc. Nati. Acad. Sci. 85, 6949-6953.Gabizon, A., Dagan., A., Goren, D., Barenholz, Y., and Fuks, Z. (1982) Liposomes as in vivocarriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice. CancerRes. 42, 4734-4739.Gabizon, A., Meshorer, A., and Barenholz, Y. (1986) Comparative long term study of thetoxicities of free and liposome associated doxorubicin in mice after intravenous injection. JNCI77, 459-469.129Gabizon, A., Price, D. C., Huberty, J., Brescalier, R. S., and Papahadjopoulos, D. (1990) Effectof liposome composition and other factors on the targeting of liposomes to experimental tumors:biodistribution and imaging studies. Cancer Res. 50, 6371-6378.Gabizon, A., Isacson, R., Libson, E., Kaufman, B., Uziely, B., Catane, R., Ben-Dor, C. G.,Rabello, E., Cass, Y., Peretz, T., Sulkes, A., Chisin, R., and BarethoLz, Y. (1994a) Clinicalstudies of liposome-encapsulated doxorubicin. Acta Oncologica 33, 779-786.Gabizon, A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., Martin, F., Huang, A.,and Barenholz, Y. (1 994b) Prolonged circulation time and enhanced accumulation in malignantexudates of doxorubicin encapsulated in polyethylene-coated liposomes. Cancer Res. 54, 987-992.Ghose, T. and Blair, A. H. (1987) The design of cytotoxic-agent-antibody conjugates. Crit. Rev.Ther. Drug. Carrier Syst. 3, 263-361.Glaves, D. (1983) Correlation between circulating cancer cells and incidence of metastases. Br.J. Cancer 48, 665-673.Hale, G, Dyer, M. J. S., Clark, M. R., Phillips, J. M., Marcus, R., Reichmann, L., Winter, G., andWaldmann, H. (1988) Remission induction in non-Hodgkin lymphoma with reshaped humanmonoclonal antibody CAMPATH- 1 H. Lancet 2, 1394-1399.Haran, G., Cohen, R., Bar, L. K., and Barenholz, Y. (1993) Transmembrane ammonium sulfategradients in liposomes produce efficient and stable entrapment of amphipathic weak bases.Biochim. Biophys. Acta 1151, 201-215.Harasym, T. 0., Tardi, P., Longman, S. A., Ansell, S. M., Bally, M. B., Cullis, P. R., and Choi,L. S. L. (1995) Poly(ethylene glycol)-modified phospholipids prevent aggregation duringcovalent conjugation of proteins to liposomes. Bioconj. Chem. 6, 187-194.Harrigan, P. R., Hope, M. J., Redelmeier, T. E., and Cullis, P. R. (1992) The determination oftransmembrane pH gradients and membrane potentials in liposomes. Biophys. 3. 63, 1336-1345.Harrigan, P. R., Wong, K. F., Redelmeier, Wheeler, J. J., and Cullis, P. R. (1993) Accumulationof doxorubicin and other lipophilic amines into large unilamellar vesicles in response totransmembrane pH gradients. Biochim. Biophys. Acta 1149, 329-338.Harrison, M. Tomlinson, D., and Stewart, S. (1995) Liposomal-entrapped doxorubicin- an activeagent in AIDS-related Karposi’s sarcoma. J. Clin. Oncol. 13, 9 14-920.Helenius, A., Fries, E., and Kartenbeck, J. (1977) Reconstitution of Semliki forest virusmembrane. J. Cell Biol. 75, 866-880.Herman, E. H., Rahman, A., Ferrans, V. 3., Vick, J. A., and Schein, P. S. (1983) Prevention ofchronic doxorubicin cardiotoxicity in beagles by liposomal encapsulation. Cancer Res. 43, 5427-5432.Hoedemakers, R. M. 3., Vossbeld, P., Daemen, T, and Scherphof, G. L. (1993) Functionalcharacteristics of the rat liver macrophage population after intravenous injection of liposomeencapsulated muramyl peptide. J. Immunother. 13, 252-260.Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Production of large unilamellarvesicles by a rapid extrusion procedure: characterization of size, trapped volume and ability tomaintain a membrane potential. Biochim. Biophys. Acta 812, 55-65.130Hope, M. J., Bally, M. B., Mayer, L. D., Janoff, A. S., and Cullis, P. R. (1986) Generation ofmultilamellar vesicles. Chem. Phys. Lipids 40, 89-107.Horowitz, A. T., Barenholz, Y., and Gabizon, A. A. (1992) In vitro cytotoxicity of liposomeencapsulated doxorubicin: dependence on liposome composition and drug release. Biochim.Biophys. Acta 1109, 203-209.Huang, C. H. (1969) Studies on phosphatidylcholine vesicles: formation and physicalcharacteristics. Biochemistry 8, 344-352.Huang, S. K., Lee, K.-D., Hong, K., Friend. D. S., and Papahadjopoulos, D. (1992) Microscopiclocalization of sterically stabilized liposomes in colon carcinoma-bearing mice. Cancer Res. 52,5135-5 143.Huang, S. K., Martin, F. J., Jay, G., Vogel, J., Papahadjopoulos, D., and Friend, D. S. (1993)Extravasation and transcytosis of liposomes in Karposi’s sarcoma-like dermal lesions oftransgenic mice bearing the HIV Tat gene. Am. J. Pathol. 143, 10-14.Huang, S. K., Stauffer, P. R., Hong, K, Guo, J. W. H., Phillips, T. L., Huang, A., andPapahadjopoulos, D. (1994) Liposomes and hyperthermia in mice: increased tumor uptake andtherapeutic efficacy of doxorubicin in sterically stabilized liposomes. Cancer Res. 54, 2186-2 191.Hubbell, W. L. and McConnell, H. M. (1971) Molecular motion in spin-labeled phospholipidsand membranes J. Am. Chem. Soc. 93, 314-319.Hunt, C. A. (1982) Liposome disposition in vivo. V. Liposome stability in plasma andimplications for drug carrier function. Biochim. Biophys. Acta 719, 450-463.Jam, R. K. (1987) Transport of molecules across tumor vasculature. Cancer and Metastasis Rev.6, 559-593.Jam, R. K. (1988) Determinants of tumor blood flow: a review. Cane. Res. 48, 2641-2658.Juliano, R. L. and Stamp, D. (1975) The effect of particle size and charge on the clearance rate ofliposomes and liposome encapsulated drugs. Biochem. Biophys. Res. Commun. 63, 65 1-658.Kampschmidt, R. F., Upchurch, H. F., and Park, A. (1966) Factors in the suspending mediawhich alter the carbon clearance rate. RES, J. Reticuloendothelial Soc. 3, 214-222.Khazaeli, M. B., Conry, R. M., LoBuglio, A. F. (1994) Human immune response to monoclonalantibodies. J. Immunotherapy 15, 42-52.Kirby, C., Clarke, J., and Gregoriadis, G. (1980) Effect of the cholesterol content of smallunilamellar liposomes on their stability in vivo and in vitro. Biochem. J. 186, 591-598.Klausner, R. D., Blumenthal, R., Innerarity, T., and Weinstein, J. N. (1985) The interaction ofapolipoprotein A-i with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine. J.Biol. Chem. 260, 13719-13727.Klibanov, A. L., Maruyama, K., Torchilin, V. P. and Huang, L. (1990) Amphipathicpolyethylene glycols effectively prolong the circulation time of liposomes. FEBS Lett. 268, 235-237.Kohn, S., Nagy, J. A., Dvorak, H. F., and Dvorak, A. M. (1992) Pathways of macromoleculartracer transport across venules and small veins. Structural basis for the hyperpemeability oftumor blood vessels. Lab. Invest. 67, 596-607.131Lasic, D. D. (1994) Sterically stabilized vesicles. Angew. Chem. mt. Ed. Engi. 33, 1685-1698.Lasic, D. D., Martin, F. J., Gabizon, A., Huang, S. K. and Papahadjopoulos, D. Stericallystabilized liposomes: a hypothesis on the molecular origin of the extended circulation times(1991) Biochim. Biophys. Acta 1070, 187-192.Lazar, G., van Galen, M., and Scherphof, G. L. (1989) Gadolinium chloride-induced shifts inintrahepatic distributions of liposomes. Biochim. Biophys. Acta 1011, 97-101.Em, 3. H. (1994) Dose-dependent pharmacokinetics: experimental observations and theoreticalconsiderations. Biopharmaceutics and Dmg Disposition 15, 1-31.Litzinger, D. C. and Huang, L. (1992) Amphipathic poly(ethylene glycol) 5000-stabilizeddioleoylphosphatidylethanolamine liposomes accumulate in spleen. Biochim. Biophys. Acta1127, 249-254.Liu, D., Mori, A., and Huang, L. (1992) Role of liposome size and RES blockade in controllingbiodistribution and tumor uptake ofGM-containing liposomes. Biochim. Biophys. Acta 1104,95-101.Liu, D., Mori, A., and Huang, L. (1991) Large liposomes containing ganglioside GMI accumulateeffectively in spleen. Biochim. Biophys. Acta 1066, 159-165.Livingston, R. B. (1994) Dose intensity and high dose therapy. Cancer 74, 1177-1183.Longman, S. L, Tardi, P., Parr, M. J., Choi, L. S. L., Cullis, P. R., and Bally, M. B. (1995)Accumulation of protein coated liposomes in an extravascular site: influence of increasingcarrier circulation lifetimes. J. Pharm. Exp. Ther. in press.Loughry, H. C., Ferraretto, A., Cannon, A., Acerbis, G., Sudati, F., Bottiroli, G., Masserini, M.,and Soria, M. R. (1993) Characterization of biotinylated liposomes for in vivo targetingapplications. FEBS Left. 332, 183-188.Madden, T. D. (1986) Current concepts in membrane protein reconstitution. Chem. Phys. Lipids40, 207-222.Madden, T. D., Janoff, A. S., and Cullis, P. R. (1990a) Incorporation of Amphotericin B intolarge unilamellar vesicles composed of phosphatidylcholine and phosphatidylglyerol. Chem.Phys. Lipids 52, 189-198.Madden, T. D., Harrigan, P. R., Tai, L. C., Bally, M. B., Mayer, L. D., Redelmeier, T. E.,Loughrey, H. L., Tilcock, C. P., Reinish, L. W., and Cullis, P. R. (1990b) The accumulation ofdrugs within large unilamellar vesicles exhibiting a proton gradient: a survey. Chem. Phys.Lipids 53, 37-46.Maruyama, K., Unezaki, S., Takahashi, N., and Iwatsura, M. (1993) Enhanced delivery ofdoxorubicin to tumor by long-circulating thermosensitive liposomes and local hyperthermia.Biochim. Biophys. Acta 1149, 209-216.Maruyama, K., Yuda, T., Okamoto, A., Kojima, S., Suginaka, A. and Iwatsuru, M. (1992)Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoylphosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). Biochim.Biophys. Acta 1128, 44-49.Mauk, M. R. and Gamble, R. E. (1979) Stability of lipid vesicles in tissues of the mouse. A y-rayperturbed angular correlation study. Prod. Natl. Acad. Sci. U. S. A. 76, 765-769.132Mayer, L., D., Hope, M. J., Cullis, P. R., and Janoff, A. S. (1985) Solute distributions andtrapping efficiencies observed in freeze-thawed multilaxnellar vesicles. Biochim. Biophys. Acta817, 193-196.Mayer, L. D., Bally, M. B., and Cullis, P. R. (1986) Uptake of Adriamycin into large unilamellarvesicles in response to a pH gradient. Biochem. Biophys. Acta 857 123-126.Mayer, L D., Bally, M. B., Loughrey, H., Masin, D., and Cullis, P. R. (1990a) Liposomalvincristine preparations which exhibit decreased drug toxicity and increased activity againstmurine L1210 and P338 tumors. Cancer Res., 50: 575-579.Mayer, L. D., Bally, M. B., Cullis, P. R., Wilson, S. L., and Emerman, J. T. (1990b) Comparisonof free and liposome encapsulated doxorubicin tumor uptake and anti tumor efficacy in theSC 115 murine mammary tumor. Cancer Lett. 53, 183-190.Mayhew, E. G., Goidrosen, M. H., Vaage, J., and Rustum, Y. M. (1987) Effects of liposomeentrapped doxorubicin on liver metastases of mouse colon carcinomas 26 and 38. JNCI 78, 707-713.Mayhew, E. G., Lasic, D., Babbar, S. and Martin, F. J. (1992) Pharmacokinetics and antitumoractivity of epirubicin encapsulated in long circulating liposomes incorporating a polyethyleneglycol-derivatized phospholipid. Intl. J. Cancer 51, 302-309.Mimms, L. T., Zainpigi, G., Nozaki, Y., Tanford, C., and Reynolds, J. A. (1981) Phospholipidvesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry20, 833-840.Minow, R. A., Benjamin, R. S., and Gottlieb, J. A. (1975) Adriamycin (NSC 123 127)-cardiomyopathy: an overview with determination of risk factors. Cancer Chem. Rep. 6, 195-201.Miyata, H., Abe, M., Takehana, K., Yamaguchi, M., Masty, J., Iwasa, K., and Hiraga, T. (1994)Two distinct types of reticular cells in the pig sheathed artery. Acta Mat. 149, 209-214.Moghimi, S. M. and Patel, H. M. (1989) Serum opsonins and phagocytosis of saturated andunsaturated phospholipid liposomes. Biochim. Biophys. Acta 984, 384-387.Mori, A., Klibanov, A. L., Torchilin, V. P. and Huang, L. (1991) Influence of the steric barrieractivity of amphipathic poly(ethylene glycol) and ganglioside GMI on the circulation time ofliposomes and on the target binding of immunoliposomes in vivo. FEBS Lefl. 284, 263-266.Nagarkatti, M., Clary, S., and Nagarkatti, P. S. (1990) Characterization of tumor-infiltratingCD4+ T cells as Th 1 cells based on lymphokine secretion and functional properties. J. Immunol.144, 4898-4905.Needham, D., McIntosh, T. 3. and Lasic, D. D. (1992) Repulsive interactions and mechanicalstability of polymer-grafted lipid membranes. Biochim. Biophys. Acta 1108, 40-48.Nichols, J. W., and Deamer, D. W. (1976) Catecholamine uptake and concentration by liposomesmaintaining pH gradients. Biochim. Biophys. Acta 455, 269-271.Ning, S., MacLeod, K., Abra, R. M., Huang, A. H., and Hahn, G. M. (1994) Hyperthermiainduces doxorubicin release from long-circulating liposomes and enhances their anti-tumorefficacy. mt. J. Radiation Oncology Biol. Phys. 29, 827-834.Oja, C., Semple, S., Chonn, A., and Cullis, P. R. (1995) Influence of dose on liposome clearance:role of blood proteins. J. Biol. Chem. submitted.133Oku, N., Mamba, Y., and Okada, S. (1992) Tumor accumulation of novel RES-avoidingliposomes. Biochim. Biophys. Aeta 1126, 255-260.Olson, F., Mayhew, E., Maslow, D., Rustum, Y., and Szoka, F. (1982) Characterization, toxicity,and therapeutic efficacy of adriamycin encapsulated in liposomes. Eur. J. Cancer Clin. Oncol.18, 167-176.Owellen, R. J., Owens, A. H., and Donigian, D. W. (1972) The binding of vincristine,vinbiastine, and coichicine to tubulin. Biochem. Biophys. Res. Commun. 47, 685-691.Owellen, R. J., Hartke, C. A., Dickerson, R. M., and Hainis, F. 0. (1976) Inhibition of tubulinmicrotubule polymerization by drugs of the Vinca alkaloid class. Canc. Res. 36, 1499-1502.Papahadjopoulos, D., Jacobson, K., Nir, S., and Isac, T. (1973) Phase transitions in phospholipidvesicles. Fluorescence polarization and permeability measurement concerning the effect oftemperature and cholesterol. Biochim. Biophys. Acta 311, 330-348.Papahadjopoulos, D., Allen, T. M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S. K., Lee,K.-D., Woodle, M. C., Lasic, D. D., Redemann, C., and Martin, F. J. (1991) Sterically stabilizedliposomes: improvements in pharmacokinetics and anti-tumor activity. Proc. Natl. Acad. Sci. U.S. A. 88, 11460-11464.Park, Y. S. and Huang, L. (1993) Effect of chemically modified GMI and neoglycolipid analogsof GM1 on liposome circulation time: evidence supporting the dysopsonin hypothesis. Biochim.Biophys.Acta 1166, 105-14.Pate!, H. M. (1984) Liposomes: bags of challenge. Biochem. Soc. Trans. 12, 333-334.Phillips, N. C. (1989) Kupffer cells and liver metastasis. Optimization and limitation ofactivation of tumoricidal activity. Cancer. Metast. Rev. 8, 231-252.Poste, G., Kirsh, R., and Kuster, T. (1984) The challenge of liposome targeting in vivo. InLiposome Technology, Vol. III. Gregoriadis, G., ed. CRC Press, Boca Raton, FL, pp 1-28.Pratten, M. K. and Lloyd, J. B. (1986) Pinocytosis and phagocytosis: the effect of size of aparticulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro.Biochim. Biophys. Acta 881, 307-3 13.Rahman, Y. E., Cerney, E. A., Patel, K. R., Lau, E. H., and Wright, B. J. (1982) Differentialuptake of liposomes varying in size and lipid composition by parenchymal and Kupffer cells ofmouse liver. Life Sciences 31, 2061-2071.Rhinehart, J. J., Lewis, R. P., and Ba!cerzak, S. P. (1974) Adriamycin cardiotoxicity in man.Ann. Intern. Med. 81, 475-478.Roerdink, F., Dijkstra, J., Hartman, G., Bolscher, B, and Scherphof, G. (1981) The involvementof parenchymal, Kupffer and endothelial liver cells in the hepatic uptake of intravenouslyinjected liposomes. Effects of lanthanum and gadolinium salts. Biochim. Biophys. Acta 667, 79-89.Rottenberg, H. (1979) The measurement of membrane potential and delta pH in cells, organelles,and vesicles. Meth. Enzymol. 55, 547-569.Senior, J. H. (1987) Fate and behavior of liposomes in vivo: a review of controlling factors. CRCCrit. Rev. Ther. Drug Carrier Syst. 3, 123-193.134Senior, J. H. (1990) Liposome in vivo: prospects for liposome based pharmaceuticals in the1990’s. Biotechnol. Genetic Eng. Rev. 8, 279-3 17.Senior, J. H. (1992) How do hydrophilic surfaces determine liposome fate in vivo. 3. LiposomeRes. 2, 307-3 19.Senior, J. H., Trimble, K. R., Maskiewicz, R. (1991 a) Characterization of positively-chargedliposomes with blood: implications for their application in vivo. Biochim. Biophys. Acta 1070,173-179.Senior, J., Delgado, C., Fisher, D., Tilcock, C. and Gregoriadis, G. (1991 b) Influence of surfacehydrophilicity of liposomes on their interaction with plasma protein and clearance from thecirculation: studies with poly(ethylene glycol)-coated vesicles. Biochim. Biophys. Acta 1062,77-82.Sessler, J. L., Magda, D., Furuta., H. (1992) Synthesis and binding properties of monomeric anddimeric guanine and cytosine amine derivatives. J. Org. Chem. 57, 8 18-826.Shroit, A. J., Jadsen, J., and Nayer, R. (1986) Liposome cell interactions: in vitro discriminationof uptake mechanism and in vivo targeting strategies to mononuclear phagocytes Chem. Phys.Lipids 40, 373-393.Silverman, B. A., Carney, D. F., Johnston, C. A., Vanguri, P, and Shin, M. L. (1984) Isolation ofmembrane attack complex of complement from myelin membrane treated with serumcomplement. J. Neurochemistry 42, 1024-1029.Silvius, J. R. and Zuckermann, M. J. (1993) Interbilayer transfer of phospholipid-anchoredmacromolecules via monomer diffusion. Biochemistry 32, 3153-3161.Storm, G., Steerenberg, P. A., Emmen, F., van Borrsum Waalkes, M., and Crommelin, D. J.(1988) Release of doxorbucin from peritoneal macrophages exposed in vivo to doxorubicincontaining liposomes. Biochim. Biophys. Acta 965, 136-145.Sung, C., Schockley, T. R., Morrison, P. F., Dvorak, H. F., Yarmush, M. L., and Dedrick, R. L.(1992) Predicted and observed effects of antibody affinity and antigen density on monoclonalantibody uptake in solid tumors. Canc. Res. 52, 377-3 84.Szoka, F. and Papahadjopoulos, D. (1978) Procedure for preparation of liposomes with largeinternal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. U.S. A. 75, 4194-4198.Szoka, F., and Papahadjopoulos. D. (1980) Comparative properties and methods of preparationof lipid vesicles (liposomes). Ann. Rev. Biophys. Bioeng. 9, 467-508.Thomas, G. D., Chappell, M. J., Dykes, P. W., Ramsden, D. B., Godfrey, K. R., Ellis, J. R. M.,and Bradwell, A. R. (1989) Effect of dose, molecular size, affinity, and protein binding on tumoruptake of antibody or ligand: a biomathematical model Canc. Res. 49, 3290-3296.Thomas, C., Nijenhuis, A. M., Dontje, B., Daemen, T., and Scherphof, G. L. (1995) Tumoricidalresponse of liver macrophages isolated from rats bearing liver metastases of colonadenocarcinoma. J. Leukocyte Biology 57, 617-623.Toth, C. A. and Thomas, P. (1992) Liver endocytosis and Kupffer cells. Hepatology 16, 25 5-266.Toyohara, A. and Inaba, K. (1989) Transport of phagosomes in mouse peritoneal macrophages.3. Cell Science 94, 143-153.135Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S., Casazza, A. M., Firestone,R. A., Helistrom, I., and Helistrom, K. E. (1993) Cure of xenografted human carcinomas byBR96-doxorubicin immunoconjugates. Science 261, 212-215.Vaage, J., Mayhew, B., Lasic, D. and Martin, F. (1992) Therapy of primary and metastatic mousemammary carcinomas with doxorubicin encapsulated in long circulating liposomes. Intl. 3.Cancer 51, 942-948.Vaage, J., Donovan, D., Mayhew, B., Uster, P., and Woodle, M. (1993) Therapy of mousemammary carcinomas with vincristine and doxorubicin encapsulated in sterically stabilizedliposomes. mt. 3. Cancer 54, 959-964.Van Meer, G., Davoust, J., and Simons, K. (1985) Parameters affecting low-pH-mediated fusionof liposomes with the plasma membrane of cells injected with influenza virus. Biochemistry 24,3593-3602.Van Rooijen, N. (1989) The liposome-mediated macrophage suicide technique. J. Immunol.Methods 124, 1-6.Viero, J. A. and Cullis, P. R. (1990) A novel method for the efficient entrapment of calcium inlarge unilamellar phospholipid vesicles. Biochim. Biophys. Acta 1025, 109-115.Vose, J. M. and Armitage, J. 0. (1995) Clinical applications of hematopoietic growth factors. J.Clin. Oncol. 13, 1023-1035.Weinstein, J. N. and van Osdol, W. (1992) Early intervention in cancer using monoclonalantibodies and other biological ligands: micropharmacology and the binding site barrier. Canc.Res. 52(Suppl), 2747s-275 is.Wong, M., Anthony, F. H., Tillack, T. W., and Thompson, T. E. (1982) Fusion ofdipalmitoylphosphatidylcholine vesicles at 4°C. Biochemistry 21, 4126-4132.Woodle, M. C. and Lasic, D. D. (1992) Sterically stabilized liposomes. Biochim. Biophys. Acta1113, 171-199.Woodle, M. C., Matthay, K. K., Newman, M. S., Hidayat, J. E., Collins, L. R., Redemann, C.,Martin, F. 3. and Papahadjopoulos, D. (1992) Versatility in lipid compositions showingprolonged circulation with sterically stabilized liposomes. Biochim. Biophys. Acta 1105, 193-200.Wu, N. Z., Da, D., Rudolf, T. L., Needham, D., Whorton, A. R., and Dewhirst, M. W. (1993)Increased microvascular permeability contributes to preferential accumulation of stealthliposomes in tumor tissue. Cancer Res. 53, 3765-3 770.Yuan, F., Leunig, M. Huang, S. K., Berk, D. A., Papahadjopoulos, D, and Jam, R. K. (1994)Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomesin a human tumor xenograft. Cancer Res. 54, 3352-3356.Zalipsky, S., Seltzer, R. and Menon-Rudoiph, S (1992) Evaluation of a new reagent for covalentattachment of polyethylene glycol to proteins. Biotechnol. Appi. Biochem. 15, 100-114.136


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items