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Biophysical and anticancer properties of mitoxantrone in programmable fusogenic vesicles Adlakha-Hutcheon, Gitanjali 1999

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B I O P H Y S I C A L A N D A N T I C A N C E R P R O P E R T I E S OF M I T O X A N T R O N E IN P R O G R A M M A B L E F U S O G E N I C V E S I C L E S by G I T A N J A L I A D L A K H A - H U T C H E O N B.Sc., Delhi University, 1988 M . Sc., G . B. Pant University of Agriculture and Technology, 1991 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E STUDIES D E P A R T M E N T O F P H A R M A C O L O G Y A N D T H E R A P E U T I C S We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA June, 1999 © Gitanjali Adlakha-Hutcheon, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P H A R M A C - Q LO^Y OAXJ Trf £ R A P f T U T / C S The University of British Columbia Vancouver, Canada Date 2b*43»*n DE-6 (2/88) ABSTRACT This thesis characterizes programmable fusogenic vesicles (PFVs) and examines their usefulness as carriers for an anticancer drug. PFVs are cationic liposomes designed to exhibit time-dependent destabilization. They consist of non-bilayer-forming lipids that are stabilized into a bilayer by exchangeable poly(ethylene glycol)-conjugated lipids (PEG-lipids). The rate at which the vesicles destabilize is determined by the rate at which the bilayer stabilizing component, PEG-phosphatidylethanolamine (PEG-PE), exchanges out of the vesicle. In turn, this exchange rate is controlled by the acyl composition of the P E G - P E . PFVs are composed of l ,2-dioleoyl -5«-glycero-3-phosphoethanolamine (DOPE), cholesterol ( C H O L ) , ArN-dioleyl-N, Af-dimethylammonium chloride ( D O D A C ) and a P E G -conjugated lipid. The morphology of PFVs was examined as a function of lipid composition using cryo-transmission electron microscopy (cryo-TEM). While predominantly unilamellar, PFVs exhibit a variety of morphologies, including spherical, discoid and invaginated shapes. In the absence of PEG-lipid, PFVs formed in distilled water are spherical unilamellar structures but aggregation/fusion is observed when these systems are placed in isotonic saline. A n essential finding is that despite the high proportion of non-bilayer-forming lipids in PFVs, these vesicles can efficiently accumulate the anticancer drug mitoxantrone in response to an imposed transmembrane proton gradient. Release of intravesicular contents of PFVs was demonstrated using radiolabeled sucrose. The rates of plasma elimination and biodistribution of PFVs and mitoxantrone-loaded PFVs were characterized as functions of P E G - P E acyl chain length. The presence of mitoxantrone did not alter the time-course of elimination of PFVs from the circulation. The rate of elimination of PFVs from the circulation depended on the chain length of PEG-lipid ii anchor. This is because long chain lengths of P E G - P E slow its loss from the P F V surface and, in turn, prolong the blood residence of PFVs. For instance, PFVs containing PEG-1,2-d i s tearoy l - s« -phosphoethanolamine (DSPE) showed the slowest exchange and the longest blood residence times among the three anchors examined. The efficacy and accumulation within tumors of mitoxantrone-loaded PFVs were examined using a human colon carcinoma (LSI80) xenograft in severe combined immune deficient (SCID) mice. PFVs prepared with a slow-exchanging PEG-l ipid delayed tumor progression more effectively than either PFVs that are rapidly eliminated from the circulation or conventional liposomes. Efficacy of mitoxantrone-loaded PFVs was also evaluated against a murine tumor model in which disease progression occurs primarily in the liver. A l l P F V formulations were significantly more active against this tumor model than free mitoxantrone or mitoxantrone-loaded conventional liposomes suggesting that PFVs are capable of releasing drug more efficiently than conventional formulations. iii T A B L E O F C O N T E N T S ABSTRACT ii T A B L E OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES ix ABREVIATIONS x ACKNOWLEDGMENTS xii DEDICATION xiv CHAPTER 1: INTRODUCTION 1 1.1 Project Overview - Programmable Fusogenic Vesicles - towards a Transformable Liposome 1 1.2 Lipids: Building Blocks of Liposomes 3 1.2.1 Chemistry and Physics of Lipids 3 Phospholipids 3 Sphingolipids 5 Cholesterol 5 1.2.2 Lipids: Structure and Behavior 6 Lipid Polymorphism 6 1.3 Types of Liposomes 9 1.3.1 Liposome Classification: Based on Size 10 Multilamellar Vesicles (MLVs) 10 Large Unilamellar Vesicles (LUVs) 12 Small Unilamellar Vesicles (SUVs) 13 1.3.2 Liposome Classification: Based on Lipid Composition 13 pH-sensitive Liposomes 14 Target-sensitive Immuno-Liposomes 14 Temperature-sensitive Liposomes 15 1.3.3 Surface Modifying Lipids 15 Poly(ethylene glycol) Lipids 15 Cationic Lipids 21 1.4 Liposomal Drug Loading 23 1.4.1 Hydrophobic Association 23 1.4.2 Passive Encapsulation 24 1.4.3 Active Loading 24 1.5 Mitoxantrone - An Anthracenedione Derivative 27 1.5.1 Structure 27 1.5.2 Biological Effects 27 Pharmacodynamics 28 Pharmacokinetics 31 • 1.5.3 Liposomal Mitoxantrone 32 1.6 Liposomes in a Biological Milieu 33 1.6.1 Liposome-Macromolecule and/ or Cell Interactions 33 1.6.2 Fate of Intravenously Injected Liposomes 36 Factors Influencing the Blood Residence Times of Liposomes 37 Mononuclear Phagocyte System 40 Passive and Active Targeting 41 1.6.3 Extravasation of Liposomes from Blood 42 Extravasation: From the Blood to the Interstitium 42 1.7 The Ideal Liposome - An Enigma 45 1.7.1 The Conceptual Dilemma 45 1.8 Novel Liposome Design 46 1.8.1 Programmable Fusogenic Vesicles 47 Composition 47 Mechanism of Action of Programmable Fusogenic Vesicles: A Hypothesis 48 1.10 Research Hypothesis 50 CHAPTER 2: MATERIALS and METHODS 2.1 Materials 52 2.2 Cell lines and Animals 53 2.3 Preparation of Programmable Fusogenic Vesicles 53 2.3.1 Establishment of pH Gradient 54 2.3.2 Establishment of an Osmotic Gradient 54 2.4 Determination of Trapped Volume of PFVs 54 2.5 Size Analysis of PFVs 55 2.6 Cryogenic-Transmission Electron Microscopy (cryo-TEM) 56 2.6.1 Interpretation of Cryo-Transmission Electron Micrographs 56 2.7 Encapsulation of Mitoxantrone 57 2.8 Release of PFV Contents 59 2.8.1 Release of Mitoxantrone from PFVs 59 2.8.2 Release of [14C]-Sucrose from PFVs 59 2.9 Animal Studies of Mitoxantrone-loaded PFVs 60 2.9.1 Plasma Elimination Studies 60 2.9.2 Tissue Biodistribution 60 2.9.3 Studies on Exchange of PEG-lipids from PFVs or Conventional Vesicles in vivo 61 2.9.4 Plasma Elimination Studies of Exchange of DODAC from PFVs in vivo 61 2.9.5 Analysis of Plasma Samples by Fast Protein Liquid Chromatography 62 2.9.6 Tumor Accumulation and Plasma Elimination Studies of Mitoxantrone-loaded PFVs 63 2.9.7 Establishment of Maximum Tolerated Dose 63 2.9.8 Antitumor Activity against Human Colon Carcinoma Xenografts 64 2.9.9 Antitumor Efficacy against Murine L1210 Leukemia 65 2.10 Statistical Analysis 65 CHAPTER 3: CHARACTERIZATION OF PROGRAMMABLE FUSOGENIC VESICLES IN VITRO 3.1 Introduction 67 3.2 Results 69 3.2.1 Morphology of PFV 69 Morphology of PFV: Influence of PEG-PE 69 Morphology of PFVs: Influence of Cationic Component, DODAC 72 3.2.2 Uptake of Mitoxantrone in PFVs 76 Uptake of Mitoxantrone in PFVs: Influence of PEG-lipid Species 76 Effect of Drug to Lipid Ratio on Uptake of Mitoxantrone in PEG-Ceramide Containing-PFVs77 Stability of Mitoxantrone Uptake in PFVs Containing PEG-Ceramide 77 3.2.3 Visualization of Mitoxantrone Encapsulated within PFVs 81 3.2.4 Programmed Release of PFV Contents: Influence of Incubation with Sink and Negatively Charged Vesicles 83 3.3 Discussion 89 v CHAPTER 4: CHARACTERIZATION OF THE SYTEMIC PROPERTIES OF PFVs: INFLUENCE OF POLY(ETHYLENE GLYCOL)-PHOSPHATIDYLETHANOLAMINE A C Y L COMPOSITION 4.1 Introduction 96 4.2 Results 97 4.2.1 Plasma Elimination of Programmable Fusogenic Vesicles and Encapsulated Mitoxantrone: Influence of PEG-PE Acyl Composition 97 4.2.2 Biodistribution of Mitoxantrone-Loaded PFVs 100 4.2.3 Plasma Concentrations of PEG-PE and PFVs: Influence of PEG-PE Acyl Chain Lengths 106 4.2.4 Plasma Elimination of PEG-PE from DSPQCHOL Liposomes: Influence of PEG-PE Acyl Chain Length 108 4.2.5 Rate of Exchange of PEG-PE from The Carrier: Influence of Vesicle Composition 112 4.2.6 In Vivo Exchange of DODAC from Programmable Fusogenic Vesicles 113 4.2.7 Distribution of PEG-PE within Blood Compartment Determined by Fast Protein Liquid Chromatography 116 CHAPTER 5: EVALUATION OF THE ANTICANCER PROPERTIES OF MITOXANTRONE ENCAPSULATED WITHIN PROGRAMMABLE FUSOGENIC VESICLES 5.1 Introduction 128 5.2 Results 132 5.2.1 Elimination of mitoxantrone and PFVs from plasma and their accumulation in solid tumor xenografts on SCID/RAG-2 mice 132 Mitoxantrone and Lipid levels in the plasma 132 Mitoxantrone and Lipid Accumulation in LSI 80 Solid Tumors 133 5.2.2 Therapeutic Activity of Mitoxantrone-Loaded PFVs 137 Efficacy of Mitoxantrone-loaded PFVs against LSI 80 human colon carcinoma model. 137 Efficacy of Mitoxantrone-loaded PFVs against Liver localized disease 140 5.3 Discussion 145 CHAPTER 6: DISCUSSION and IMPLICATIONS 6.1 Significance of Results 151 6.2 Implications 152 REFERENCES 155 vi LIST O F F I G U R E S Figure 1.1 Structures of Common Lipids 4 Figure 1.2 Lipid Polymorphism 7 Figure 1.3 Types of Liposome 11 Figure 1.4 Structures of Surface-modifying Lipids 19 Figure 1.5 Active Drug Encapsulation in Liposomes 26 Figure 1.6 Structure of Mitoxantrone Hydrochloride 30 Figure 1.7 Liposome-Cell Interactions 34 Figure 1.8 Liposome in a Biological Milieu: Target Site Accumulation 39 Figure 1.9 Capillary Endothelial Structure and Extravasation 43 Figure 1.10 Mechanism of Action of Programmable Fusogenic Vesicles: A Hypothesis.. 49 Figure 2.1 Interpretation of a 3D object as a 2D image 58 Figure 3.1 Cryo-transmission electron micrographs of PFVs in the absence of P E G - P E . . . 73 Figure 3.2 Cryo-transmission electron micrographs of P F V of different compositions.... 75 Figure 3.3 Uptake of mitoxantrone in PFVs: Influence of PEG-lipid species 78 Figure 3.4 Uptake of mitoxantrone by PFVs: Influence of drug to lipid ratio on uptake of mitoxantrone in PFVs containing PEG-Ceramide 79 Figure 3.5 In vitro release of mitoxantrone from PEG-Ceramide-PFVs: effect of drug to lipid ratio 80 Figure 3.6 Cryo-transmission electron micrograph of "empty" programmable fusogenic vesicles and mitoxantrone-loaded PFVs 82 Figure 3.7 In vitro release of aqueous contents from PFVs: Influence of concentration and acyl composition of P E G - P E 88 vii Figure 4.1 Plasma elimination of mitoxantrone-loaded PFVs after i.v. administration: Influence of P E G - P E acyl composition 103 Figure 4.2 Biodistribution of PFVs and mitoxantrone in the liver of BDF-1 mice 105 Figure 4.3 Plasma elimination of P E G - P E and PFVs after i.v. administration: Influence of P E G - P E acyl composition 107 Figure 4.4 Elimination of P E G - P E and liposomes from plasma: influence of P E G - P E acyl chain composition 110 Figure 4.5 Loss of P E G - P E from liposomes in blood I l l Figure 4.6 Plasma elimination of the cationic lipid, D O D A C from PFVs: influence of P E G -P E composition 114 Figure 4.7 Separation of liposomes from plasma components using Fast Protein Liquid Chromatography: distribution of P E G - P E 120 Figure 5.1 Plasma elimination of mitoxantrone and mitoxantrone-loaded PFVs from tumor bearing mice 135 Figure 5.2 Tumor accumulation of mitoxantrone and liposomal lipid in the human LSI80 solid tumor xenograft 136 Figure 5.3 Mitoxantrone mediated LS180 solid tumor growth inhibition 138 Figure 5.4 Therapeutic activity of mitoxantrone-loaded PFVs against a murine L1210 leukemia model 143 viii LIST O F T A B L E S Table 1. Types of liposomes 10 Table 2. Mean diameter of P F V from quasielastic light scattering and actual measurements from the micrographs 71 Table 3. A comparison of release of the aqueous contents of PFVs as a function of concentration and P E G - P E chain length on day 2 in the presence of 10-fold sink 85 Table 4. A comparison of release of the aqueous contents of PFVs as a function of concentration and P E G - P E chain length on day 7 in the presence of 30-fold sink 87 Table 5. Mean area under the curve (AUC) for mitoxantrone and P F V lipid in plasma and other selected tissues 104 Table 6. Mean area under the concentration-time curves for programmable fusogenic vesicle and conventional vesicles ( D S P C : C H O L ) 137 Table 7. L1210 Antitumor activity of free and mitoxantrone-loaded PFVs in BDF-1 mice. 144 ix A B R E V I A T I O N S A U C area under the curve C H O L cholesterol C H E cholesteryl hexadecyl ether C n V conventional liposomes c ^ m a x tumor concentration levels cryo-TEM cryogenic transmission electron microscopy D D A B N, Af-dimethyl-A7, A^-dioctadecylammonium bromide D O D A C N, TV-dioleyl-Af A^-dimemylammonium chloride D O P C 1,2-dioleoyl-.s«-glycero-3 -phosphatidy choline D O P E 1,2-dioleoyl-5«-glycero-3 -phosphatidy lethanolamine D S P C 1,2-distearoyl-5/7-glycero-3 -phosphatidylcholine E D T A ethylenediaminetetraacetic acid E P C egg phosphatidylcholine F A T M L V frozen and thawed L U V s F P L C fast protein liquid chromatography G M i monosialoganglioside G M 1 H n hexagonal phase H B S HEPES-buffered saline H D L high density lipoprotein H E P E S A^-2-hydroxyethylpiperazine-7V-2-ethane-sulphonic acid i.p. intraperitoneal i.v. intravenous L D L low density lipoproteins L U V s large unilamellar vesicles M e P E G monomethoxypoly(ethylene glycol) M L V s multilamellar vesicles M P S mononuclear phagocyte system M T D maximum tolerated dose P A phosphatidic acid P C phosphatidylcholine P E phosphatidylethanolarnine P E G poly(ethylene glycol) PEG-Cer poly(ethylene glycol) ( 2 0 0 0 )-modified ceramide PEG-CerC20 1 -0-(2' -(w-methoxypolyethyleneglycol(2000)succinoyl)-2-A /-arachidoylsphingosine P E G - P E poly(ethylene glycol) ( 2 0 0 0 )-modified phosphatidylethanolarnine P E G - D M P E poly(ethylene glycol)(2000)-dimyristoylphosphatidylethanolamine P E G - D P P E poly(ethylene glycol)(2000)-dipalmitoylphosphatidylethanolamine P E G - D S P E poly(ethylene glycol)(2000)-disteroylphosphatidylethanolamine P E G - D M P E - P F V P F V prepared with P E G - D M P E P E G - D P P E - P F V P F V prepared with P E G - D P P E P E G - D S P E - P F V P F V prepared with P E G - D S P E P F V programmable fusogenic vesicle P G phosphatidylglycerol PI phosphatidylinositol POPS 1 -palmitoyl, 2-o leoyl - i , «-glycero-3-phosphat idylchol ine Q E L S quasielastic light scattering RES reticulo-endothelial system s.c. subcutaneous SCID severe combined immune deficient mice S M sphingomyelin SSL sterically stabilized liposomes S U V s small unilamellar vesicles T * c transition temperature V L D L very low density lipoproteins A C K N O W L E D G M E N T S " O M N A M O SHIVAI" It is with a lot of joy that I begin this section by offering my humble thanks to God and the powers that be, for giving me the perseverance to do as I dreamt. I am grateful to Dr. Tom Madden, for giving me a chance just when I almost quit. I would also like to thank Tom for making me strive beyond my own capabilities. I am indebted to Dr. Marcel Bally, for his support and for encouragement par excellence, thank you. This thesis would not have been possible without the competent help of Dana Masin towards all the animal work. Dr. Katarina Edwards provided long-distance assistance with Cryo-transmission electron microscopy. Dr. Edward Choice helped with the running of the F P L C . I would like to express my appreciation to Mac, Cliff and all the fun folks in the Cullis and the Bally lab, respectively, for sharing their lab space and for keeping me confused about where I really belonged! I gratefully acknowledge the G . R . E . A . T award from The Science Council of British Columbia and University Graduate Fellowship from The University of British Columbia for financial support. I am hard pressed for words to acknowledge my family members who believed in me unconditionally. To my two guardian angels, my mothers, Prema whose cancer made me seek the answers and Adarsh, whose prayers helped me find them; Papa, for being a true 'marshal', constantly motivating me to march forward; my little sister, Ami , for being my xii pillar of strength and above all, my friend, my husband, Bruce for being there in science and in spirit - a heartfelt thank you. While I am putting down thank you's in black and white, friends who I could not have done without: Vijaya, for 'following up on me'; Siri, for being the 'chicken soup for my soul'; Carine, for cheering me on; Betty, for acquainting me with "Oh Henry's" and providing me with a home away from home; fellow Mahikarians for keeping me in the "light", a BIG thank you. A n amazing source of inspiration was Nelson Mandela and I thank him for writing Long Walk to Freedom. Lastly, I wish to acknowledge my virtual friend: "e-mail" for keeping me connected to my husband every day of the 4 years, x months and days. xiii D E D I C A T E D T O my husband, my second conscience B R U C E C H A P T E R 1: I N T R O D U C T I O N 1.1 Project Overview - Programmable Fusogenic Vesicles - towards a Transformable Liposome The potential use of liposomes in the delivery of bioactive agents was recognized shortly after the discovery of liposomes (Bangham, 1968; Sessa and Weissman, 1968 and Gregoriadis, 1973). Technological advances towards optimizing liposomes as drug carriers have culminated in several of these systems being approved for human use or undergoing clinical trials. While these advances are encouraging, they are unable to meet the ultimate goal of an ideal delivery vehicle - that of optimal bioavailability, which is probably best achieved by directly delivering the entrapped agent into the cytoplasm of the target cell. One advantage associated with liposomal carriers for anticancer drugs, such as adriamycin, includes reduced toxicity over the free drug to healthy tissue, even for very simple systems (Rahman, 1982; Olson, et al., 1982). The reduced cardiac toxicity of doxorubicin in the liposomally encapsulated form allowed the administration of higher doses of doxorubicin over free drug (Gabizon et al., 1982; Mayer et al., 1989; Bally et a l , 1990a). A second advantage is derived from the decreased plasma elimination rate of liposomally entrapped drug as compared to the free drug. The reduced elimination from plasma due to prolonged circulation lifetimes of the carrier has been strongly correlated with "passive targeting" or the tendency of liposomes to accumulate at disease sites (Gabizon and Papahadjopoulos, 1988; Gabizon et al., 1990; Gabizon, 1992). It is believed that this selective accumulation reflects extravasation of liposomes across immature or fenestrated capillary endothelium within rapidly growing tumors (Wu et al., 1993; Bally et al., 1994). 1 Preferential accumulation of liposomes allows liposomes to act as reservoirs for release of drug into the affected tissue (Mayer, et al., 1990a; Huang, et al., 1992; Gabizon, 1992). Improvements in the circulation longevity of the carrier have been achieved by refinements such as incorporation of long-chain saturated lipids, reduced liposome size, neutral surface charge and incorporation of surface-modifying agents such as poly(ethylene glycol) (PEG)-lipid conjugates. However, the degree to which carrier accumulation at the target site will reflect drug delivery is determined by the rate of drug released as well as the rate of accumulation in the tumor. For example, carrier systems which display higher levels of drug retention will promote enhanced drug delivery to the tumor. An inherent contradiction therefore arises, in the optimization of a conventional liposomal drug delivery system. In selecting drug retention properties that minimize systemic exposure of agents encapsulated in long circulating liposomes and maximize tumor delivery, drug bioavailability may be severely limited (Mayer et al., 1994; reviewed by Bally et al., 1998). After carriers have accumulated in the tumor interstitial space, encapsulated drug must be released in order to be taken up by the surrounding neoplastic cells. These considerations imply that significant advances in the use of liposomes for therapeutic purposes will require the ability to formulate "transformable" systems that exhibit different characteristics depending on where they reside following intravenous administration. The objective of this thesis was to characterize a novel, transformable liposome formulation, programmable fusogenic vesicles (PFVs), which should help to address the long-standing problem of compromised bioavailability of drugs encapsulated within liposomes. 2 1.2 Lipids: Building Blocks of Liposomes Liposomes are closed spheres with one or more lipid bilayers containing a defined aqueous core. In general, liposomes are made up of phospholipids and cholesterol. Advances in this carrier technology have included the use of surface-modifying hydrophilic polymers and cationic lipids. These vesicle components will now be discussed in the context of this thesis. 1.2.1 Chemistry and Physics of Lipids Phospholipids The importance of phospholipids is perhaps best described in the words of Ludwig Buechner (1827-1899) "Ohne phosphor, kein gedanke" - "Without phosphorus there would be no thoughts"(Bangham, 1983). The major class of naturally occurring phospholipids are the glycerophospholipids or phosphoglycerides. Like other membrane lipids, phosphoglycerides are amphiphilic molecules made up of a polar headgroup and one or more hydrophobic acyl chains attached to a central glycerol backbone. The characteristics of each lipid molecule are determined by the chemical nature of the headgroup and tails (a summary of phospholipid structures is shown in Figure 1.1 A) which, in turn, dictate the physical properties of the lipid bilayer. The headgroups of phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylinositol (PI) are anionic, while those of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are zwitterionic. This translates into a negative surface charge at physiological pH for liposomes containing PS, PA, PG or PI. 3 B Sphingolipid Phosphatidylcholine (PC) R-CH 2 CH 2 N(CH 3 ) 3 Phosphatidylethanolarnine (PE) + C Z z j ^ > Headgroup > Glycerol Backbone R-NH C = £ > Acyl chain R-CH 2 CH 2 NH 3 R-H H + R C H — C - N H 3 Phosphatidic acid (PA) Phosphatidylserine (PS) n u n . - — _ ioo-Phosphatidylglycerol (PG) R-CH 2 CH(OH)CH 2 OH Phosphatidylinositol (PI) OR" R = H; R" = H Sphingosine R = COR"; R' = H Ceramide (Cer) R = COR"; R' = phosphocholine Sphingomyelin (SM) (R" = hydrocarbon) Some naturally occurring fatty acids Laurie (12:0) CH,(CH,),0COOH Myristic(14:0) C r y c H^coOH Palmitic (16:0) C R , ( C H , ) „ C O O H Stearic (18:0) C H , ( C H , ) „ C O O H Palmitoleic (16:1,A9) C H , ( C H J ) 5 C H = C H ( C H , ) 7 C O O H Oleic (18:1. A9) CH,(CH3)7CH=CH(CHj)7C00H Linoleic (18:2, A9'1 2) CH J(CH,)4CH=CHCH2CH=CH(CH J)7C00H Unolenic(18:3,A9'12'15) CHjCHjCH-CHCHjCHoCHCHjCH'CHfCHjJjCOOH Cholesterol (Choi) Figure 1.1 Structures of Common Lipids 4 Sphingolipids Lipid derivatives of the long chain amino alcohol, sphingosine, constitute the second major class of membrane lipids. A class of sphingolipids referred to as ceramides are formed when a fatty acid is linked via an amide linkage to the amino group of sphingosine (Figure L I B ) . One of the most common membrane sphingolipids is sphingomyelin (SM). Sphingomyelin has a phosphocholine group esterified to the 1-hydroxyl group of a ceramide. Cholesterol Cholesterol is an amphipathic molecule that constitutes the principal neutral lipid component of eukaryotic biological membranes. While its polar 3-hydroxyl group is oriented towards the lipid-water interface, its rigid ring structure lies buried within the hydrophobic interior with the phospholipid acyl chains. Cholesterol has the ability to decrease the rigidity or order of gel phases and increase the order of liquid-crystalline phases (see below, section above their transition temperature (T c , Oldfield and Chapman, 1972; Demel and de Kruijiff, 1976). A reduction in the enthalpy of the gel to liquid-crystalline phase transition ensues on increasing the cholesterol content above 7%. In addition, at 33% cholesterol and higher, the phase change becomes undetectable (Hubbell and McConnel, 1971; Demel and de Kruijiff, 1976; Linseisen et al., 1993). As a rule, the permeability and order parameter of a bilayer are inversely related. Thus, the addition of cholesterol to liquid-crystalline bilayers increases the molecular packing of the membrane and subsequently decreases its permeability while the reverse is true for gel phase membranes (Bittman and Blau, 1972; reviewed by Yeagle, 1985). Cholesterol content is one of the variants of liposome structure that could 5 control retention of drugs within the vesicle. The role of cholesterol in improving the retention of solutes by liposomes within a biological milieu was first investigated by Kirby and coworkers (1980). Since the inclusion of cholesterol in lipid bilayers helps to reduce solute permeability (Demel et al., 1972; Papahadjopoulos et al, 1973; Kirby et al., 1980), cholesterol has been incorporated in liposomes formulated for systemic drug delivery. The stability of cholesterol-containing liposomes has been attributed to low lipid exchange with lipoproteins (Scherphof, 1979) and limited interactions with plasma proteins (Moghimi and Patel, 1988; Semple et al., 1996). Prolonged circulation lifetimes are a characteristic of cholesterol-stabilized vesicles (Kirby et al., 1980; Senior and Gregoriadis, 1982; Semple et al., 1996). 1.2.2 Lipids: Structure and Behavior The primary structural characteristics of lipids not only depend on the lipid species but also upon the surrounding environmental conditions, such as pH and temperature of the medium. Upon hydration, lipids adopt specific conformations, the most common of which is the lipid bilayer; however, other non-bilayer organizations (hexagonal, H n and isotropic phases) have also been characterized. Lipid Polymorphism Lipid polymorphism is defined as the ability of lipids to adopt different phases such as the lamellar or bilayer, the hexagonal, H n phase and other non-bilayer phases grouped together as the "cubic" phases (Cullis et a l , 1986). B LIPIDS Lysophospholipids Detergents PHASE Micellar MOLECULAR SHAPE • Inverted Cone Phosphatidylcholine Sphingomyelin Phosphatidylserine Phosphatidylinositol Phosphatidylglycerol Phosphatidic Acid Cardiolipin Digalactosyldiacylglycerol If i l Bil 11 111 ayer Cylindrical Phosphatidylethanojamine Cardiolipin - Ca Phosphatidic Acid - Ca Phosphatidic Acid (pH<3.0) Phosphatidylserine (pH<4.0) Monogalactosyldiacylglycerol Hexagonal (H||) Cone Figure 1.2 L ip id Polymorphism (A) Factors influencing the bilayer to hexagonal phase transition (B) Polymorphic phase behavior of lipids (Figures 1.2A and 1.2B were adapted from Gruner et al., 1985 and Cullis et al., 1985, respectively). 7 Various intrinsic and extrinsic factors influence lipid polymorphism. The intrinsic factors include the nature of the lipid head group and/or acyl chains. The p H and temperature of the medium, the presence of ions, and lipids or proteins constitute external factors affecting the polymorphic behavior of lipids (Figure 1.2). The subject of lipid polymorphism has been the topic of many reviews (Cullis and de Kruijff, 1979; Gruner, et al., 1985; Lindblom and Rilfors, 1989; Seddon, 1990). The molecular basis of lipid polymorphism stems from the shape of the lipid molecule which in turn is dependent on the three-dimensional volume occupied by the hydrophilic head group and the hydrophobic tail (Israelachvili et al., 1980; Cullis et al., 1986). A cylindrical molecular shape arranged in a planar organization is associated with bilayer forming lipids. Dispersions of lipids that demonstrate this packing behavior are P C , PS (pH > 4.0), PI, P A , P G (pH > 3.0), cardiolipin (CL) and sphingomyelin (SM). Other lipids are believed to have a "cone" shaped arrangement because they have a small head group volume relative to their acyl chains, for example unsaturated P E , PS (pH < 4.0) and P A (pH < 3.0). Such lipids have the tendency to organize themselves into a tubular arrangement with the hydrophilic head groups facing inwards, towards an aqueous channel and outwards facing the aqueous medium while the hydrophobic tails interact with the acyl chains of other lipid molecules (Tanford, 1980). In contrast, lipids such as detergents and lysophospholipids (which contain only one acyl chain) possess a large head with respect to the tail portion and adopt an "inverted cone" geometry which results in micelle formation. Modulation of factors such as p H , temperature and addition of counter-ions allows alteration of the preferred phase of a given lipid. The addition of micelle-forming species like detergents (Madden and Cullis, 1982) or 20-50 moi % of P C or PS (pH > 4.0) into P E systems can stabilize the H„ phase into 8 a bilayer conformation (Cullis et al., 1985; Seddon, 1990). A summary of factors that influence the inter-conversion of bilayer to H n phase is shown in Figure 1.2A. On the basis of their phase transition temperature (T c), lipids can adopt either a 'frozen' gel, solid (L p ) phase or a liquid crystalline ( L J phase. T c is principally dependent on the length and saturation of the acyl chain. In general, shorter, unsaturated acyl phospholipids have a lower phase transition temperature than longer, saturated acyl chains (reviewed by Cullis and Hope, 1991 and Fenske, et al., 1995). The gel state is characterized by a rigid, ordered acyl chain organization, and shorter or unsaturated acyl chains disrupt this organization, thereby lowering the T c . A n order parameter "S" often defines the acyl chain motion, where S = 1 represents no motion and S = 0 stands for rapid isotropic motion. Above the T c , the more fluid liquid-crystalline state exists, exhibiting decreased order and an increased movement of the membrane components: lateral diffusion, rotation around the long molecular axis and trans-gauche isomerization occurs. In addition, membranes are more permeable to a variety of solutes and solvents at or above T c than below (Bittman and Blau, 1972). 1.3 Types of Liposomes In general, hydration of dried, bilayer-forming lipids in aqueous solutions above the phase transition temperature of the lipid components results in liposome formation (Bangham, et al., 1965). These liposomes are typically referred to as multilamellar vesicles (MLVs) . Further processing of M L V s can produce liposomes that result in uniform and small size. Such liposomes can be categorized on the basis of size and lamellarity, or lipid composition (Table 1.1). Table 1.1 Types of Liposomes Basis of classification Size M L V s (1 pm - 100 pm) LUVs (0.05 pm - 1.0 pm) SUVs (< 0.05 nm) Lipid Composition Conventional: base carrier Second generation: "Stealth", incorporation of PEG-lipids Third generation: surface-associated targeting information (antibodies) Function pH-sensitive liposomes Target-sensitive immuno-liposome (antibodies attached) Temperature-sensitive Contents release triggered based on the lipid T c 1.3.1 Liposome Classification: Based on Size Vesicles of different sizes can be obtained by varying the mode of preparation of liposomes. Such types of vesicles include multilamellar vesicles (MLVs), large unilamellar vesicles, (LUVs) and small unilamellar vesicles (SUVs) as shown in Figure 1.3. Multilamellar Vesicles (ML Vs) Liposomes formed by the dispersion of lipid films or powder in aqueous solution by mechanical agitation consist of concentric bilayers and are referred to as multilamellar vesicles. 10 c Figure 1.3 Types of Liposomes Schematic representation and freeze fracture micrographs of (A) M L V s , (B) LUVs , and (C) SUVs prepared from egg phosphatidylcholine. The bar in the upper left corner in micrograph A represents 200 nm in length. (Freeze fracture micrographs were courtesy of Cullis and Ostro, 1989). 11 Typically these M L V s are heterogenous in size, ranging from 1pm to 100 pm. M L V s also exhibit variation in lamellarity such that less than 10% of the total lipid is present in the outermost bilayer of most M L V s (Mayer et al., 1985a). Low aqueous trap volumes are commonly associated with M L V s which makes them poor candidates for delivering drugs. Sequential freezing and thawing of M L V s is one way of increasing the trap volume because the number of internal bilayers are thus reduced, yielding the so-called frozen and thawed M L V s (FATMLVs, Mayer et al., 1985a). Alternatively, incorporating charged lipids increases the interbilayer spacing by enhancing the charge repulsion between lamellae, thereby increasing the trap volumes (Hope et al., 1986). Large Unilamellar Vesicles (LUVs) Second in size to M L V s are large unilamellar vesicles (LUVs) which range from 0.05 pm to 1.0 pm in diameter. Techniques employed to formulate LUVs include ether injection (Deamer and Bangham, 1976), reverse phase evaporation (Szoka and Papahadjopoulos, 1978), ethanol injection (Chen and Schullery, 1979), detergent dialysis (Mimms et al., 1981; Madden, 1986) and extrusion through polycarbonate filters (Olson et al., 1979; Hope et al., 1985). LUVs are the most commonly used systems for delivering drugs because they are stable and have higher trapped volumes (1-3 pl/pmol lipid) than M L V s (0.5 ul/umol lipid), allowing greater drug encapsulation (reviewed by Cullis et al., 1987). In addition, L U V s have relatively long circulation half-lives as compared to M L V s (Juliano and Layton, 1980; Abra and Hunt, 1981; Allen and Everest, 1983). Of all the methods listed above for preparing LUVs , the most commonly used is the process of extrusion due to its ease of usage, 12 simplicity, speed and reproducibility. In addition, the problems of residual detergent and/or organic solvent are avoided. In brief, the technique relies on repeatedly (ten times) forcing M L V s or F A T M L V s through polycarbonate filters of a defined pore size (Olson et al., 1979; Mayer et al., 1985a; Hope et al., 1985; Hope et al., 1986). Extrusion is applicable over a wide variety of lipid compositions and concentrations, producing well defined vesicles with diameters close to those of the filter pores. Small Unilamellar Vesicles (SUVs) Unilamellar vesicles possessing the smallest diameter (typically < 50 nm) fall into the class of small unilamellar vesicles (SUVs). SUVs can be prepared directly from M L V s by sonication (Huang, 1969) and/or French press (Barenholz et al., 1979). The packing constraints induced by the small radius of SUVs limits the maximum curvature that the bilayer can assume, thus generating unstable preparations subject to fusion (Lichtenberg et al., 1981; Wong et al., 1982). Concomitant with this instability are low trapped volumes (Huang, 1969; Barenholz et al., 1979) and lipoprotein-induced leakage of contents (Scherphof and Morselt, 1984), all of which make SUVs unsuitable as drug delivery vehicles. 1.3.2 Liposome Classification: Based on Lipid Composition Another way of classifying liposomes is on the basis of their lipid composition. Conventional liposomes have been defined as "un-derivatized membrane bilayers composed of naturally occurring lipids" (Bally et al., 1998). Alterations in the basic lipid composition of conventional liposomes generate surface-modified or derivatized liposomes. Most changes in liposomal lipid components were intended to optimize a specific liposomal 13 attribute. F o r instance, attempts to formulate fusogenic l iposomes such as pH-sens i t ive and target-sensitive immuno- l iposomes were intended for the cy toplasmic de l ivery o f entrapped contents. V e r y often the pr imary l i p i d component o f these fusogenic l iposomes was a l i p i d , l ,2-dioleoyl- .yH-grycero-3 -phosphatidylethanolarnine ( D O P E ) , w h i c h is non-bi layer fo rming and fusogenic under phys io log ica l condit ions. Temperature-sensitive l iposomes were the result o f l iposome designs a imed at cont ro l l ing the release o f encapsulated contents. A l t h o u g h pH-sens i t ive , target-sensitive and temperature-sensitive l iposomes were conceptual ly advanced over convent ional l iposomes, their appl icabi l i ty was l im i t ed in vivo. Some o f these l imita t ions are detailed be low. Surface properties o f l iposomes can be mod i f i ed b y the inc lus ion o f polymers such as poly(ethylene g lyco l ) and/or cat ionic l ip ids . These l ip ids w i l l be covered i n greater depth i n section 1.3.3. pH-sensitive Liposomes pH-sens i t ive l iposomes typ ica l ly consist o f D O P E stabi l ized by a pH-sens i t ive fatty acy l amino ac id or fatty ac id such as o le ic acid. P r i m a r i l y these carriers employ a variety o f protonable l ip ids to trigger vesicle destabil izat ion under ac idic condit ions tak ing advantage o f endosomal ac id i f ica t ion ( Y a t v i n , 1980; C o n n o r and Huang , 1985; C o n n o r et a l . , 1986). H o w e v e r , the disadvantage o f such pH-sensi t ive l iposomes stems f rom their tendency to aggregate and become leaky i n the presence o f serum (Connor et a l . , 1986). Target-sensitive Immuno-Liposomes Target-sensitive immuno- l iposomes are s imi lar to the class o f pH-sens i t ive , D O P E -containing l iposomes but differ i n that these are s tabi l ized direct ly by an acylated m o n o c l o n a l 14 antibody (Huang et al., 1982; Ho et a l , 1986). The monoclonal antibody was included to confer target cell specificity. The binding of the antibody to the target cell antigen caused aggregation of the stabilizer in the contact region with the cell, thereby removing the stabilizers from the rest of the immuno-liposome. Transient increases in the encapsulated drug at the target cell surface have been reported by this mechanism (Ho et al., 1986; reviewed by Holmberg and Huang, 1989). A disadvantage associated with antibody-coated liposomes was their rapid clearance from the blood compartment (Leserman et al., 1983; Loughrey etal., 1993). Temperature-sensitive Liposomes Temperature-sensitive liposomes are designed to undergo a lipid phase transition at a temperature slightly above 3 7 ° C , resulting in leakage of the encapsulated drug. Localized heating of a tumor site should therefore trigger selective drug release (Weinstein et al., 1980). Although the feasibility of this approach can be demonstrated in experimental models, it does not address the major difficulty associated with cancer, namely that of treating widespread metastases. 1.3.3 Surface-Modifying Lipids Poly (ethylene glycol) Lipids There is a wealth of data available on polymer-coated nanoparticles and other polymer-coated spheres that persist in the circulation longer than uncoated particles (reviewed by Brannon-Peppas, 1995). Longer blood residence of such particles has been attributed to reduced protein binding due to steric stabilization. These data were applied to refining liposome formulations towards achieving longer circulation half-lives. Interest in the development of liposomes exhibiting enhanced circulation lifetimes was renewed after the initial successes with the inclusion of monosialoganglioside G M , (Allen et al., 1985). However, one of the major drawbacks to the use of G M , for enhancing vesicle circulation longevity was its cost. Therefore, alternatives to G M , were sought and some of the other polymers used to modify the vesicle surface characteristics have included PEG-cholesterol (Allen et al., 1991; Vertut-Doi, 1996), amphiphilic poly(acrylamide) and poly(vinylpyrrolidone) (Torchilin et al., 1994b), oligosaccharides and polysaccharides (Lasic, 1994) and thiolytically cleavable PEG-polymers (Kirpotin et al., 1996). However, all of these polymers were shown to be less effective than poly(ethylene glycol) PEG-phosphatidylethanolamine (PEG-PE) (reviewed by Harris and Zalipsky, 1997). The focus of much interest in the liposome community, therefore, is the polymer polyethylene glycol), or PEG (-[0-CH 2-CH 2] n-) (Figure 1.4). This is primarily because PEG is unusually effective at excluding other polymers such as proteins from its presence in an aqueous environment (Harris, 1992). This property translates into protein rejection, relatively weakened antigenicity and immunogenicity. PEG appears to be a simple molecule. It is a neutral polyether available in a variety of molecular weights and is soluble in water and most organic solvents. In addition, PEG lends itself very well to conjugation to other molecules. Abuchowski and coworkers (1977) observed that conjugates formed by covalent attachment of PEG to proteins are active and have enhanced serum lifetimes. Mori et al. (1982) reported that covalent attachment of PEG to surfaces retards protein adsorption to these surfaces. These observations led to the use of PEG polymers coupled to proteins and 16 drugs for systemic use. While Senior et al. (1991b) have described a method to coat PEG on the outer surface of pre-formed liposomes, the preferred method of incorporation is the inclusion of PEG in the original formulation mixture. Surface modification of liposomes with PEG conjugated to lipid anchors has evolved as one of the most successful techniques to enhance the circulation half-life of liposomes (reviewed by Harris, 1992; Harris and Zalipsky, 1997). The properties of PEG that promote steric stability in polymer-grafted lipid bilayer systems include its ability to extend 5 nm from the surface of the bilayer (Needham et al., 1992). In addition, the flexible hydrophilic PEG (molecular weight 2000) layer is able to maintain a separation of 4 nm between bilayers containing 4 moi % PEG2 0 0o-PE (Needham et al., 1992; Zalipsky, 1993). The scaling laws that first enabled the calculation of polymer conformation and the repulsive forces above the grafted polymer surface were described by Alexander (1977); and deGennes (1980). These have been reviewed by Milner (1991); Lasic and Martin (1995). The conformation of the PEG layer is determined by the polymer content such that, at higher levels, a "brush model" best describes the properties of the PEG layer while a "mushroom model" is more appropriate at low polymer densities (deGennes, 1980, 1987). Recent experimental data and theoretical simulations by Rex and coworkers (1998) suggest that throughout the range (0-10 mole% PEG200o-PE or 0-6 mole % PEG5 0 0 0-PE) of polymer densities the grafted PEG chains exist in a mushroom conformation. In either model of polymer conformation, a certain loss of translational, rotational and configurational entropy occurs (Janin and Chothia, 1978; Silvius and Zuckermann, 1993) which is probably responsible for the reduced surface adsorption of protein to PEG-coated surfaces (Arakawa and Timasheff, 1985). While in vitro evidence points to a direct correlation between an 17 increased polymer density, chain length and higher repulsive forces (Kuhl et al., 1994; Lasic, 1994), optimal circulation levels in plasma in vivo are observed with short chain lengths with a molecular weight of 2,000 Da (approximately 46 monomeric residues) at 5 mole %. Liposomes containing PEG4ipids in the bilayer have often been referred to as "sterically stabilized liposomes" (SSL). Although the mechanism underlying liposome circulation longevity remains unresolved, one school of thought believes it to be a contribution of three factors, namely; high lipid bilayer rigidity (Kirby et a l , 1980; Senior et al., 1985), absence of surface charge and high surface hydrophilicity (Maruyama et al., 1991; Mori et al., 1991; Senior et al., 1991b and reviewed by Lasic, 1994). However, Blume and Cevc (1993) suggest that liposome longevity is controlled by the mobility and size of the polar head group of P E G . A refined view of the surface characteristics of liposomal P E G -phosphatidylethanolamine (PE) suggest it to be a combination of steric and dynamic properties of the polymer. P E G is envisioned not only as an initial steric barrier, inhibiting the close approach, but also a sweeping action inhibiting protein binding near the liposome surface (reviewed by Lasic, 1994). A theoretical model of a "statistical cloud" has also been described to explain the reduced bilayer adsorption in the presence of P E G (Torchilin et al., 1994a). The molecular origin of PEG4iposome stability in blood is probably a consequence of its extended conformation and reduced interactions with plasma proteins. There is evidence for decreased serum interactions in vitro (Senior et al., 1991a; Blume and Cevc, 1990) as well as in vivo (Chonn et a l , 1992, Semple et al., 1996 and Oja, 1998). PEG-Ceramide (C14) DDAB Figure 1.4 Structures of Surface-modifying Lipids (A) Polyethylene glycol)-lipids: PEG-Ceramide(C14) and PEG-DMPE (B) Cationic lipids: DODAC and D D A B 19 It is of interest, however, that little attention has been paid to the extent to which the PEG coating remains associated with the liposome, in view of the enhanced rates of spontaneous exchange exhibited by PEG-lipid conjugates compared to the non-derivatized lipid (Silvius and Zuckermann, 1993). Parameters that regulate the transfer half-life of PEG-lipids include the length and degree of saturation of the lipid fatty acyl chains and the size of the PEG moiety (Silvius and Zuckermann, 1993). Consequently, liposomes composed of shorter or unsaturated acyl chains (POPE-PEG 2 0 0 0) were found to have a shorter circulation life in vivo than the longer, saturated PEG derivatives (DSPE-PEG 2 0 0 0 ; P a r r e t al., 1994). This aspect of transfer of PEG-lipid conjugates from a vesicle is addressed further in this thesis in Chapter 4. Although polymer-coated liposomes exhibit increased circulation half-lives, this attribute of the carrier does not necessarily ensure an improved bioavailibility of the encapsulated drug (Mayer et al., 1994). In addition, Lim et al. (1997) have demonstrated no increase in efficacy of drug encapsulated within longer circulating, non-derivatized, conventional liposomes made of DSPC:CHOL over that of free drug. These observations taken together suggest that although an improvement in the blood residence time of a carrier enhances its selective accumulation at a disease site, this does not translate into an increase in drug bioavailibility. The lack of a direct correlation between prolonged carrier circulation, enhanced delivery of carrier to disease site and efficacy could mean one of two things: (a) entrapped drug remains within the liposome, or (b) all the drug has leaked out before carrier accumulation takes place at the target site. Parr et al. (1997) have shown equivalent levels of doxorubicin accumulation in the Lewis lung carcinoma solid tumor model with doxorubicin encapsulated within DSPC:CHOL (conventional liposomes) or PEG-containing sterically 20 stabilized liposomes. Their results rule out the option (b) above. Therefore, in improving the delivery of a drug-loaded vesicle, the release of drug from the carrier was being compromised. This thesis seeks to find a solution to this problem. Cationic Lipids The use of charged lipids as liposomal components is another way to modify surface properties. Biological cationic lipids are extremely rare; only sphingosine occurs in nature. The first use of cationic lipids such as stearylamine as a component of liposomes came about in the early seventies (Gregoriadis, 1973; Gregoriadis and Neerunjun, 1974; Juliano and Stamp, 1975) but did not progress further, as positively charged vesicles exhibited more rapid clearance in vivo compared to neutral liposomes (Juliano and Stamp, 1975; Senior, et al., 1991b). Another factor curtailing their use was the evidence that positively charged liposomes containing stearylamine were toxic in vivo (Adams et al., 1977). Interest in the development of gene transfer techniques capable of delivering genetic material in sufficient concentrations to appropriate sites of expression has been revived with the possibility of human gene therapy. Although viral vectors are a popular choice, they are very often associated with the significant problem of being immunogenic. This drawback of viral vectors catalyzed the search for alternative vectors. It was not until the report of the first successful cationic lipid-mediated D N A transfection in vitro that the technology of using cationic liposomes as non-viral vectors became widespread (Feigner et al., 1987). While A^A'-dioleyl-A^A^-dimethylammonium bromide (DODAB), (Kunitake and Okahata, 1977) and l,2-dioleolxy-3-(trimethylammonio) propane (DOTAP), (Eibl and Wooley, 1979) were the first two cationic lipids to be synthesized, the utility of positively charged lipids as 21 components of non-viral vectors further provided an impetus for the synthesis of other novel cationic lipids. Cationic liposomes have been widely used in mediating the delivery of D N A (Feigner et al., 1987; Pinnaduwage et a l , 1989; Rose et al., 1991; L i u et al., 1997; Templeton et al., 1997), R N A (Malone et al., 1989; Weiss et al., 1989), oligonucleotides (Bennett et al., 1992; Wagner, 1994; Litzinger et al., 1996) and proteins (Debs et al., 1990; Walker et al., 1992) into living cells. Therefore, efforts in the synthesis of cationic lipids have included enhancing the properties desirable for good transfection agents, for example, lower toxicity, ability to transfect several cell lines and biodegradability. It should be noted that a transfection agent typically consists of a cationic amphiphile and an accompanying "helper" or co-lipid, usually D O P E . The appropriate selection of either lipid plays a crucial part in determining the potency of such agents. To date, two hypotheses for the mechanism of entry of lipid-based transfection agents into transfecting cells have been put forward. The first route is via membrane fusion, while the second is by way of endocytosis. Earlier work on cationic liposomes of different formulations has demonstrated the ability of these vesicles to undergo membrane fusion with anionic vesicles (Stamatatos et al., 1988; Duzgunes et al., 1989; Leventis and Silvius, 1990). The lipid components of both cationic (Leventis and Silvius, 1990) and anionic target membranes determine the extent of membrane fusion (Bailey and Cullis, 1997). However, recent evidence suggests that the initial mode of entry of l ip id-DNA or liposome-DNA complexes occurs by endocytosis (Farhood et al., 1995; Wrobel and Collins 1995; Stegmann and Legendre, 1997). The rationale behind the use of cationic liposomes as transfection agents is that, since most cell surfaces contain negatively charged residues, these anionic biological surfaces could be targeted through charge interaction by a cationic delivery system (Feigner and 22 Ringold, 1989). Bearing this rationale in mind, the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC; Figure 1.4 B) was utilized as a component of programmable fusogenic vesicles. The synthesis of DODAC drew inspiration from N,N-dimethyl-Af Af-dioctadecylammonium bromide (DDAB), a saturated, bromide quarternary ammonium lipid (Ansell et al., 1998). DODAC is a monovalent, unsaturated amphiphilic, chloride quaternary ammonium lipid. 1.4 Liposomal Drug Loading Passive and active trapping are the two basic techniques for encapsulating drugs within liposomes. Whereas the drug is combined with the lipid at the time of liposome formation in the passive entrapment method, active trapping involves encapsulation of the drug in pre-formed liposomes exhibiting a transmembrane ion gradient. Hydrophobic association of drug to the liposomal bilayer constitutes a third category and applies mostly to lipophilic drugs. This form of encapsulation of hydrophobic agents is essentially a variation of passive encapsulation. 1.4.1 Hydrophobic Association Amphotericin B is an example of a non-polar drug bound by hydrophobic interactions to the vesicle bilayer by including it in the original lipid mixture before the preparation of the liposome. This technique can yield high efficiencies of incorporation depending upon the capacity of the bilayer to solubilize the agent while maintaining vesicle structure. Low levels of cyclosporin incorporation have been reported within vesicle membranes (Ouyang et al., 23 1995) and the potential application of such lipophilic drugs inserted into the vesicle bilayer is limited by the drug often exchanging into other lipid membranes (Choice et al., 1995). 1.4.2 Passive Encapsulation Water-soluble drugs can be entrapped within the liposome aqueous core by passive means; however, the extent of entrapment varies with the trapped volume of the vesicles. Efficiency of entrapment may vary anywhere from 1% (in SUVs; Szoka and Papahadjopoulos, 1980) to 80% (in F A T M L V s and L U V s ; Mayer, et al., 1985a). The leakage of drugs subsequent to passive encapsulation is frequent and occurs rapidly. For example, between 20 - 60% of passively loaded doxorubicin is released from egg phosphatidylcholine (EPC): cholesterol vesicles by 1 hour (Gabizon et al., 1982). In general, the retention rates can be improved significantly with the incorporation of cholesterol and long-chain, saturated acyl lipid species. 1.4.3 Active Loading The classes of drugs that can be successfully entrapped by active loading include: antineoplastics that are biogenic amines, local anesthetics, antiarrhythmics, antimalarial and antidepressant agents (surveyed by Madden et al., 1990b). Most of these drugs are lipophilic cations or anions with an ionizable amino or carboxyl functional group, respectively. Active entrapment commonly involves the use of a pH-gradient across pre-formed bilayers to drive the drug into the liposome (Mayer et al., 1986a; 1986b; 1986c; Bally et al., 1988) and takes advantage of the higher membrane permeability of the neutral form of the drug (Addanki et al., 1968; Rottenberg, 1979). For example, drugs such as doxorubicin and vincristine that are 24 weak bases will accumulate inside liposomes with acidic interiors. In an analogous manner, liposomes with a basic interior can be used to encapsulate weakly acidic drugs (Eastman et al., 1991). The mechanism of accumulation is based on the Henderson-Hasselbalch equation and is shown in Figure 1.5. The loading of doxorubicin by active means is far greater than that predicted by the residual p H gradient; this is likely due to the concentration of the drug exceeding its solubility product and precipitating within the vesicle interior (Madden et al., 1990b; Mayer, et al., 1990a; 1990c; 1990d; Harrigan, et al., 1993). In addition, dissipation of the p H gradient (ApH) caused either by decreasing the external p H (Mayer et al., 1990d) or by the addition of proton gradient uncouplers led to a reduction in doxorubicin retention. Also, in contrast to release of doxorubicin entrapped passively, less than 5% of actively loaded doxorubicin was released from egg phosphatidylcholine: cholesterol ( C H O L ) liposomes by 24 hours (Mayer et al., 1986a; 1986b). The leakage of vincristine too appears to be related to the loss of A p H (Boman et al., 1993). The use of ammonium sulphate to generate a p H gradient is a variation of the active loading by p H gradient (Haran et al., 1993). Other methods of active encapsulation of drugs include ion gradients such as a valinomycin-dependent K + diffusion gradient with a negative intraliposomal potential (Mayer et al., 1985b; Bally et al., 1988) and the formation of manganese-drug complexes (Cheung et al., 1998). 25 Outside pH 7.5 Inside pH 4.0 K = [B]0[H+]0 [BH+]0 At equillibrium, if: Then: K — [B],[H+], [BH+], [BL = P I [BH+], = [H], [BH+]0 [H+]0 Figure 1.5 Active Drug Encapsulation in Liposomes Redis t r ibu t ion o f a weak base ( l ipoph i l i c amine) i n response to a transmembrane p H gradient ( A p H ) , across the l iposomal bi layer . O n l y the neutral, uncharged fo rm o f the base is capable o f cross ing the l i p i d bi layer . (Figure taken f rom Parr, 1995) 26 1.5 Mitoxantrone - An Anthracenedione Derivative 1.5.1 Structure Mitoxantrone is a synthetic anthracenedione that lacks the amino sugar moiety and tetracyclic A-ring of the more commonly used drug in oncology, doxorubicin. Mitoxantrone or Novantrone®, (l,4-dihyroxy-5,8-bis (2-[(2-hydroxyethyl)-amino] ethyl) amino-9,10-anthracycenedione dihydrochloride) is an antitumor agent that owes its synthesis to two independent groups of researchers. Serendipity led to mitoxantrone synthesis for one group (Zee-Cheng and Cheng, 1978). The other group synthesized it from anthraquinone chemicals used as blue dyes in the fabric industry based on rational drug design (Murdock et. al., 1979; Figure 1.6). Mitoxantrone is a less cardiotoxic analog of the widely used antineoplastic anthracycline drug doxorubicin (reviewed by Smith, 1983; Weiss, R. J3., 1989; Birchall, 1991). It had been postulated that doxorubicin derived its cardiotoxic side effects, but not its antitumor activity, from the amino-sugar moiety (Adamson, 1974). Mitoxantrone was therefore added to the therapeutic armamentarium of the practicing oncologist with the promise of a better therapeutic index than doxorubicin. 1.5.2 Biological Effects The activity of mitoxantrone has been tested against a wide spectrum of experimental tumor models such as P388 and L1210 leukemias, B16 melanoma, colon and mammary adenocarcinomas and transitional bladder carcinoma (Johnson et al., 1979; Wallace et al., 1979; Corbett et al., 1982; Fujimoto and Ogawa, 1982; Schabel et al., 1983a, 1983b; Ballou and Tseng, 1986). The efficacy of mitoxantrone has been determined against Hodgkin's and non-Hodgkin's lymphomas (Bajetta, et al., 1988; Hiddemann et al., 1991; Ho et al., 1990 and 27 L i m , et al., 1992), melanoma (Arseneau et al., 1986) and multiple myeloma (Alberts et al., 1985). Mitoxantrone has been used systemically either alone or in combination with other chemotherapeutic agents in the treatment of advanced breast cancer (Allegra et al., 1985; Leiby et al., 1986; Broun et al., 1993), acute leukemia (Bezwoda et al., 1990; Amadori et al., 1991; Archimbaud et al., 1991; Hiddemann et al., 1991; Wahlin et al., 1991) and hepatocellular carcinoma (Dunk et al., 1985; Lai et a l , 1989; Yoshida et al., 1988; Colleoni et al., 1992). Palliative-care patients with hepatocellular carcinoma (Civalleri et al., 1996), advanced breast cancer (Roberston et al., 1989) or prostate cancer (Tannock et al., 1996) have also been treated with mitoxantrone. Pharmacodynamics Mechanism of Action There is evidence to attribute the antineoplastic activity of mitoxantrone to at least three modes of action. First, mitoxantrone interferes with the D N A rejoining step catalyzed by the enzyme topoisomerase II. Topoisomerase enzymes catalyze the severing and reannealing of D N A strands through enzyme-DNA complexes. Two types of topoisomerases are known - topoisomerse I and II for single and double strand breaks, respectively (reviewed by Wang, 1987). Second, mitoxantrone produces protein-associated D N A strand breaks by stabilizing the DNA-topoisomerase II complex (Nelson et al., 1984; Tewey et al., 1984) and intercalating with D N A (Johnson et al., 1979; Durr, 1984). The side chains of mitoxantrone interact electrostatically with anionic phosphate groups exposed on the D N A helix causing D N A aggregation and compaction (Foye et al., 1982; Lown et al., 1984). Finally, the oxidative activation of mitoxantrone by free radical generation induces non-protein-associated D N A strand breaks (Fisher and Patterson, 1989). Mitoxantrone produces 28 concentration- and time-dependent delays in cell cycle progression. It is most cytotoxic in the late S phase and inhibits D N A in both proliferating and resting cells (Drewinko et al., 1983) . The dose-limiting toxicity of mitoxantrone is myelosuppression. As compared to doxorubicin, its spectrum of toxicity is advantageous in that it causes less alopecia and can be administered for a longer period of time (reviewed by Weiss, 1989 and Faulds et al., 1991). In patients for whom alopecia or cardiotoxicity are a concern, mitoxantrone has a toxicity advantage over doxorubicin as shown by comparative trials (Allegra et al., 1985, Neidhart et al., 1984). The cumulative dose equivalent of mitoxantrone as a single agent theoretically permits 12 doses of 14 mg/m 2 vs. 9 doses of doxorubicin at 60 mg/m 2 (Dukart and Barone, 1984) . In addition, Tham et al. (1987) have shown that while doxorubicin toxicity is progressive and cumulative, mitoxantrone administered subsequent to doxorubicin exhibits either stabilization or regression of cardiomyopathy. Free Radical Formation In contrast to doxorubicin, mitoxantrone produces few, i f any semiquinone free radicals via the N A D P H cytochrome P-450 reductase pathway which in turn mediate non-protein-associated D N A breaks and membrane lipid peroxidation (Basra et al., 1985; Durr, 1984; Kharasch and Novak, 1985; Novak and Kharasch, 1985; Vile and Winterbourn, 1989). However, mitoxantrone undergoes an oxidative activation of an irreversible peroxidative type, yielding a cyclic compound which further oxidizes to an unstable diimino compound, finally forming a corresponding radical cation (Kolodziejczyk et al., 1988). Figure 1.6 Structure of Mitoxantrone Hydrochloride In the present studies, radiolabeled mitoxantrone was used (Mitoxantrone[1 4CJ). The asterisk denotes the position of the radiolabeled carbon atoms as per Alberts et. al. (1985) 30 Resistance The possible mechanisms of development of multidrug resistance by cancerous cells include decreased intracellular drug accumulation, enzymatic changes reducing susceptibility to D N A damage or enhancing D N A repair. Decreased intracellular drug accumulation is often accompanied by overexpression of P-glycoprotein ( M D R 1) which mediates increased drug efflux. Overexpression of P-glycoprotein has been observed in some mitoxantrone-resistant human tumor cell lines (Dalton et a l , 1986, 1988; Jensen et al., 1989). In a human colon carcinoma cell line, decreased intracellular mitoxantrone accumulation not associated with an increase in efflux of mitoxantrone has been observed (Wallace et al., 1987). Alteration in levels of topoisomerase II activity has been suggested to mediate resistance in a human promyelocytic leukemia cell line (Harker et al., 1989). Taken together, these observations point towards a multifactorial mechanism of resistance in response to mitoxantrone as with most anticancer drugs. Tumor cell lines exhibiting resistance to mitoxantrone in vitro display an atypical resistance profile, showing only partial cross-resistance to other intercalating agents (reviewed by Faulds, et al., 1991). Hazelhurst et al. (1998) have recently reported a non-transport-mediated mechanism of drug resistance to mitoxantrone in the M C F 7 breast cancer cell line. They suggest that resistance is mediated in part by M D R 1, multidrug resistant protein (MRP), altered D N A topoisomerase II and non-transport-mediated drug efflux. Pharmacokinetics The recommended administration of mitoxantrone is as an intravenous infusion as it is poorly absorbed orally. Tissue uptake and distribution are rapid and elimination is slow 31 with a prolonged terminal half-life (Savaraj et al., 1982; Alberts et a l , 1985). Following intravenous administration, the elimination of mitoxantrone generally displays triphasic kinetics. Despite extensive studies having been conducted on mitoxantrone pharmacokinetics in cancer patients, technical difficulties have yielded a wide range of pharmacokinetic values for the rapid initial (a) intermediate distribution (P) and the slow elimination phase (y) with half-lives of 3-10 minutes, 0.3-3.1 hours and up to 12 days, respectively (reviewed by Ehninger et al., 1990). Mitoxantrone has a large volume of distribution of approximately 2248 L / m 2 , suggesting that much of the drug is sequestered in the body tissues. It binds extensively to plasma proteins (78%), therefore less free form of the drug is bioavailable. Excretion of mitoxantrone is primarily via biliary and fecal routes (Savaraj et al., 1982; Alberts et al., 1985). The urinary metabolites of mitoxantrone include mono- and di-carboxylic acid derivatives (Chicccarelli et al., 1986) and mitoxantrone is detoxified in the liver via glucuronide and glutathione conjugation (Savaraj et a l , 1982). 1.5.3 Liposomal Mitoxantrone Although mitoxantrone has been the subject of pre-clinical and clinical studies since its synthesis in 1978 and 1979, it was not until 1990 that Madden et al. (1990b) reported loading of mitoxantrone within egg phosphatidylcholine liposomes by the "remote loading" technique (which is the same as active loading defined earlier, section 1.4.3). A number of investigators have conducted studies on encapsulation of mitoxantrone in multilamellar vesicles of various surface charges and lipid compositions (Law et al., 1991; Beck et al., 1993). Encapsulation of mitoxantrone within large unilamellar vesicles driven by a transmembrane p H gradient has been demonstrated by L i m et al., 1997; Chang et al., 1997. 32 On the other hand, Schwendener et al. (1991, 1994) have examined antitumor efficacy and acute toxicity using liposomal mitoxantrone formulations prepared using an anionic lipid-mitoxantrone complex. These investigators included mitoxantrone along with a negatively charged phospholipid (phosphatidylserine) before generation of liposomes i.e. mitoxantrone was loaded by passive means (section 1.4.2). 1.6 Liposomes in a Biological Milieu 1.6.1 Liposome-Macromolecule and/ or Cell Interactions Four mechanisms of interaction between liposomes and cells in vitro have been identified, namely: stable adsorption, endocytosis, fusion and lipid transfer or exchange (Figure 1.7, Pagano and Weinstein, 1978; Pagano et al., 1981). Stable adsorption is defined as the association of intact liposomes with the cell surface and generally is mediated by hydrophobic, non-specific electrostatic or other forces. The uptake of liposomes into endocytotic vesicles, mostly resulting in liposome delivery to lysosomes, is referred to as endocytosis. Fusion is the merging of the vesicle bilayer with the plasma membrane and concomitant release of liposomal contents into the cytoplasm. The transfer of individual lipid molecules between vesicles and the cell surface without the cell-association of aqueous contents is the process of lipid exchange (reviewed by Pagano and Weinstein, 1978; Pagano et al., 1981). In addition, these investigators provide criteria for distinguishing the various mechanisms (Pagano and Weinstein, 1978). While there are several lines of evidence that support adsorption, endocytosis and lipid exchange, there is a greater difficulty associated with obtaining unequivocal evidence for fusion. 33 Lipid Exchange Adsorption Fusion Endocytosis Figure 1.7 Liposome-Cell Interactions Lipid exchange, adsorption, endocytosis and fusion are four known possible interactions between cells and liposomes 34 Liposomes bind rapidly and irreversibly to proteins present within blood upon intravenous injection (Juliano and Lin, 1980; Juliano, 1983). The adsorption of specific proteins (opsonins) modifies the liposomal surface properties which further enhances their phagocytic elimination. Destabilization and leakage of vesicle contents corelates well with increased protein binding and is dependent upon lipid charge and composition (Hermandez-Caselles et al., 1993). A direct relationship has been established between the amount of protein bound in vivo and the uptake of liposomes by the reticuloendothelial cells using the spin column isolation procedure (Chonn et al., 1992; Semple et al., 1996). Protein-liposome interactions are influenced by liposome characteristics such as phospholipid composition, cholesterol content, surface charge and hydrophobicity. Liposomes containing phospholipids with saturated, long-chain fatty acids (e.g. D S P C , SM) experience decreased protein binding, reduced reticuloendohelial system (RES) uptake and decreased permeability (Gregoriadis and Senior, 1980; Senior and Gregoriadis, 1982b). This effect has been attributed to the close spacing of phospholipid headgroups with reduced acyl chain spreading. Liposomes containing negatively charged lipids are cleared rapidly by the R E S (Gregoriadis and Neerunjun, 1974; Juliano and Stamp, 1975; Chonn et al., 1992) and experience significant blood protein interactions. Examples of phospholipid exchange, especially from negatively charged liposomes to lipoproteins followed by membrane disruption (very low density lipoproteins ( V L D L s ) , low density lipoproteins (LDLs) and high density lipoproteins (HDLs)) have been documented (Damen et al., 1980; Tall and Green, 1981; Shahrokh and Nichols, 1982). Furthermore, a variety of apolipoproteins have been found to adsorb to liposomes in vitro and in vivo, including Apo AI , A l l , A I V , B and E (Nichols et al., 1978; Guo et al., 1980; Tall and Green, 35 1981; Mendez et al., 1988; Chonn et al., 1995). The leakage of liposomal contents following liposome-lipoprotein exchange has been attributed to both pore formation (Kirby and Gregoriadis, 1981) and the complete destruction of liposome structure (Scherphof and Morselt, 1984). It was speculated that lipid exchange may play a role in membrane biogenesis, in transporting lipids and other lipophilic molecules such as drugs and toxic compounds. Furthermore, exchange processes may contribute to transbilayer lipid asymmetry of the plasma membrane of cells (Pagano et al., 1981). Although examples of adsorption, lipid exchange and endocytosis are common mechanisms of liposome interactions with cells and/or macromolecules, fusion appears to be a rare event in vivo (New et al., 1990; Jones and Chapman, 1995). 1.6.2 Fate of Intravenously Injected Liposomes Important among the variables governing the fate of liposomes in vivo are gross compositional differences including different phospholipids or other lipids, neutral or charged. However, before discussing these factors it is essential to consider the interaction of liposomes with "compartments" in vivo that are distinct physiologically (Figure 1.8). Liposomes in the biological milieu would primarily encounter three "compartments", namely, the central blood compartment, the interstitial space and the cell. Within the central blood compartment, liposomes first are exposed to numerous circulating proteins and cellular components, several of which may interact with the lipid bilayer and destabilize the liposome or trigger biological reactions that further increase liposome leakage or elimination via the mononuclear phagocytic system (MPS). In order to exit the blood compartment, liposomes must traverse the vascular endothelium. This poses a prime barrier for liposomes to gain 36 access to a disease in an extravascular site and also allows differentiation between normal and diseased tissue. In the case that liposomes are able to cross the endothelial barrier, they enter into the interstitial space. The liposome movement and distribution within this compartment is hindered by factors such as interstitial volume and pressure and the presence or absence of the lymphatic system. Significant variability is observed within the interstitium both within the normal tissue in different organs and between normal and diseased tissue. The cells into which liposomes are taken up constitute the final physiological compartment. Included within this compartment are the intracellular organelles responsible for processing the administered agent. The cell membrane thus becomes the next critical hurdle for the development of therapeutically optimized liposomal agents, second only to the vascular endothelium (reviewed by Bally et al., 1998). Factors Influencing the Blood Residence Times of Liposomes Physical characteristics that influence circulation lifetimes of liposomes are size, composition, charge and dose. Enhanced retention of liposomes within the bloodstream can be achieved if the liposomes are smaller than 200 nm in size since large vesicles (> 1pm diameter) are cleared more rapidly, typically within 15 minutes. Juliano and Stamp (1975) have reported that M L V s exhibit four-fold shorter half-lives than L U V s (< 50 nm) of identical lipid composition and dose. Larger vesicles can be retained very effectively in the reticular filter structures (Claassen and Van Rooijen, 1984). Spleen is the organ principally responsible for mechanically trapping larger vesicles (Klibanov et al., 1990 and 1991; Litzinger and Huang, 1992). To a lesser degree, larger vesicles are eliminated by the lung, 37 where they can become trapped in the small capillaries (Abra et al., 1984). Very small vesicles (SUVs) too are cleared very quickly from circulation (Roerdink et al., 1981). Higher circulation levels have also been attributed to higher administered doses. At higher doses, at a given time point, lower liver uptake and higher circulation levels in terms of injected dose are observed (Poste et al., 1984). This effect is thought to be due to the saturation of liver macrophages which then results in 'spillover' into the spleen, followed by further spillover into the lung and subsequently into the bone marrow. Alterations causing a reduction in the effectiveness of liver phagocytosis also increase the concentration of liposomes in the blood and result in a 'spillover effect' with increased uptake by the spleen (Bradfield, 1974). Such changes occur in biodistribution since the amount of liposomes initially available to the spleen are dependent on the amount taken up by the liver. This effect has been observed upon increasing the lipid dose or predosing with liposomes (Abra et al., 1980; Abra and Hunt, 1981; Dave and Patel, 1986) or the use of long-circulating liposomes or sterically stabilized liposomes (Litzinger and Huang, 1992 and Klibanov et al., 1991). For example, at saturating pre-doses of M L V s (11 g/kg in mice) followed by an injection an hour later, Abra et al. (1980) have demonstrated a 5-fold reduction in the extent of liver uptake and a 4-fold increase in uptake by the spleen. However, more recent work suggests that the saturation phenomena observed at high doses of liposomes actually represent a depletion of certain blood opsonins (Oja et al., 1996). 38 Figure 1.8 Liposome in a Biological Milieu: Target Site Accumulation To allow for passive accumulation to the target site appropriately designed liposomal carriers must be retained in the blood compartment for extended time period (A). While in the blood compartment, liposomes interact with the cells lining the endothelium (B) or with specific target cells (C). Passive targeting is dependent on the presence of altered vascular endothelium, alterations that permit extravasation of circulating macromolecules (D). Following extravasation, liposomes can release drug while residing in the interstitial space (E) or can be taken up by tumor associated macrophages (F). The potential to achieve specific interactions with target cells (active targeting) through the use of targeting ligands is also feasible (G). (Figure taken from Harasym, 1997). 39 Mononuclear Phagocyte System The mononuclear phagocyte system consists of monocytes, macrophages and their precursor cells resident within the blood, liver, spleen, lungs, bone marrow and other sites in the body. MPS was traditionally referred to as the reticuloendothelial system (RES) and constitutes the host-defense system. The new name is a direct consequence of the recognition that circulating monocytes play a role in the clearance mechanism of liposomes (Senior, 1987). Cells of the MPS are involved in the removal of dead, senescent, foreign or altered cells, particulates such as latex beads (Pratten and Lloyd, 1986), regulation of other cell functions, presentation and processing of antigens in immune reactions, participation in inflammatory reactions, destruction of microorganisms and neoplastic cells. Given the aforementioned functions, it is not surprising that opsonized liposomes from the circulation are primarily taken up by the liver and the spleen. Macrophages in the liver (Kupffer cells) line the sinusoids, either attached to or embedded within the endothelium, in an ideal location to take up liposomes (Roerdink et al., 1981; Poste et al., 1982) or carbon particles (Kampschmidt et al., 1966) and other molecules. The extent of participation of parenchymal cells (hepatocytes) is determined by the dose and size of the injected liposomes. In general, uptake by hepatocytes is substantial for smaller vesicles (<50 nm) which can extravasate easily through the pores of the discontinuous endothelium (Roerdink et a l , 1981; Poste et a l , 1982; Spanjer et al., 1986). Elimination of liposomes by the liver is dependent on their interaction with plasma proteins. In a rat liver perfusion model, limited uptake was observed for plasma-free liposomes (Kiwada et a l , 1986 and 1987). In addition, various organ-specific opsonins 40 which are proposed to be responsible for the varied uptake by the liver and the spleen have been identified by Moghimi and Patel (1988 and 1996). The spleen plays a less significant role compared to the liver in particle elimination because of its small size and the relatively low blood flow despite the fact that on a per cell basis more liposomes are taken up by the spleen. Its role in vesicle uptake becomes more significant for larger vesicles (Klibanov et al., 1990 and 1991; Liu et al., 1991; Litzinger and Huang, 1992) because they are retained in the reticular filter structure more effectively (Claassen and Van Rooijen, 1984). Liposome uptake by the MPS is useful when the macrophage population is the intended biologic target as for example, in the liposomal delivery of macrophage activators to enhance the tumoricidal activity of macrophages (Fidler et al., 1982). However, the MPS is an impediment if alternative target sites are being considered. Passive and Active Targeting Passive targeting is a term coined to define the accumulation of liposomes at disease sites such as loci of infection, inflammation and tumors. It takes advantage of the fenestrations within the endothelium characteristic of diseased tissue and has been demonstrated for liposomes with long circulation lifetimes (Gabizon and Papahadjopoulos, 1988; Gabizon et al., 1990; Gabizon, 1992). Passive targeting allows up to 10-100 times more liposomal drug to be directed to the disease site as compared to an injection of the same amount of free drug (reviewed by Bally et al., 1998). On the other hand active targeting involves the attachment of specific ligands such as antigen-specific antibodies onto the liposome surface and thus targets specific cell populations. Therefore, active targeting often 41 requires long circulation times and premium passive targeting capabilities to allow penetration to the disease site. However, it has been a general observation that antibody-coated liposomes are eliminated more rapidly than their non-targeted counterparts (Leserman et al., 1983; Loughrey et al., 1993). 1.6.3 Extravasation of Liposomes from Blood Extravasation: From the Blood to the Interstitium Extravasation is the process by which the drug carrier crosses the vascular barrier to a defined interstitial space. Thus for both non-targeted and targeted systems, the accumulation at a disease site is believed to depend upon the time taken for elimination of liposomes from blood. Barriers to Extravasation In principle, endothelial cells lining the interior surface of all blood vessels constitute a primary barrier for circulating liposomes having access to the disease/target site. A characteristic of endothelial cells of normal vasculature is the presence of intact intercellular junctions. It is as a result of these junctions that only small molecules can permeate across capillaries. It is also for this reason that one must consider the physical structure of the capillary walls that limit the extravascular movement of liposomes (shown in Figure 1.9). 42 1 3a 2 5a 5b Continuous Capillary 1 3a 2 4a 4b 5a 5b Fenestrated Capillary 1 2 3b 5a 5b Discontinuous Capillary Figure 1.9 Capillary Endothelial Structure and Extravasation The three classes of capillary endothelial vascular structure (continuous, fenestrated and discontinuous) and the mechanisms of transport possible across them: direct diffusion across endothelial cell; (2) lateral membrane diffusion; (3) interendothelial transport (a= narrow, b= wide); (4) endothelial fenestrae (a= closed, b= wide) and (5) vesicular transport (a= transcytosis; b= vacuolar-vesicular organelles (channels)). (Adapted from Jain, 1987, Parr, 1995 andOja, 1997) Lipophilic solutes: 1, 2, 3 and 4; Hydrophilic solutes: 3, 4 and 5; Macromolecules: 3, 4, and 5 4 3 Types of Capillaries In general, on the basis of the endothelium and basement membrane (Poste et al., 1984; Jain, 1987), the normal capillary blood vessel structures can be classified into three: namely, continuous, fenestrated and discontinuous. Continuous capillaries are found in most tissues including skeletal, cardiac and smooth muscle; these are lined with a continuous layer of endothelial cells and an uninterrupted basement membrane. On the other hand, a thin membrane lines fenestrated capillaries that are interrupted by 20-100 nm wide fenestrae. Such fenestrated capillaries are commonly found in endocrine glands, the intestinal vil l i and kidney glomerulus. Only in the discontinuous capillaries of the liver, spleen and bone marrow are gaps as wide as 100 nm found in the endothelial cell layer; in addition, the underlying basement membrane is discontinuous, i f present, or it may be absent. In disease states such as tumors and infections, large intercellular openings and fenestrations are found in the capillaries. Mechanisms of Transport Modes of transport across these classes of vessels include the direct diffusion of molecules across the endothelial cell, lateral membrane diffusion and movement via vesicular mechanisms across the endothelial fenestrae and through interendothelial junctions (Jain, 1987; Kohn et al., 1992). While the permeability of normal vasculature is tightly regulated, hyperpermeability to circulating macromolecules is associated with tumor vasculature. Increased permeability may be, in part, due to fenestrations or widened interendothelial junctions within the endothelial layer. The release of the endothelial cell specific mitogen, the vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF), by tumor cells further mediates vascular permeability or the development of neovasculature (Dvorak et 44 al., 1991; Stein et al., 1995). Within sites of tumor growth, hydrostatic pressure is elevated relative to the vascular pressure, leading to a pressure gradient that impedes the movement of molecules from the blood into the interstitium (Baxter and Jain, 1989). A n extravascular trapping phenomenon has also been observed in the absence of a developed lymphatic system in conjunction with the large openings in the endothelial lining (Butler et al., 1975). In addition, in the absence of lymphatic drainage, molecules would escape from the disease site by diffusion, the rate of which would be dependent on molecule size: large molecules exiting more slowly than small molecules. 1.7 The Ideal Liposome - An Enigma While researchers have jumped through several technical hoops and refined liposomal parameters such as size, charge and circulation lifetimes, the ideal liposome that delivers the encapsulated agent into the cytoplasm of the target cell still remains elusive. 1.7.1 The Conceptual Dilemma It has been accepted that it is the ability of liposomes to accumulate preferentially at disease sites, including tumors, that has led to their use as drug carriers (Proffitt et al., 1983; Ogihara et al., 1986; Ostro and Cullis, 1989; Allen, 1997). This selective accumulation is believed to reflect extravasation of the liposomes due to immature or fenestrated capillary endothelium within the rapidly growing tumor. The extent of accumulation within tumors is largely determined by the circulation lifetime of the carriers, which can be enhanced by minimizing recognition by cells of the reticuloendothelial system (Wu et al., 1993). It is indeed very intriguing to find that as a consequence of liposome-mediated changes in drug pharmacokinetic and biodistribution characteristics, incremental broadening of the narrow therapeutic index of antineoplastic agents can be achieved. First, circulating levels of liposomal drug can be enhanced over those of free drug (Lim et al., 1997). Second, free drug exhibited twenty-five fold lower peak plasma levels versus liposomal drug (Parr et al., 1997). Why then are the liposomal drugs not more toxic and/or more efficacious? Is this because the optimization approach prevalent until now in the design of lipid-based drug carriers has relied on maximizing retention of drug to facilitate localization of the drug at disease sites? In the process, perhaps the drawback of such formulations has been ignored: that the drug sequestered inside the liposomes is not efficiently delivered into tumor cells. Having highlighted the problem of compromised bioavailability (section, the following section shifts in focus to the working assumptions which form the basis for the emerging technology of programmable fusogenic vesicles (PFVs). 1.8 Novel Liposome Design The inability to differentially control drug release rates in the plasma compartment and disease site is perhaps the most significant limitation of presently available liposomes. It is to meet the requirements of a delivery system that would contain specific features exhibited by conventional liposomes (such as attributes for stability to blood components, controlled circulation half-lives and the ability to localize at sites of disease progression) and in addition have the ability to undergo structural modifications facilitating release of drug, that the transformable liposome was designed. The central premise for the design of such multifunctional carriers is that properties required during the delivery phase of treatment can 46 be differentiated from those required for therapy. In this regard, the liposomal carrier should display properties that favor enhanced plasma residence times and good drug retention in the circulation, but should then transform into a leaky or fusogenic vesicle following extravasation. Such attributes may be obtained through a new technology that relies on programmable fusogenic vesicles. 1.8.1 Programmable Fusogenic Vesicles Programmable fusogenic vesicles (PFVs) are transformable liposomes that consist of lipids that, in isolation, adopt non-bilayer structures but which, when combined with a specific stabilizing component, are able to exist in a bilayer organization. The bilayer stabilizing component is designed, however, to be exchangeable and can be lost from the vesicle over time. Loss of this component results in vesicle destabilization and unmasks the inherent fusogenic character of the system. Furthermore, the rate at which the stabilizing component is lost, and hence the rate at which fusion activity is recovered, can be controlled by modifying the length of the acyl chains which "anchor" this component within the bilayer, hence the term programmable fusogenic vesicles. Composition In the present studies, PFV systems contain the non-bilayer-forming lipids 1,2-dioleoyl-^«-glycero-3-phosphoethanolamine (DOPE), and cholesterol, together with a positively charged lipid N-dioleyl-N, N-dimemylammonium chloride (DODAC). This lipid mixture is stabilized in a bilayer organization by the inclusion of a lipid conjugate consisting of the hydrophilic polymer, poly(ethylene glycol2 0 0o) (PEG) coupled to 47 phosphatidylethanolamines (PE) of varying acyl composition. The cationic lipid, DODAC, is included in the formulation to promote PFVs binding to cells due to electrostatic attraction once the PEG-PE on the vesicle surface is lost. Mechanism of Action of Programmable Fusogenic Vesicles: A Hypothesis Principles underlying the function of programmable fusogenic vesicles are illustrated in Figure 1.10. It is important to recognize that each component within the PFV formulation plays a specific role. As illustrated in Figure 1.10, loss of PEG-PE results in PFV destabilization: the subsequent fate of the carriers will then be determined, to a certain extent, by their location. In the case of PFVs still residing in the circulation, exposure of the cationic bilayer surface should result in protein binding and subsequent recognition and clearance of the carrier by phagocytic cells of the MPS; in addition, destabilization may promote drug leakage. In the case of PFVs that have exited the circulation, for example at tumor sites, destabilization will allow either fusion with adjacent tumor cells or leakage of encapsulated drug (mitoxantrone) within the interstitial space. Either mechanism can enhance local delivery of the antineoplastic agent to the tumor cells as a consequence of selective carrier accumulation at this disease site. The extent to which PFVs accumulate at tumor sites will be determined by their residence time in the blood and hence the rate of PEG-PE loss should largely dictate their therapeutic activity. Ideally, PEG-PE exchange should be sufficiently slow to ensure good drug retention in the circulation, slow elimination from the blood compartment and hence, efficient tumor accumulation. Subsequent carrier destabilization at the tumor site will then allow full drug bioavailability to the tumor cells. 48 Blood Vessel Figure 1.10 Mechanism of Action of Programmable Fusogenic Vesicles: A Hypothesis The first step is the loss of PEG-lipid leading to P F V destabilization. This critical step would be followed by one of three outcomes: (A) the elimination of the destabilized P F V s via the reticuloendothelial cells of the body (B) the release of the contents of PFVs in the vicinity of the target cell or (C) the fusion of the destabilized PFVs with the target cell, releasing the encapsulated drug into the cytoplasm of the cell 49 1.9 Summary of Research Objectives The overall objective of the research carried out for this thesis was to increase the bioavailability of mitoxantrone encapsulated within PFVs. Steps towards this goal were: (1) To develop a well-defined, transformable liposome, (programmable fusogenic vesicle) and characterize this carrier in vitro in terms of its morphology, ability to encapsulate mitoxantrone and to demonstrate the programmed release of its aqueous contents. (2) To characterize in vivo the rates of plasma elimination and biodistribution of PFVs and mitoxantrone-loaded PFVs as a function of PEG-PE acyl chain length. (3) To characterize the exchangeability of PEG-lipids from PFVs and the influence of this process on circulation lifetime of PFVs. (4) To determine tumor accumulation and efficacy of mitoxantrone-loaded PFVs using a human tumor xenograft. The steps mentioned above also form the outline of this thesis. After a Methods section (Chapter 2), I introduce step (1) as Chapter 3. Steps (2) and (3) form the subject matter of Chapter 4. And the final step (4) is taken in Chapter 5. Chapter 6 will provide conclusions and a glimpse of future work. 1.10 Research Hypothesis The primary hypothesis is that the bioavailability and thus the efficacy of mitoxantrone can be improved by using programmable fusogenic vesicles as a vehicle to deliver mitoxantrone to target tumor tissue. I predict that by the appropriate selection of the 50 PEG-PE component of PFV, properties of PFV in circulation can be modulated to achieve increased accumulation at tumor sites. Following accumulation at a disease site, controlled vesicle destabilization will ensure higher availability of encapsulated mitoxantrone to target cells. 51 CHAPTER 2: MATERIALS and METHODS 2.1 Materials A l l phospholipids including PEG-PE conjugates were purchased from Avanti Polar Lipids, (Alabaster, AL) . DODAC was a generous gift from Inex Pharmaceuticals Corporation, Vancouver, B.C., Canada. [^CJ-A^TV-dioleyl-A^A^-dimethylammonium chloride ([ 1 4C]-DODAC) was custom synthesized by Stephen Ansell of Inex Pharmaceuticals (Vancouver, B.C). [3H]-Cholesteryl Hexadecyl Ether ([ 3H]-CHE) was purchased from Amersham (Oakville, Ontario, Canada). Novantrone® (mitoxantrone hydrochloride) was obtained from the British Columbia Cancer Agency and is a product of Wyeth Ayerst (Montreal, PQ) and [1 4C] mitoxantrone used as a tracer was generously donated by Wyeth Ayerst (Montreal, PQ). 1,2-dimyristoyl (9',10'-di-3H)-^«-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2 0 0 0] (PEG 2 0 0 0 -DMPE[ 3 H]); 1,2-dipalmitoyl (9' ,10'-di- 3 H ) -5«-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2 0 0 0] (PEG 2 0 0 0-DPPE[ 3H]) and 1,2-distearoyl (9', 10' -di- 3H)-5«-glycero-3 -phosphoethanolamine-N- [poly(ethy lene glycol) 2 0 0 0] (PEG 2 0 0 0-DSPE[ 3H]) were custom synthesized and radiolabeled by Xue M i n Zhou of Northern Lipids Inc. (Vancouver, B.C). Solvable was obtained from N E N Research Products (DuPont Canada, Mississauga, Ontario, Canada). Other reagents, HEPES, citric acid, cholesterol and Sephadex G-50 (medium) were purchased from Sigma, (St. Louis, MO). RPMI-1640 medium was purchased from Stem Cell Technologies (Vancouver, B.C., Canada). 52 2.2 Cell lines and Animals Human colon carcinoma cells, LSI80, were obtained from A T C C (Manassas, V A ) . The L1210 tumor cell line was originally obtained from the National Cancer Institute tumor repository (Bethesda, M D , U S A ) and cells were harvested from ascites fluid generated weekly by passage in BDF-1 mice. Female BDF-1 mice (8-9 weeks old, 18.0-23.0 g) were purchased from Charles River Laboratories (Ontario, Canada). Severe combined immune deficient mice, SCID/RAG-2 , were bred at the Joint Animal Facility, B . C Cancer Agency and B . C Research Center. A l l of the mice used in the study were raised and kept in a pathogen-free environment and handled according to Institute guidelines. A l l procedures were done in accordance with Canadian Council on Animal Care guidelines ( C C A C ) . 2.3 Preparation of Programmable Fusogenic Vesicles Lipid components (DOPE:Cholesterol:DODAC:PEG-lipid, 30:45:15:10 molar ratio) containing the non-exchangeable, non-metabolizable lipid label (Stein et al., 1980) [ 3H]-Cholesteryl Hexadecyl Ether ([ 3 H]-CHE) or [ 1 4 C ] - C H E were solubilized in 1-2 ml of benzene:methanol (95:5, v/v) and then lyophilized for a minimum of 5 h at a pressure of <60 m Torr using a Virtis lyophilizer equipped with a liquid nitrogen (N 2) trap. For PFVs prepared for in vitro studies, the concentration of lipids was 30 m M and for in vivo studies the concentration was 80 m M . Multilamellar vesicles were prepared by hydrating the dry lipid mixtures in 300 m M citric acid (pH 4.0). To assist lipid hydration, the suspension was vortexed and then frozen (liquid N 2 ) and thawed (37°C) five times (Mayer et al., 1985a). The resulting multilamellar vesicles were then extruded 10 times through two polycarbonate filters with 0.1 pm diameter pores at 3 7 ° C (Olson et a l , 1979; Hope et al., 1985). 53 2.3.1 Establishment of p H Gradient A transmembrane pH gradient was established across the vesicle bilayer by passing an aliquot of the PFV suspension (internal and external pH 4.0) down a Sephadex G-50 (medium) column (1.0 cm x 15.0 ml) equilibrated with degassed 150 m M NaCl, with 25 m M HEPES buffer (pH 7.4). Fractions (0.5 ml) were collected and the most concentrated vesicle fractions combined. 2.3.2 Establishment of an Osmotic Gradient A hypo-osmotic gradient was established by 1:1 dilution of aliquots of PFVs prepared in 150 m M NaCl, with 25 m M HEPES buffer (pH 7.4) into a buffer of lower salt concentration (25 m M NaCl and 25 mM HEPES buffer; Mui et al., 1993). The diluted samples were allowed to stand at 22°C for 10 minutes before the samples were processed for visualization by cryogenic transmission electron microscopy (cryo-TEM). 2.4 Determination of Trapped Volume of PFVs The internal aqueous volume of PFVs was determined by preparing PFVs as described in section 2.3, with the exception that hydration of the lyophilized mixture of lipids (radiolabeled with [3H]-CHE) was carried out in HEPES-buffered saline (pH 7.4) containing 1 m M sucrose, radiolabeled with [14C]-sucrose, as the impermeable trapped volume marker at 3 pCi/ml (specific activity = 615 mCi/mmol). After extrusion of the hydrated lipid solution, 100 pi of the sample was counted to determine the [14C]-sucrose and [ 3H]-CHE lipid counts. From the rest of the extruded vesicle preparation, the unentrapped [14C]-sucrose was removed by passing the vesicle suspension down a Sephadex G-50 (medium) column (1.0 cm 54 x 15.0 ml) equilibrated with degassed 150 m M NaCl containing 25 m M H E P E S buffer (pH 7.4). Fractions (0.5 ml) were collected and the most concentrated vesicle fractions combined. The eluant of the mini columns was then assayed by dual label scintillation counting for [14C]-sucrose and [ 3 H]-CHE lipid levels. The entrapped sucrose volume (in pi) was calculated by the formula: 100 x B / A , where B equals sucrose counts eluted from the spin column and A represents the [14C]-sucrose counts in the initial liposome sample (100 pi). Lipid concentration was determined as per the formula: C / D , where C represents the lipid counts eluted from the mini column and D equals the specific activity of the [3H]-liposomes used (Hope et al., 1985). Entrapped volume was expressed as pi of trapped [1 4C]-sucrose/ pmole of lipid given by the formula: (100 x B/A) / (C/D). 2.5 Size Analysis of PFVs Size distributions of P F V were determined by quasielastic light scattering (QELS) using a Nicomp 270 submicron particle sizer (Nicomp Instruments, Goleta, C A ) operating at 632.8 nm and 5 mW, as described previously (Berne and Pecora, 1976; Madden et al., 1988). It is possible to determine the mean diameters of vesicles by Q E L S since this technique estimates the fluctuations in scattered light intensity generated by the diffusion of particles in solution. The measured diffusion constant is then used to obtain the hydrodynamic radius and hence the mean diameters of vesicles. Data were recorded at 2 2 ° C over a period of 10-20 min as mean ± intrasample standard deviation (S.D) fitting a Gaussian distribution of the diameters. Data are representative single samples. Average vesicle diameters were also determined by direct measurement from cryo -TEM micrographs. The diameters of vesicles that appeared elongated were taken to be the longest side measured. 55 2.6 Cryogenic-Transmission Electron Microscopy (cryo-TEM) Cryogenic-transmission electron microscopy has been described in detail by Dubochet et al., 1988 and Bellare et al., 1988. In brief, sample films were prepared in a custom-built environmental chamber under controlled temperature ( 2 5 ° C ) and humidity conditions. Care was taken to minimize water loss from the sample by evaporation in order to avoid osmotic effects on vesicle morphology. Thereafter, the films were vitrified by quick freezing in liquid ethane and transferred to a Zeiss E M 902 transmission electron microscope for examination. The specimens were kept below 108 K during both the transfer and viewing procedures to prevent sample perturbation and ice formation. Micrographs were taken under zero-loss, bright-field mode at an accelerating voltage of 80 kV. Cryo-transmission electron microscopy studies were conducted in the laboratory of Dr. Katarina Edwards at the Department of Physical Chemistry, Uppsala University, Sweden. The electron microscope was run by Goran Karlsson. 2.6.1 Interpretation of Cryo-Transmission Electron Micrographs For the interpretation of the cryo-TEM pictures, it is important to realize how the two-dimensional representation of a three-dimensional bilayer aggregate depends on the projected thickness of the depicted aggregate. As illustrated in Figure 2.1, for a closed, spherical liposome the projected thickness of the bilayer shell is greatest at the edges. The image of the liposome will therefore appear as a circular disc with markedly enhanced contrast around the rim. The two-dimensional projection of a bilayer disc, however, will appear even in contrast right up to the edge. For a flattened liposome, finally, the contrast difference between the rim and the bulk of the circular object, observed in the two-dimensional 56 projection, will vary depending on the amount of compression of the liposome. The lowest contrast difference will be observed for a completely flattened liposome, whereas only slightly distorted liposomes will show a contrast reminiscent of that obtained from a perfectly spherical liposome. 2.7 Encapsulation of Mitoxantrone Mitoxantrone was entrapped in PFVs using a transmembrane p H (A pH) loading procedure described elsewhere (Madden et al., 1990b; Chang et al., 1997). Briefly, vesicles exhibiting a transmembrane p H gradient (pH 4.0 i n/pH 7.40Ut) were added to mitoxantrone (2.0 mg/ml), (pH of mitoxantrone solution was pre-adjusted to 7.4 with 1 M N a O H solution) to achieve a drug: lipid molar ratio of 0.1: 1.0. This mixture of PFVs containing P E G - P E was adjusted to the desired final mitoxantrone concentration by addition of 150 m M N a C l , 25 m M H E P E S (pH 7.4) and incubated for 1 hour at 4 5 ° C (PFVs containing PEG-Ceramide (PEG-Cer) were incubated at 3 7 ° C ) . Kinetics of mitoxantrone uptake were determined by taking aliquots (100 pi) of this mixture at various times and passing them down 1 ml Sephadex G-50 (medium) mini columns (Pick, 1981) to remove the unencapsulated drug. The eluant of the mini columns was then assayed by dual label scintillation counting for drug and lipid levels. 57 I A B C Figure 2.1 Interpretation of a 3D object as a 2D image (I) Schematic illustration showing how the 2D image of a spherical liposome (A), a flattened liposome (B), and a bilayer disc (C) all appear as circular objects in the c r y o - T E M micrographs. The contrast obtained in the bulk and at the rim of the circular objects depends on the projected thickness of the bilayer(s) in the original 3D structure. (II) A projection of a three dimensional volume as a two dimensional image. 58 2.8 Release of PFV Contents 2.8.1 Release of Mitoxantrone from PFVs The kinetics of mitoxantrone release from PFVs in vitro were determined by dialysis. Mitoxantrone-loaded PFVs were transferred to dialysis tubing (Spectra /Por 2, moi. wt. cut off 12-14,000) and dialyzed against 500 volumes of 150 mM sodium chloride, 25 mM HEPES, pH 7.4 at 37°C with constant stirring. At various times up to 24 h, aliquots were removed from the dialysis bag and assayed for lipid and mitoxantrone by dual label liquid scintillation counting. 2.8.2 Release of [ , 4C]-Sucrose from PFVs The initial volume of [14C]-sucrose entrapped within PFVs was measured as described in Section 2.4 as a function of the acyl composition of the PEG-PE component of PFVs. The kinetics of [14C]-sucrose release from PFVs was monitored by assaying the change in the contents of trapped [l4C]-sucrose under various conditions. PFVs differing only in the PEG-PE composition were incubated with a 10 or 30-fold excess of sink (neutral acceptor vesicles, composed of DOPC:Cholesterol, 55:45, molar ratio) and at various times (1, 6 days) the variation in [14C]-sucrose content was measured. The release of [14C]-sucrose from PFVs was also ascertained after incubating them for 24 hours in the presence of vesicles containing the negatively charged phospholipid POPS, 1-palmitoyl, 2-oleoyl-s«-glycero-3-phosphoserine (DOPE:Cholesterol: POPS, 40:45:15, molar ratio) following their incubation with sink for various lengths of time. In all cases, the trapped volume was determined by running the samples, control (PFVs in buffer alone), PFVs with sink or PFVs in the presence of sink and 59 negatively charged liposomes down mini columns (Pick, 1981). The levels of lipid and sucrose in the eluant were assayed by dual label scintillation counting. The trap volume calculations were the same as described in Section 2.4. 2.9 Animal Studies of Mitoxantrone-loaded PFVs 2.9.1 Plasma Elimination Studies Plasma elimination studies in mice were performed by administering single intravenous doses via the lateral tail vein at 10 mg/kg mitoxantrone (22.5 pmoles/kg mitoxantrone and 225 pmoles/kg total lipid) in an injection volume of 200 pi. Control liposomes (no mitoxantrone) were administered at the same lipid dose as that of the drug-loaded PFVs . At 1, 4 and 24 hours, four animals from each experimental group were anaesthetized (160 mg/kg ketamine, 20 mg/kg Xylazine). Blood samples, collected by heart puncture using a 25 gauge needle, were placed into EDTA-coated microtainer (Becton Dickinson) tubes maintained on ice. Liposomal lipid and/or mitoxantrone concentrations in plasma were then assayed by dual label liquid scintillation counting (Beckman LS3801). A plasma correction factor was used in the calculation of tissue liposome and drug levels. Plasma and tissue levels of mitoxantrone given in the results will include radiolabeled metabolites of mitoxantrone. 2.9.2 Tissue Biodistribution Organs were collected from animals following the heart puncture, ensuring that the animals were killed by cervical dislocation prior to removal of the organs. Lungs, liver, spleen, kidney, heart and skeletal muscle were removed and placed into pre-weighed glass 60 tubes. After the tissue.weight was determined, a 10 % (w/v) homogenate was prepared in distilled water using a Brinkmann Polytron homogenizer (Kinematica, Switzerland). Tissue homogenates (200 pi) were digested with 500 pi of "Solvable" (Du Pont, Inc.) for 3 h at 5 0 ° C . After cooling, ethylenediamine tetraacetic acid ( E D T A ) (50 pi of a 200 m M stock solution) was added followed by 30 % H 2 0 2 (200 pi) and 10 N HC1 (25 pi). Samples were incubated at room temperature for 1 hour. Following the addition of scintillation cocktail (Ultima Gold, Packard, Meriden, CT) , samples were counted. 2.9.3 Studies on Exchange of PEG-lipids from PFVs or Conventional Vesicles in vivo The rate of exchange of PEG-lipid from PFVs or conventional vesicles was determined in female BDF-1 mice following a single i.v. injection (225 pmoles total lipid/kg in an injection volume of 200 pi) administered via the lateral tail vein. The initial ratio of the exchangeable P E G - P E to liposomal lipid was 0.1:1.0 and 0.05:1.0 for PFVs and conventional liposomes, respectively. At various times (0.5, 1, 2, 4, 8 and 24 hours), the mice were sacrificed and blood withdrawn by cardiac puncture and collected in microtainer tubes containing E D T A . The plasma was isolated by centrifugation at 500 x g for ten minutes. Liposomal lipid ( [ 1 4 C]-CHE) and PEG-PE[ 3 H] levels in plasma were determined by dual label scintillation counting. 2.9.4 Plasma Elimination Studies of Exchange of D O D A C from PFVs in vivo Based on the P F V preparation procedure described in Section 2.3, [ 1 4 C ] - D O D A C was incorporated in PFVs labeled with [ 3 H]-CHE as the vesicle marker. Plasma elimination studies to determine the rate of loss of DODAC from PFVs were conducted as described in Section 2.9.3. 2.9.5 Analysis of Plasma Samples by Fast Protein Liquid Chromatography Plasma samples were isolated from BDF-1 mice injected with PFVs containing PEG-DMPE[ 3 H] or PEG-DPPE[ 3H] or D S P C C H O L liposomes containing PEG-DMPE[ 3 H] after 1 and 8 hours, respectively. Radiolabeled Cholesteryl Hexadecyl Ether ([ 1 4C]-CHE) was used as a marker for liposomal lipid. Liposomes and PEG-lipids were separated from lipoproteins and other plasma proteins within plasma by Fast Protein Liquid Chromatography (FPLC). Resolution of plasma samples on FPLC was carried out by Dr. Edward Choice. The FPLC instrument consisted of a L K B brommer pump (Germany) linked to a BioRad Econo System Controller running a Pharmacia Superose 6 H.R. (10 mm diameter X 30 cm length) column and a Pharmacia 12 prep grade (16 mm diameter X 50 cm length) column in series. The flow rate was set at 0.5 ml/min at 3 bar pressure run on a pump for a total run time of 240 minutes. (Conversions: 1 bar = 14.696 p.s.i. = 1 atmosphere = 0.1 Mpa = 760 mm Hg). The BioRad Econo System Controller (BioRad, Canada) was set up to collect 60 fractions of 1 ml each. The eluting fractions were also monitored by a spectrophotometer attached in series to measure the absorbance of the eluant at 280 nm. The running buffer (mobile phase) consisted of 150 mM NaCl, 10 m M Tris, 0.03 % sodium azide (NaN3) at pH 7.4. The absorbance of each fraction was read at 280 nm and the lipids tracked by dual label scintillation counting on a Perkin Elmer LSI80 instrument. A detailed description of the methods used in the separation of liposomes and individual lipoprotein fractions is available from Choice et al. (1999). 62 2.9.6 Tumor Accumulation and Plasma Elimination Studies of Mitoxantrone-loaded PFVs Severe combined immune deficient mice (SCID/RAG-2, 18-22g, female mice, 4 per group, B . C . Cancer Agency) were inoculated bilaterally with 1 x 106 human colon carcinoma, LSI80 cells subcutaneously on the hind regions of the back. Mice were injected with a 10 mg/kg dose of free mitoxantrone or mitoxantrone-loaded P E G - D M P E - P F V s or P E G - D S P E -PFVs when the tumors had reached a size measurable by calipers. At 1,4, 24 and 48 hours, animals were terminated by C 0 2 asphyxiation and whole blood collected via cardiac puncture and placed into EDTA-coated tubes. Plasma was isolated following centrifugation of whole blood at 500 x g for 10 min. Aliquoted plasma samples (100 pi) were mixed with 5 ml scintillation fluid (Packard, Meriden, CT) and counted for [3H] and [ l 4 C]. Isolated tissues were processed as outlined in Section 2.9.2 with the exception that the entire tissue was dissolved in Solvable and then decolorized and counted as described earlier. 2.9.7 Establishment of Maximum Tolerated Dose Limited dose-ranging studies on the LSI80 tumor model were performed to determine the maximum tolerated dose (MTD) of mitoxantrone. These studies were completed using protocols approved by the University of British Columbia Animal Care Committee and were done in accordance with the guidelines of the Canadian Council on Animal Care ( C C A C ) . It should be noted that only two of 25 animals (SCID/RAG-2 mice) used in this experiment died as a consequence of drug-related toxicity and no animal lives had to be terminated as a result of unacceptable suffering. The C C A C does not permit formal L D i 0 or L D 5 0 studies in which death is used as an end-point, so the M T D was approximated 63 using a small number of animals and a limited number of drug doses. At the end of the 30-day period, animals were sacrificed by C 0 2 asphyxiation and necropsies were completed to identify any additional toxicities. For efficacy experiments, mitoxantrone was injected at the lower dose of (2 mg/kg). 2.9.8 Antitumor Activity against Human Colon Carcinoma Xenografts On day zero, SCID/RAG-2 mice (B.C. Cancer Agency) were inoculated s.c. on the right and left posterior flanks with 1 x 106 human colon carcinoma, LSI80 cells in a volume of 0.1 ml RPMI 1640 media. Mice were then randomized to groups and numbered. In treatment groups (4 mice per group), free mitoxantrone or mitoxantrone in PFV was administered i.v. at 2.0 mg/kg on days 2, 6 and 10 after tumor cell inoculation. Tumor size was determined every other day by measuring the longest (a) and shortest (b) dimensions of each individual tumor in millimeters. Ellipsoidal tumor volumes were estimated using the formula of Tomayko and Reynolds (1989) who have reported the ellipsoidal tumor volume to be the most accurate measure of actual tumor mass (correlation coefficient of 0.93). Tumor volume (cm3) = 7tab2/6 The volumes of all the tumors in each individual treatment group were summed. The cumulative sum of tumor volumes was then divided by the number of mice. Antitumor efficacy data are presented as cumulative tumor volumes for all animals in each group to account for both number of tumors and number of mice alive. This normalization is also the reason for the lack of error bars in Figure 5.3. Animals were sacrificed when tumors had reached diameters greater than about 1.2 cm (volume approx. 2.4 cm3) and tumor size on the day of termination was used in subsequent cumulative totals. The day of death of each 64 mouse was recorded and experiments were ended 40 days after injection of tumor when all remaining mice were killed. Heart, lungs, liver, spleen and kidneys from each mouse were examined for any histopathological changes. Data are representative of two separate experiments. 2.9.9 Antitumor Efficacy against Murine L1210 Leukemia Antitumor efficacy of free mitoxantrone or mitoxantrone encapsulated in PFVs was determined using the murine L1210 leukemia model. BDF-1 mice (8 per group) were inoculated intravenously with 104 L1210 murine tumor cells derived from the ascites fluid of a previously infected BDF-1 mouse. The indicated doses of free mitoxantrone and P F V -loaded mitoxantrone were administered (i.v.) 24 h after tumor inoculation. Animal weights and mortality were monitored daily. 2.10 Statistical Analysis In order to detect differences in the results obtained after administration of the P F V formulations and free mitoxantrone, analysis of variance ( A N O V A ) was conducted at the last time point (usually 24 hours). If a significant difference between means was detected by A N O V A , a post hoc Scheffe's test was used to compare the different groups pairwise. If a group differed from all the other groups, it was denoted by an asterisk symbol in the figures. Differences were considered significant at P < 0.01. Similar analysis of variance followed by Scheffe's test was also used to evaluate differences between groups for studies on exchange of PEG-lipids from PFVs or conventional liposomes. For the LI210 efficacy studies, survival times (in days) were ranked and statistically analyzed using a Cox's F test. 65 Compar i sons indicated as hav ing statistical significance had P values f rom the C o x ' s F test o f < 0.01. M e a n area under the curve analysis was performed us ing trapezoidal integration. 66 CHAPTER 3: CHARACTERIZATION OF P R O G R A M M A B L E FUSOGENIC VESICLES IN VITRO 3.1 Introduction As outlined in Chapter 1, programmable fusogenic vesicles are designed as multi-component systems that yield a multifunctional liposomal drug carrier. As systemic drug delivery systems, they are intended to retain an encapsulated drug, exhibit prolonged circulation half-lives after intravenous injection, and then later transform into leaky, fusogenic carriers. Each lipid constituent is intended to make a specific contribution to the overall PFV function. In the present work, systems composed of CHOL and the non-bilayer-forming lipid DOPE were stabilized in a lamellar organization by inclusion of PEG-PE or PEG-Ceramide. In addition, a cationic lipid, DODAC, was included to promote apposition of PFVs with negatively charged surfaces such as cell plasma membranes. Accumulation of pharmaceutical agents within lipid vesicles exhibiting a proton gradient is a fairly general phenomenon for drugs that are lipophilic amines and has been referred to as "remote loading" (Madden et al., 1990b, see section 1.4.3). The ability to load drugs into pre-formed liposomes employing a transmembrane pH gradient has greatly facilitated development and characterization of liposomal drug carriers. In the case of anticancer agents, for example, studies have demonstrated that drug toxicity to normal, healthy tissues and organs can be reduced by their administration in such carriers (Rahman et al., 1980, Mayer et al., 1994). In addition, increased antitumor efficacy has been reported for vincristine encapsulated in a liposomal carrier (Mayer et a l , 1990c; Boman et al., 1994). In the case of mitoxantrone, liposomal encapsulation of the drug employing a proton gradient 6 7 results in formulations that exhibit reduced drug toxicity and significantly enhanced antitumor efficacy (Chang et al., 1997; L i m et al., 1997). It should be noted that some earlier studies with a liposomal formulation of mitoxantrone involved a complexation of the drug to anionic lipids. These systems were not stable in the circulation and rapidly released the drug (Schwendener et al., 1991, Schwendener et al., 1994). Previous studies have also examined the influence of lipid composition on the accumulation of anticancer drugs by liposomes. In particular, it has been reported that lower drug to lipid ratios and encapsulation efficiency are seen for doxorubicin when loaded into cationic carriers (Rahman et a l , 1980; Shinozawa et al., 1981; Herman et al., 1983). Again, however, it should be noted that these earlier studies examined formulations in which drug binding to the liposome occurred predominantly through electrostatic interactions (Gabizon et al., 1982) rather than through encapsulation of the agent within the liposome aqueous core. The characteristics of PFVs in vitro were examined with regard to their ability to release entrapped contents. The goal of the present studies was to demonstrate that loss of PEG-lipids from PFVs to sink would sufficiently destabilize PFVs to expose D O D A C and subsequently trigger electrostatic interactions with negatively charged vesicles. Therefore, the release of P F V contents as effected by the incorporation of P E G - D M P E or P E G - D S P E into the P F V bilayer at different molar ratios was examined in the absence or presence of negatively charged vesicles. Radiolabeled sucrose was used as a marker for the aqueous contents of PFVs because it is a recognized trapped volume marker (Zborowski et al., 1977; Allen and Cleland, 1980; Hunt, 1982) due to its impermeability through vesicular membranes as compared to glucose which is membrane permeable (Mui et al., 1993). 68 In this chapter, the technique of cryogenic transmission electron microscopy (cryo-TEM) was utilized to delineate PFV morphology and the influence of PFV composition on vesicle structure. In addition, loading of mitoxantrone into PFVs was characterized employing a pH gradient, and cryo-TEM was used to visualize the location of encapsulated drug within PFVs. Furthermore, the proposed hypothesis of programmed release of PFV contents was examined using [14C]-sucrose as a marker for PFV contents. 3.2 Results 3.2.1 Morphology of P F V Morphology of PFV: Influence of PEG-PE The influence of individual lipid components on PFV morphology was examined by cryo-TEM. The first series of experiments was performed to characterize the morphology of PFVs and the influence of the bilayer-stabilizing component, PEG-PE. Vesicles composed of DOPE:CHOL:DODAC could not be prepared in 150 m M NaCl, 25 m M HEPES, pH 7.4 and were instead prepared in distilled water. Electron micrographs revealed unilamellar, spherical vesicles with an average size of approximately 90 nm (Figure 3.1 A and Table 3.1). When the medium ionic strength was modified to contain 150 m M NaCl, large aggregate structures were observed (Figure 3.IB) which may contain some hexagonal (H n) phase similar to those observed by Gustafsson et al. (1995). In contrast to systems lacking PEG-lipid, PFV containing this bilayer-stabilizing component can readily be prepared in saline solution. When PFVs consisting of DOPE:CHOL:DODAC:PEG-DSPE, (30:45:15:10 molar ratio) were visualized by cryo-TEM under iso-osmotic conditions, a variety of morphologies could be seen. Many of the vesicles 69 appeared to be discoid while others were spherical or tubular. Although some of the vesicles appear to contain internal lamellae, this may represent invagination of a single bilayer. Similarly, the tubular structures seen may represent discoid vesicles observed side-on. The morphological consequences of increasing P E G - P E content from 10 mole% to 20 mole% are shown in Figure 3.2B. In addition to some of the vesicular structures seen in Figure 3.2A, open bilayer discs are observed in edge-on and face-on projections. Most of the discs appear to be circular with smooth edges. Open bilayer discs have also been observed in preparations of conventional liposomes ( D S P C : C H O L ) containing greater than 10 mole% P E G - D S P E (Edwards, 1997). Average vesicle diameter, as determined from cryo -TEM measurements, is not significantly different between systems prepared with P E G - P E or without this bilayer stabilizing component (in distilled water, Table 3.1). In the case of PFVs prepared with 20 mole% P E G - D S P E , however, a small reduction in mean diameter is found but this is well within the standard deviation of the results. In comparing vesicle diameters determined by direct measurement from cryo -TEM electron micrographs with sizes determined by quasielastic light scattering (QELS), it is notable that the Q E L S data give consistently larger sizes. This discrepancy has been reported previously for large unilamellar vesicles prepared by extrusion (Mayer et al., 1986c) and will be commented on in the Discussion. 70 Table 3.1 Mean diameter of PFVs from quasielastic light scattering and actual measurements from the micrographs. Sample Mean Diameter (nm)a Mean Diameter (nm)b PEG-CerC20-PFV DOPE:CHOL:DODAC:PEG CerC20 (30:45:15:10) 105.4 (28.7) 74.2(17.2) PEG-DSPE-PFV DOPE:CHOL:DODAC: PEG-DSPE (30:45:15:10) 103.5 (26.4) 89.2 (25.4) PEG-DSPE-PFV DOPE:CHOL:DODAC:PEG-DSPE (20:45:15:20) 112.8(21.9) 78.3 (34.0) No PEG-PFV DOPE:CHOL:DODAC (40:45:15)c 137.3 (45.7) 91.7(13.3) No DODAC-PFV DOPE:CHOL:PEG-DSPE (45:45:10) 124.9 (21.1) 86.3 (12.0) a Mean diameter as determined by quasielastic light scattering Mean diameter as measured from cryo-transmission electron micrographs, an average of 10 measurements and corrected for a magnification of 120,000. The number in brackets denotes the intrasample standard deviation. c Prepared in distilled water 71 Morphology of PFVs: Influence of Cationic Component, DODAC PFV morphology was also examined in the absence of the cationic lipid, D O D A C , (DOPE:CHOL:PEG-DSPE, 45:45:10, molar ratio). Again, a variety of vesicle shapes were observed by cryo-TEM (Figure 3.2C). In addition to spherical, discoid and tubular morphologies, what appear to be highly invaginated structures can be observed. Liposomes are osmotically sensitive and relaxation of non-spherical morphologies to a spherical shape would be opposed by osmotic forces (Mui et al., 1993; 1995). Therefore, the next step was to examine whether dilution of PFVs into a hypo-osmotic solution resulted in the vesicles assuming a more spherical morphology. As illustrated in Figure 3.2D and 3.2E, PFVs prepared with or without DODAC can be generally seen to adopt a more spherical appearance in hypotonic solution although invaginated structures can still be observed (cf. Figure 3.2A and 3.2C). This tendency to adopt a more spherical morphology results from water influx as the vesicles tend to maximize their volume to surface area ratio (Mui etal., 1993). 72 Figure 3.1 Cryo-transmission electron micrographs of PFVs in the absence of P E G - P E PFVs composed of DOPE:CHOL:DODAC (40:45:15, molar ratio) in distilled water (a) or in 150 m M NaCl, 25 m M HEPES, (pH 7.4) for 10 min (b). The arrow in micrograph (b) points to hexagonal (H n) phase. The bar represents 100 nm. 73 Figure 3.2 Cryo-transmission electron micrographs of PFV of different compositions (following page) Cryo- t ransmiss ion electron micrographs o f (a) P F V s composed o f D O P E : C H O L : D O D A C : P E G - P E , (30:45:15:10, molar ratio) i n 150 m M N a C l , 25 m M H E P E S , p H 7.4. (b) P F V s composed o f D O P E : C H O L : D O D A C : P E G - D S P E , (20:45:15:20, mola r ratio). The arrows denoted E and F point out bi layer discs as v i e w e d edge-on and face-on, respectively. (c) P F V s i n the absence o f the cat ionic l i p i d component, D O D A C ( D O P E : C H O L : P E G -D S P E , 45:45:10, mola r ratio), made i n H E P E S - b u f f e r e d saline (150 m M N a C l , 25 m M H E P E S , p H 7.4). The arrows denote invaginated structures v i e w e d face-on. (d) P F V s i n the absence o f the cat ionic l i p i d component, D O D A C , d i lu ted 1:1 i n hypo-osmot ic (25 m M N a C l , 25 m M H E P E S , p H 7.4) solut ion. (e) P F V s ( D O P E : C H O L : D O D A C : P E G - P E , 30:45:15:10, molar ratio) d i lu ted i n hypo-osmot ic buffer ( 25 m M N a C l , 25 m M H E P E S , p H 7.4). The bar represents 100 n m . 74 3.2.2 Uptake of Mitoxantrone in P F V s Uptake of Mitoxantrone in PFVs: Influence of PEG-lipid Species. Transmembrane p H gradients have previously been used to encapsulate a variety of drugs, including mitoxantrone, within pre-formed vesicles (Madden et al., 1990b). Furthermore, this technique can be applied to load conventional liposomes of varying lipid composition (Mayer et al., 1989). In view of the fact that PFVs are designed to be relatively unstable, it was essential to examine whether they could support a transmembrane proton gradient and subsequently accumulate mitoxantrone added to the external solution. In these drug-loading studies, P F V formulations containing a variety of bilayer-stabilizing PEG-lipids were compared. As shown in Figure 3.3A, P F V containing PEG-Ceramide and exhibiting a p H gradient (3.4 p H units) rapidly accumulate mitoxantrone on incubation at 3 7 ° C . Similar uptake is seen for PFVs containing PEG-Ceramides with N-palmityl (C16, PEG-CerC16) or N-arachidyl (C20, PEG-CerC20) hydrocarbon chains. Complete mitoxantrone uptake occurs within 40 minutes and this uptake is stable with no loss of accumulated drug for up to 2 h at this temperature. Similar drug loading efficiencies are observed for PFVs containing P E G -P E (Figure 3.3B). Mitoxantrone encapsulation was rapid with essentially complete uptake seen by 30 minutes on incubation at 45°C. Again, P F V formulations containing either P E G -D M P E , (dimyristoylphosphatidylethanolamine) or P E G - D S P E (distearoylphosphatidylethanolamine) exhibited similar loading properties. 76 Effect of Drug to Lipid Ratio on Uptake of Mitoxantrone in PEG-Ceramide Containing-PFVs The efficiency of drag-loading into liposomes exhibiting a p H gradient is influenced by the drug-to-lipid ratio (Mayer et al., 1989). Therefore, the effect of this parameter on mitoxantrone loading into PFVs ( D O P E : C H O L : D O D A C : P E G - C e r C 2 0 ) was examined. At initial drug:lipid ratios of 0.1:1 and 0.2:1, essentially complete (100%) mitoxantrone loading is observed (Figure 3.4). At higher drug ratios (0.3:1) loading is incomplete, with about 80% of the added drug being accumulated. This observation is consistent with previous reports, and reflects depletion of the intravesicular buffering capacity on protonation of the accumulated drug, with subsequent increase in the internal p H (Mayer et al, 1990d). In comparing mitoxantrone uptake at varying drag:lipid ratios, it is also clear that the kinetics of loading are slower at higher ratios (Figure 3.4). Stability of Mitoxantrone Uptake in PFVs Containing PEG-Ceramide Having demonstrated that mitoxantrone can be efficiently encapsulated within PFVs, the length of time for which mitoxantrone was stably retained within PFVs was determined next. Release of mitoxantrone from PFVs ( D O P E : C H O L : D O D A C : P E G - C e r C 2 0 ) during dialysis at 37°C was monitored for vesicles of varying drug:lipid molar ratio (Figure 3.5). In the case of vesicles loaded at 0.1:1 dragdipid ratio, no mitoxantrone leakage was observed over 24 hours. At 0.2:1 molar ratio, slow release was observed with approximately 85% of the encapsulated drug retained at 24 hours. At the highest molar ratio tested, 0.3:1, only 80%> drag loading is achieved and efflux of the free drug is observed over the first 4 hours. Subsequent efflux of mitoxantrone is much slower up to 24 hours. 77 o '•+-« CO l _ •g Q. 76 CD (0 -I—< Q. 3 o ro •g CD "D 120 100 r-ro -»—' Q. 3 60 ^ 40 20 -oi o B 30 60 90 120 Time (minutes) Figure 3.3 Uptake of mitoxantrone in PFVs: Influence of PEG- l ip id specie Mitoxantrone was loaded at a drug to total lipid molar ratio of 0.1:1.0 into PFVs exhibiting a transmembrane p H gradient (pH 4.0 i n/pH 7.40Ut). Panel A : P F V s containing P E G -CeramideC16 (•) or PEG-CeramideC20 (•) at 37 °C. Panel B: PFVs containing P E G -D M P E (•), or P E G - D S P E (•,) at 45 ° C . The data points represent a mean ± standard deviation, n = 3. Mitoxantrone and lipid levels were determined by dual label liquid scintillation counting as described in Chapter 2. 78 120 0 20 40 60 80 100 Time (minutes) Figure 3.4 Uptake of mitoxantrone by PFVs: Influence of drug to lipid ratio on uptake of mitoxantrone in PFVs containing PEG-Ceramide Mitoxantrone was encapsulated in response to a transmembrane p H gradient in PFVs containing PEG-Ceramide (C20) at drug to total lipid molar ratios of 0.1:1.0, (•), 0.2:1.0 (•) and 0.3:1.0 (A), mean ± standard deviation, n = 3. 79 Figure 3.5 In vitro release of mitoxantrone from PEG-Ceramide-PFVs: effect of drug to lipid ratio Release of mitoxantrone from PFVs containing PEG-Ceramide (C20) at drug to lipid molar ratio of 0.1:1.0 (•), 0.2:1.0 (•), and 0.3:1.0 ( A ) under dialysis conditions at 3 7 ° C . Mitoxantrone and lipid were assayed as indicated under Materials and Methods using dual label scintillation counting. Mean ± standard deviation, n = 3. 80 3.2.3 Visualization of Mitoxantrone Encapsulated within PFVs Previous studies on liposomal mitoxantrone have employed formulations in which the cationic drug is electrostatically complexed with anionic lipids in a vesicle membrane. This interaction is not stable in vivo resulting in rapid release from the liposome and clearance from the circulation (Schwendener et al., 1991; 1994). To ascertain the suitability of PFVs for systemic delivery of mitoxantrone it was necessary to confirm that accumulated drug was encapsulated in the vesicle interior and not simply associated through hydrophobic or electrostatic interactions with the P F V bilayer. Mitoxantrone-loaded PFVs containing either P E G - D S P E or PEG-CerC20 as their bilayer-stabilizing lipid were examined by cryo -TEM. Electron micrographs shown in Figure 3.6 demonstrate that both empty and mitoxantrone-loaded PFVs exhibit vesicle shapes ranging from spherical to discoid or tubular (average size approximately 100 nm, see Table 3.1). In contrast to empty PFVs , mitoxantrone-loaded vesicles showed a distinct dark band within the intravesicular aqueous compartment (Figure 3.6B and 3.6D). This dark band likely represents mitoxantrone that has precipitated within the vesicle (Madden et al., 1990b). Furthermore, the appearance of mitoxantrone-loaded PFVs is similar to that reported by Lasic et al. (1992; 1995) for liposomes containing the related anthracycline, doxorubicin. Subtle differences in morphology are seen between PFVs containing either P E G - D S P E or PEG-CerC20, for both empty and mitoxantrone-loaded systems. In general, PFVs containing PEG-Ceramide appear more spherical compared to PFVs containing P E G - D S P E which show a preponderance of tubular structures, possibly reflecting differences in overall net charge on the PFVs. P E G - D S P E carries a net negative charge and will therefore partly compensate for the positive charge on D O D A C . 81 Figure 3.6 Cryo-transmission electron micrograph of "empty" programmable fusogenic vesicles and mitoxantrone-loaded PFVs PFVs were made of D O P E : C H O L : D O D A C : P E G - l i p i d (30:45:15:10, P E G - D S P E (a, b) or PEG-CeramideC20 (c, d), respectively). Mitoxantrone-loaded PFVs containing P E G - D S P E and PEG-CeramideC20 are shown in panels (b) and (d), respectively. The bar represents 100 nm. 82 In contrast, PEG-Ceramides are uncharged and therefore PFVs stabilized with this component will carry the full positive charge contributed by D O D A C . This increase in surface charge, and hence charge repulsion, may contribute to the more spherical morphology of PEG-Ceramide-containing PFVs. 3.2.4 Programmed Release of P F V Contents: Influence of Incubation with Sink and Negatively Charged Vesicles The final set of experiments was designed to demonstrate release of vesicular contents of PFVs in vitro, following destabilization. PFVs differing in only the acyl chain length of the bilayer stabilizing lipid, P E G - P E , were prepared in HEPES-buffered saline (HBS) containing [14C]-sucrose as a marker for the intravesicular contents (Chapter 2). It should also be mentioned that the initial volume trapped within PFVs lies in the range of 1.64 and 1.92 pi/umole lipid. Wasan et al. (1997) have reported an internal volume of 1.45 pl/pmole lipid for D O P E : D O D A C vesicles. Similar trapped volumes have also been reported for conventional vesicles (Hope et al., 1985). Before describing the results, it should be pointed out that PFVs were designed to be capable of transforming into destabilized vesicles with the loss of PEG-lipids. Earlier research from our laboratory has shown in vitro that loss of PEG-lipids from PFVs can occur via exchange to neutral "acceptor" vesicles (Holland et al., 1996b). Based on those studies, it was expected that the presence of neutral vesicles would serve as a sink for the loss of P E G -P E from PFVs. In addition, the loss of P E G - P E would unmask the fusogenic lipids and in doing so sufficiently destabilize the PFVs to trigger release of P F V contents. In order to test this hypothesis in vitro, changes in [14C]-sucrose content within PFVs were investigated as a 83 function of the concentration of PEG-lipids (5 or 10 mole%) and the acyl composition of PEG-lipids ( P E G - D M P E and PEG-DSPE) . Experiments involving two-step incubations were conducted to record the variation in the aqueous contents of PFVs . Vesicles were first incubated with a concentration gradient of sink (first step) with the rationale of promoting loss of PEG-lipids. Negatively charged vesicles containing the phospholipid, 1-palmitoyl, 2-o leoy l -^«-g lycero-3 -phosphoser ine (POPS) were then added for 1 day (second step) to trigger electrostatic interactions with the positively charged component of PFVs. Two sets of these experiments were carried out having different durations for the first step of incubation. In the first set of experiments, release of sucrose from PFVs was determined after incubation for 1 day with a 10-fold sink of neutral vesicles ( D O P E : C H O L , 55:45 molar ratio). As shown in Table 3.2, the loss of sucrose content from PFVs containing 5 mole% P E G - D M P E in the presence of sink was equivalent to 23.6% of the initial trapped sucrose. The extent of sucrose release increased to 59% upon incubation of the same PFVs with negatively charged vesicles. Upon incubating PFVs containing P E G - D S P E (5 and 10 mole%) with negatively charged vesicles no further increase in release of sucrose was observed over that of sink vesicles (Table 3.2). As a control for the second incubation step with anionic vesicles, PFVs already exposed to sink were incubated with a solution of aspartic acid. It was observed that the presence of negative charge in solution did not cause a substantial increase in sucrose released from vesicles. For instance, the percentages of sucrose released from P E G - D M P E - P F V s after incubation with sink or sink plus aspartic acid were 20 % and 25% respectively. I5D a St u U PM I PM -O fl « fl «t s-fl U fl o o fl <s .2 • + H •S § « at ^ a> cs PH © fl fl o u M i s o s « H-< o V « "3 fl o SB « a S o u + 1 - f i + > * PS (- CN >> s s o PS (-a in CN o PS (-Q o © © © + o +1 © +1 •a <D +1 ON +1 ON vesicl m ro OO vesicl CN OX vesicl o *~' o in ON in - g * o o O O c/3 CN o o © © >, +1 +1 +1 +1 X ca ON o O NO o Q ro <N NO CN in CN (N o © © o o © ' © © 5 H +1 +1 +1 +1 m Q oo in t--Q C/3 +1 c ON ro CN r-,3 O O o o o ' f t O o © © > +1 +1 +1 +1 rO •st 00 CN CO %-» "o 00 vo r-; ON ' f l fl .3. in /•—s cr- O in O > PFV( > o -PF PFV( -PF FV MPE SPE-MPE PE-P Q P Q ;G-DS 6 6 6 ;G-DS W w w ;G-DS PH PH Ui PH C/2 c/3 CU > <D CJ) o cj > c 13 o o V OH fl" O m •=tt s 85 In the second set of experiments, the loss of aqueous contents of PFVs was determined after 6 days of incubation with sink at a thirty-fold concentration differential (Table 3.3 and Figure 3.7). The experimental design was altered in an attempt to simulate the large sink available in vivo. The length of the first incubation (6 d) was increased to facilitate a greater loss of the PEG-lipids. With respect to initial trapped sucrose, release of sucrose was observed from PFVs containing P E G - D M P E or P E G - D S P E in the presence of acceptor vesicles (30-fold). This loss was more pronounced in the case of P E G - D M P E - P F V s (45.5% when 5 mole% P E G - D M P E was included in PFV) . PFVs formulated with 10 mole% of PEG-l ipid showed a relatively lower release of sucrose compared to control, in the presence of 30-fold sink, 30% and 18% for P E G - D M P E - P F V and P E G - D S P E - P F V , respectively. Upon the subsequent incubation of PFVs containing P E G - D M P E or P E G - D S P E with anionic vesicles, a further release of sucrose contents was observed. This release of sucrose was approximately 1.5-fold higher than that observed with sink alone. For instance, sucrose released from P E G - D M P E - P F V s (5 mole% P E G - D M P E ) increased from 45.5% to 70.5% in the presence of sink plus POPS-containing vesicles compared to sink alone (Table 3.3). The influence of P E G - P E concentration in PFVs became apparent when sucrose release from PFVs containing different mole % P E G - P E was compared. As shown in Figure 3.7 and Table 3.3, as much as 70% of the initial sucrose trapped within P E G - D M P E - P F V s (5 mole% P E G - D M P E ) was lost from the vesicles in contrast to 46% loss from P E G - D M P E -PFVs (10 mole%) after 7 days (incubation with sink + POPS-containing vesicles). Sucrose released from P E G - D S P E - P F V s after exposure to sink plus negatively charged vesicles (48% and 23.5%) for 5 and 10 mole% PEG-DSPE-containing PFVs, respectively) was lower than in the presence of sink alone. 86 Ml a cu .g '3 J S u w I O w PH T3 O (3 a o I. a o a o w o a m .2 ~ 09 CO > «n o a -** a o CJ cn S O CU 3 O" cu cu CA cu "a* C o ce U rt a a o CJ CO > * PS (-ay 7 S ro s CN S i n t--o PS (-Q O O p © + © © © +1 CO +1 +1 +1 vesicl vesicl i n 00 OX vesicl © © © m -a * '—1 o © XSii o © © © XSii >? +1 +1 +1 +1 XSii a Os ,—1 o Q 0\ o ro o ro VO CN o p p i — | p rffei © © © © rffei & +1 +1 +1 +1 m Q o o\ oo vq r-vq u S.D n +i ro CN ap Vi iT O o o o ap Vi 'a, © © © © ap Vi +1 +1 +1 +1 Tr ro 00 Tr "o oo vq Os nitial c *—< i—< »—< nitial c nitial ^° i n O (%0 i n —^' -PFV PFV( -PFV PFV( MPE SPE-MPE SPE-Q Q Q Q 6 6 6 6 W pa w PJ CH P H PH PH CO =tfc CO CD CO > -a o > U fl CU .2 c in .a • =*fc 87 •g o. s 2.0 ro -i—> o +-> o E 0) E O > Q. ro •g ro -*—• o -<—• 0) o E E O > ti-ro 1.5 1.0 o 0.5 0.0 X PEG-DMPE (5%) PEG-DMPE (10%) S 2.0 -1.5 ^ 1.0 o 0.5 0.0 B PEG-DSPE (5%) PEG-DSPE (10%) Figure 3.7 In vitro release of aqueous contents from PFVs: Influence of concentration and acyl composition of P E G - P E The volume of [14C]-sucrose-labeled buffer remaining within PFVs (pl/pmole lipid) ± S.D measured before and (clear bars) after incubation with H B S (150 m M N a C l , 25 m M H E P E S , p H 7.4, diagonally hatched bars); 30X sink (neutral vesicles, D O P C : C H O L , 55:45, molar ratio, cross hatched bars) and sink + negatively charged vesicles ( D O P E : C H O L : P O P S , 40:45:15, molar ratio, shaded bars) on day 7. Panels A and B show the release of aqueous contents from P E G - D M P E or P E G - D S P E - P F V s , (5 and 10 mole%), respectively. 88 The effect of incubation conditions on release of sucrose from PFVs was evaluated statistically by analysis of variance ( A N O V A ) . Scheffe's post hoc test showed release of sucrose to be significantly higher in the presence of sink plus anionic vesicles from all other incubations conditions for PFVs containing P E G - D M P E (5 and 10 mole%; Tables 3.2 and 3.3) and P E G - D S P E (5 mole%; Table 3.2), respectively. Results were significant at P < 0.01. 3.3 D iscuss ion On the basis of results presented in this chapter, three important inferences can be drawn: (a) PFVs are of various shapes, as observed in cryo-transmission electron micrographs, (b) PFVs readily accumulate and retain mitoxantrone, and (c) the release of aqueous contents of PFVs occurs as a function of P E G - P E acyl composition in the presence of sink and negatively charged vesicles. When PFVs are prepared in H B S , they adopt predominantly non-spherical morphologies. These vesicle shapes at least partly result from the method of preparation as indicated by the observation of spherical vesicles when such systems are prepared in distilled water. Liposomes are osmotically active and are generally unable to transform from non-spherical to spherical vesicles due to the consequent changes in intravesicular osmolality that such a shape change would induce. A sphere represents the highest possible volume to surface area ratio and therefore "rounding-up" of non-spherical vesicles would require influx of water thereby creating an osmotic gradient. In the absence of a non-permeant solute, however, such an osmotic gradient is not created and relaxation to a sphere is possible. It has previously been reported that large unilamellar vesicles prepared by extrusion are non-89 spherical (Mui et al., 1993; Mui et al., 1995). In the present work, it was demonstrated that when such extruded vesicles are placed in hypo-osmotic solutions they are able to round-up as a consequence of water influx. The morphology of PFVs is therefore not unique but has been suggested by previous studies of extruded vesicles. While many non-spherical vesicles can be the result of the extrusion process, the lipid composition of PFVs may also make subtle contributions to morphology. It has recently been reported that incorporation of cholesterol into egg phosphatidylcholine vesicles at more than 50 mole%, contributes to the formation of elongated vesicles (Edwards et a l , 1997). In this regard, the presence of 45 mole% cholesterol in PFVs, together with other non-bilayer-forming lipids, may promote non-spherical morphologies. Vesicles prepared in the absence of the cationic lipid D O D A C often exhibit an invaginated appearance or appear to consist of vesicles within vesicles. Whether all of the observed bilamellar structures represent enclosed vesicles, or might instead consist of two opposed vesicles observed from above, is unclear. In some instances, c r y o - T E M micrographs appear to show examples of vesicle-vesicle apposition, however, such apposition would be surprising, given the general ability of PEG-lipids to provide a steric barrier (Lasic et al., 1991; Needham et al., 1992; Torchilin et al., 1994a). Liposome morphology is also strongly influenced by area imbalances between the inner and outer monolayers (Seifert et al., 1991; Farge and Devaux, 1992; M u i et al., 1995). A n excess inner monolayer surface area tends to promote an invaginated morphology while an excess outer monolayer area results in vesicle "blebbing" (Mui et al., 1995 and references therein). In the case of PFVs, such area imbalances could result from loss of PEG-l ipid from the outer monolayer. As noted in Chapter 1, PFVs are designed to undergo destabilization through loss of PEG-lipids via exchange to other binding sites. This exchange can be 90 demonstrated in vitro by incubation of PFVs in the presence of "acceptor" liposomes (Holland et al., 1996b), and will readily occur in vivo through binding of PEG-lipids to lipoproteins or cell membranes. Although "acceptor" systems were not present for the cryo-T E M studies, it is possible that some PEG-lipid desorption occurs by the formation of P E G -lipid micelles following P F V preparation resulting in formation of invaginated vesicles. Such desorption might simply reflect the monomer solubility of PEG-lipids and/or result from the relatively high initial mole ratio of these lipids in the P F V bilayer. The capacity of a lipid bilayer to accommodate PEG-lipids is determined by the mean area occupied by this component. At mole ratios lower than 5 mole%, the polymer chains are able to occupy a larger surface area and sweep out a "mushroom-shaped" volume. At higher mole ratios (>5%), the P E G chains are constrained by their proximity to each other and align to form a "brush border" extending much further out into the aqueous medium (deGennes, 1980, reviewed by Milner, 1991). Rex et al. (1998) provide experimental and theoretical evidence for a mushroom conformation of P E G 2 0 0 0 - P E ranging from 0-10 mole%. When the capacity of the bilayer to accommodate P E G - P E is exceeded, however, membrane destabilization is triggered with the formation of PEG-lipid micelles or mixed micelles (Kenworthy et al., 1995). Edwards et al. (1997) have demonstrated that increasing the P E G -lipid component beyond 10 mole% initiates the formation of open bilayer discs in liposomes composed of D S P C : C H O L . They attributed these structures to the presence of surface-active agents such as PEG-lipids, which can easily be accommodated into the highly curved monolayer of lipids required for the stabilization of the disc edge. In view of the fact that PFVs contain predominantly unsaturated lipids (DOPE and D O D A C ) that exhibit a larger mean area/molecule than vesicles composed of saturated lipids, such as D S P C , P F V s would 91 therefore be expected to accommodate a higher mole ratio of PEG-lipid. In the current studies it was observed that stable PFVs could be prepared with 10 mole% PEG-lipid but, in agreement with Edwards et al. (1997), open bilayer discs were also observed when PFVs were prepared with 20 mole % P E G - D S P E . As noted under Results (Table 3.1), when a comparison is made of mean vesicle size determined by c r y o - T E M and by quasielastic light scattering, a significant discrepancy is apparent. Vesicle size determined by Q E L S is consistently greater with a difference of about 20-40 nm. A similar discrepancy was previously noted when Q E L S measurements were compared to vesicles sizes determined from freeze-fracture electron micrographs (Mayer et al., 1986c). A number of factors may contribute to the different size estimates provided by these techniques. First, vesicle morphology complicates size determinations obtained by direct measurement of cryo-TEM micrographs. From a two dimensional image, it is necessary to extrapolate to a three dimensional shape in order to estimate vesicle diameter (refer to Section 2.6.1, Figure 2.1). In the case of Q E L S , however, this technique measures the vesicle diffusion rate and then calculates size based on this estimate of mass assuming that the vesicles are unilamellar and spherical. Furthermore, Q E L S measures all vesicles within the sample including any large liposomes that may be present. As a result of the influence of vesicle size on light scattering intensity, a small percentage of large liposomes can significantly influence the calculated mean vesicle diameter. In contrast, c r y o - T E M can potentially underestimate the occurrence of large (> 1 pm) liposomes as they can be excluded from the thin aqueous layers that are visualized under the electron microscope. A possible consequence of P F V design is that the vesicle bilayer might not provide an adequate permeability barrier allowing establishment of a p H gradient and/or retention of an 92 accumulated drug. As the current research demonstrates, this is not the case and mitoxantrone can be efficiently and stably loaded into PFVs employing a proton gradient. The physical state of anticancer drugs loaded within liposomes using this technique has been the subject of debate over the past few years. It was initially noted that the apparent intracellular concentration of many liposomally loaded antineoplastics greatly exceeded their aqueous solubilities suggesting that drug might undergo precipitation within the intravesicular core (Madden et al., 1990b). A n alternative proposal was that accumulated drug could partition into the inner monolayer, thereby reducing drug concentration in the aqueous phase (Harrigan et al., 1993; Cullis et al., 1997). Analysis of doxorubicin-loaded liposomes by cryo-electron microscopy revealed a "coffee bean" appearance which was interpreted as precipitated or "gel" phase drug within the intravesicular core (Lasic et al., 1992; 1995). This debate on the physical state of liposomally encapsulated doxorubicin was recently resolved with the publication of a study combining cryo-EM and X-ray diffraction to characterize such systems. This study demonstrates that doxorubicin forms bundles of fibers in the aqueous core with no significant perturbation of the vesicle bilayer (Li et al., 1998). In the current study, similar electron opaque structures were observed within mitoxantrone-loaded PFVs . These structures appear generally to span the long axis of the vesicle but do not otherwise distort vesicle morphology. Recently, Johnsson et al. (1999) reported that loading of water soluble acridine and phenantridine derivatives at high concentration into sterically stabilized vesicles ( D S P C : C H O L : P E G - D S P E , 55:40:5, molar ratio) may result in rupture of the carriers; no evidence of P F V disruption by loaded mitoxantrone was observed in the present work. 93 The final point in the discussion concerns the relationship between the release of P F V contents in vitro and P E G - P E acyl composition. In brief, the results indicate that greater release of contents was observed from PFVs containing P E G - D M P E . Sucrose release from P E G - D S P E - P F V s was greater on day 7 compared to day 2, which is consistent with the expectation that PFVs containing a longer acyl chain P E G anchor would take longer to destabilize and release contents. The importance of anchor composition in determining the rate of PEG-l ipid exchange has been reported (Silvius and Zuckermann, 1993). In addition, the greater loss of vesicular contents of PFVs containing P E G - D M P E or P E G - D S P E , observed in the presence of 30-fold as compared to a 10-fold excess of acceptor vesicles may reflect exchange of a higher proportion of the initial PEG-lipids. The observation that incubation of PFVs with sink plus an anionic solution (aspartic acid) as opposed to anionic vesicles led to a relatively lower release of contents was expected. For example, Stamatatos and coworkers (1988) have reported the loss of cationic liposome contents upon fusion with anionic vesicles. In similar in vitro studies, the role of the target anionic vesicle membrane has been shown by Bailey and Cullis, (1997). Thus it is speculated that enhanced release in the presence of anionic vesicles is mediated by a fusion event and hence is not seen in the presence of an anion such as aspartic acid. It was recognized that the use of [14C]-sucrose would serve as a surrogate marker for encapsulated drug but was used in order to demonstrate the mechanism of action of PFVs. Several investigators have shown the tendency of liposomes to destabilize in the presence of serum (Scherphof et al., 1978; Kirby et al., 1980; Allen and Cleland, 1980). Others have also shown a correlation between destabilization of liposomes and the release of liposomal contents by exchange of phospholipids from the vesicle to lipoproteins (Scherphof et al., 94 1978; Finkelstein and Weissman, 1979; Damen et al., 1981; Scherphof and Morselt, 1984). Most of these studies were conducted using multilamellar vesicles containing entrapped aqueous space markers. A complete loss of PFV contents would be the ideal proof of principle of PFVs. Even though sucrose was used because it is impermeable through vesicle membranes, some leakage of [14C]-sucrose could occur, possibly by diffusion (less than 10 % was observed). Given that even a 30-fold concentration gradient of sink in vitro may not be a true representative of the large 'tissue sink' available in vivo, the loss of greater than 70% aqueous contents from PEG-DMPE-PFVs was very encouraging (Table 3.3). In conclusion, the present studies demonstrate that PFVs are closed vesicular structures and that PEG-lipids are required to prevent aggregation/fusion in physiological saline. The morphology of PFVs is influenced not only by the extrusion process but also by their lipid composition. Despite the high proportion of non-bilayer-forming lipids present, PFVs can maintain a transmembrane pH gradient and can efficiently accumulate mitoxantrone. The accumulated drug appears to exist as a precipitate within the vesicle aqueous core. Greater release of PFV contents was observed from PFVs-containing PEG-DMPE as compared to PEG-DSPE. In Chapter 4, the stability of PFVs in the circulation is characterized by examining the plasma elimination behavior of mitoxantrone-loaded PFVs as a function of PEG-lipid exchange rate. 95 CHAPTER 4: CHARACTERIZATION OF THE SYSTEMIC PROPERTIES OF PFVs: INFLUENCE OF POLY(ETHYLENE GLYCOL)-PHOSPHATIDYLETHANOLAMINE A C Y L COMPOSITION 4.1 Introduction Attempts to develop a variety of liposomal formulations with controlled release capabilities have been made in an effort to meet one of the major requirements of drug delivery, namely, cytoplasmic delivery. Examples of such liposomes include pH-sensitive (Yatvin et al., 1980; Connor et al., 1984; Connor and Huang, 1985; Liu and Huang, 1989), temperature-sensitive (Weinstein et al., 1980; Gaber et al., 1996) and fusogenic liposomes (Gitman and Loyter, 1984, reviewed by Szoka, 1990). Fusogenic liposomes are one of the most popular candidates for systemic delivery of gene-based drugs and are primarily composed of DOPE and cationic lipids (Feigner et al., 1987; Feigner and Ringold, 1989; Zhu et al., 1993). While the activity of pH-sensitive and thermo-sensitive vesicle formulations has been demonstrated in well-defined in vitro systems, a major drawback of such liposomes is their limited applicability in vivo due to poor stability in the circulation (Connor et al., 1986; Mahatoetal., 1995). Silvius and Zuckermann (1993) have demonstrated in vitro, that intervesicular exchange of hydrophilic molecules conjugated to phospholipid anchors occurs on biologically relevant time scales. Previous studies have shown that the inclusion of polyethylene glycol can regulate fusion between liposomes (Holland et al., 1996b). Furthermore, the work of Parr et al. (1994) demonstrated the importance of acyl chain length of the lipid anchor of PEG in regulating the rate of transfer of PEG-lipids from conventional liposomes (DSPC:CHOL). 96 Given the existing need for a liposomal carrier that displays multifunctional characteristics, such as initial stability in the circulation with good retention of contents and destabilization into a fusogenic, leaky entity, this chapter will assess the systemic stability of programmable fusogenic vesicles. As outlined in Chapter 1, the spontaneous exchange of PEG-lipid from the liposome surface forms the basis for the design and mechanism of action of PFVs. The studies presented in this chapter are primarily focused on four areas of investigation. First, the plasma elimination and biodistribution characteristics of mitoxantrone-loaded PFVs is examined. Second, the rate of exchange of PEG-lipid conjugates as a function of the composition of the lipid anchor is determined. Furthermore, this exchange is compared to that of conventional liposomes sterically stabilized by the inclusion of amphipathic PEG-conjugated lipid. The rate of loss of the cationic component of PFVs, DODAC, is also examined. The last question addressed in this chapter is the role of plasma components such as lipoproteins in taking up the PEG-lipids lost from the surface of a liposome. This issue was explored with the use of Fast Protein Lipid Chromatography (FPLC). 4.2 Results 4.2.1 Plasma Elimination of Programmable Fusogenic Vesicles and Encapsulated Mitoxantrone: Influence of PEG-PE Acyl Composition PFVs were prepared with PEG-PE of varying acyl composition (DMPE, DPPE, DSPE) and loaded with mitoxantrone using a pH gradient (Chapter 2). As shown in Figure 4.1, the PFV elimination curves are dramatically influenced by the acyl composition of the PEG-PE component. It should be stressed that this represents the only difference between 97 the three formulations which are otherwise equivalent in terms of vesicle size, lipid composition and mitoxantrone incorporation. PFVs prepared with the shortest acyl chain PEG-PE, PEG-DMPE (14:0, l,2-dimyristoyl-5«-glycero-3-phosphoethanolamine), are rapidly eliminated from the circulation (80 % of the injected dose has been eliminated from the plasma within 1 hour) consistent with the expected rapid exchange of this stabilizing component out of the bilayer (Silvius and Leventis, 1993; Silvius and Zuckermann, 1993; Holland et al., 1996a). When the PE acyl chain length was increased to sixteen carbons (PEG-DPPE, 16:0, l,2-dipalmitoyl-5«-glycero-3-phosphoethanolamine), vesicle clearance from the blood was considerably slower, with about 50% of the lipid dose remaining in circulation at 4 hours. Increasing acyl chain length to eighteen carbons (PEG-DSPE, 18:0, l,2-distearoyl-5«-glycero-3-phosphoethanolamine) resulted in an additional increase in circulation longevity with approximately 40% of the injected lipid dose remaining in the plasma even at 24 hours. These differences are reflected in the estimation of the mean liposomal lipid concentration area under the curve (AUC) for the plasma, where values of 11, 43 and 62 pmole/ml/h were calculated for PFVs prepared with PEG-DMPE, PEG-DPPE and PEG-DSPE, respectively (Table 4.1). The mean A U C obtained for DSPE-PEG-PFV was 6-and 2-fold greater than that obtained following administration of mitoxantrone-loaded DSPC:CHOL and DSPC:CHOL:PEG-DSPE conventional liposomes. It should be noted that the AUCs reported for DSPC:CHOL and DSPC:CHOL:PEG-DSPE vesicles containing mitoxantrone are taken from Chang et al. (1997) and the drug to lipid ratio in their studies was 0.416 (10 mg/kg mitoxantrone to 54 pmole phospholipid/kg). Furthermore, it is of interest that the plasma elimination behavior of the PFVs was not affected by the presence of encapsulated mitoxantrone: essentially the same plasma elimination rates are found for 98 "empty" and mitoxantrone-loaded PFVs (Figure 4.1). This observation is important in that it demonstrates that differences in vesicle clearance as a function of PEG-PE acyl composition are not related to cytotoxic effects on phagocytic cells of the MPS. Additional insight into carrier destabilization is provided by plasma elimination rates for mitoxantrone (Figure 4.IB; note plasma drug levels are shown on a log scale). As would be expected from the data in Figure 4.1 A, mitoxantrone plasma elimination is fastest for PEG-DMPE-containing carriers and slowest after injection of PFVs containing PEG-DSPE (Figure 4.IB). As shown in Table 4.1, the mean AUCs were estimated to be 0.18, 1.56, 4.20 umole/ml/h for PFVs prepared with PEG-DMPE, PEG-DPPE, and PEG-DSPE, respectively. Importantly, the mean mitoxantrone AUCs obtained for the PEG-DSPE-PFV formulation were less than or equal to those estimated for conventional DSPC:CHOL or DSPC:CHOL:PEG-DSPE liposomes. Since the mean liposomal lipid AUCs obtained for PFV were greater than those measured for the conventional liposomes, differences in mean drug A U C are a consequence of differences in drug release. By comparing plasma PFV lipid concentrations and mitoxantrone concentrations at various times after injection, for the three PFV compositions, it is also possible to determine how much of the initially encapsulated drug is retained within the carrier. This determination is possible because free mitoxantrone is rapidly cleared from the blood compartment (Figure 4.IB, and Lim et al., 1997). In Figure 4.1C are shown the leakage rates for mitoxantrone from the three PFV formulations in the circulation. As anticipated, drug loss from PFVs was related to PEG-PE exchange rate. Among the three PFV formulations, PFVs prepared with PEG-DSPE released drug slowly, with approximately 40% of the entrapped agent remaining within the circulating vesicles at 24 hours. In contrast, the drug release rates were faster for PEG-DMPE- and PEG-DPPE-99 containing PFVs. This is best illustrated by an evaluation of the ratio of mean mitoxantrone A U C (AUC D ) / mean liposomal lipid A U C (AUC L ) . Values of 0.016, 0.037 and 0.068 were obtained for PFVs containing PEG-DMPE, PEG-DPPE and PEG-DSPE, respectively. It should be noted that the A U C D / A U C l ratio for DSPC:CHOL and DSPC:CHOL:PEG-DSPE conventional liposomes was 0.39 and as has already been pointed out, the starting drug to lipid ratio in these studies was 0.416 (Chang et al., 1997). Taken together, these results suggest that these conventional vesicles retain drug to a greater extent than PFVs. Studies of liposomal mitoxantrone formulations have shown that therapeutic activity is dependent on drug release (Lim et al., 1997). 4.2.2 Biodistribution of Mitoxantrone-Loaded PFVs In view of the rapid elimination of PFVs containing PEG-DMPE from the plasma, a corresponding accumulation in the liver and spleen would be expected i f removal from the blood compartment occurred as a consequence of carrier recognition by the MPS. As shown in Figure 4.2, this anticipated uptake in liver of PEG-DMPE-containing PFVs is observed for both the lipid carrier and mitoxantrone. In the case of the PFV carrier, almost 50% of the injected dose accumulates in the liver at 1 hour and only a small further increase is seen at later times (Figure 4.2). This behavior is in sharp contrast to the liver accumulation seen for PFVs containing PEG-PE of longer acyl chain length: both PEG-DPPE- and PEG-DSPE-containing systems exhibit very little liver uptake at 1 hour. Paralleling plasma elimination curves, PFVs stabilized with PEG-DPPE show a marked increase in liver accumulation between 1 and 4 hours, and a small further increase at 24 hours. In the case of PEG-DSPE-100 containing PFVs, much slower uptake by the MPS is observed and even at 24 hours less than 15% of the injected dose is present in the liver. Mean liposomal lipid AUCs for the liver were estimated to be 50, 35 and 10 umole/g/h for PFVs prepared with PEG-DMPE, PEG-DPPE and PEG-DSPE, respectively (Table 4.1). Mean mitoxantrone AUCs were consistent with these results, so that drug levels in the liver peaked after 1 hour for PEG-DMPE-PFVs at approximately 30% of the injected drug. Even after 24 hours, only 10% of the injected drug was measured in this tissue following injection of PEG-DSPE-PFV (Figure 4.2B). Mean A U C analysis of liver mitoxantrone levels indicate step-wise decreases as the acyl chain length of the PEG-PE stabilizing component was increased from PEG-DMPE-PFV to PEG-DSPE-PFV (Table 4.1 and Figure 4.2B). The mean drug AUCs estimated following administration of conventional liposomal mitoxantrone formulations were between 5-and 10-fold greater than those obtained following administration of the PFVs. Again, it should be noted that while the initial level of mitoxantrone encapsulated within DSPC:CHOL or DSPC:CHOL:PEG-DSPE liposomes was the same (10 mg/kg), the level of lipid was 4-fold lower than that of PFVs (Chang et a l , 1997). The mean drug AUCs for liver following administration (i.v.) of free drug were substantially lower then those measured following administration of the liposomal formulations. Table 4.1 also shows mean drug and liposomal lipid AUCs for spleen, lung and kidney. A comparison of mean liposomal lipid AUCs for these tissues indicated that the presence of entrapped mitoxantrone did not significantly alter lipid accumulation in the tissues. 101 Figure 4.1 Plasma elimination of mitoxantrone-loaded PFVs after i.v. administration: Influence of PEG-PE acyl composition (following page) Mitoxantrone-loaded programmable fusogenic vesicles composed of DOPE:CHOL:DODAC:PEG-PE (30:45:15:10) were injected via the lateral tail vein into BDF-1 mice (4.5 pmole total lipid/mouse). At various times, the mice were sacrificed and plasma isolated. The marker [ 3H]-CHE was used to determine PFV recovery and mitoxantrone levels were monitored with [14C]-mitoxantrone. (A) Plasma levels of PFVs. (B) Plasma levels of mitoxantrone. (C) Mitoxantrone:lipid ratio in circulating PFVs. Symbols for all panels are; PFVs containing PEG-DSPE, (•); PFVs containing PEG-DPPE, (•); PFVs containing PEG-DMPE, (•) and free mitoxantrone, (open diamond). In panel A , the open symbols represent the equivalent "empty" PFVs (no mitoxantrone). Bars, S.E. of the mean, n = 4. * signifies P < 0.01 at 24 h, see Methods. 102 Time (hours) Vi S Vi Vt — "3 Vj U <U H-» o fl « a VI St "Si ' a ta PH -B fl « « fl O )H fl « o H-> a s-.o U < CU s u <u pfl S-~a fl s « CU s-« fl cu H <u o -fl CU CU ° .3 s o cu 6 -o - O cd _ cj ~ -fl fl H -a ^ S rt •5 o «« .2 fi -S .2 2 3 g 3. o cu o > cu o cu OH CD bO 00 fl. o rt1 in OH CN X CN O -fl T 3 OH CU ^ OH P H O cu 60 CU fl cd 2 § & bO fi bo T3 o fl rt o •1=! cd ~ £ rt « t3 cu rt o cu cu fl o CO CU a o CO o OH -fl CU -a * CU co N <-rfl ta - O P H >r> cd -3 -2 cu g sS bo a>"bb ^ a cu R o cu cd fl o CU fl o o W P H I o pq PH C+H o fl o ° rt s fl co m ^ CN S <N g co O 5 2 .2" cu -fi co o 2 T 3 fi O I H ^ O  CU ta ° * -g fl * s .o cu a> fl c o o s s g £ & OH X X u S ' s l bO o Q o < o T i -ON T t ro CN o o O o O o o a A. bO o OO o CN o ON VO i n r--oo CN s CN O o o mol/g/h) Q u ON vq i n r--CN vo i n CN ON 00 ro A. CU fl u < vo ON OS oo VO i n Kid C-O CN nol/g/h) o < o 00 CN ro vo VO c> ro 00 m CN ro ro i f l . fl cu u ro oo rt i n r--ON m rt "El 00 < ON r - i n CN i n ON r-- o -s? u < m o oo 00 o as vo ON vo CN vo ON as C3 fl fl fl. cu > o 00 oo 00 CN CN CN ro O m VO ro VO VO ON nol/ml/h) Q u < CN O o o i n ON O oo o vo i n rt CN fl. a CO ta u < i n CN 00 CN VO CN CN VO w OH CO <D U i UH O X o u Q * 6 w PH o o u PH Ui Q * > PH 1 w Q 1 a w PH > PH I w p< PH Q I a w PH > PH I w PH Ui Q I a PH VO o co cd O td i-l a O o OH r-ON ON cd H-> cu bO -fl U a o cc B <& •a -+-» S3 eu -e o OH cu kH cd td Q 104 60 CD > CD 00 o Q •g C L "D CD O CD CD > CD CO o Q CD c o i— -4—" c cc X o 40 S 20 0 60 40 | 20 T J CD O CD *S> 0 B 1 Hour 4 Hour 24 Hour Figure 4.2 Biodistribution of PFVs and mitoxantrone in the liver of BDF-1 mice Accumulation of P F V (A) and mitoxantrone (B) in the liver after i.v. administration. The mitoxantrone dose was 22.5 pmole/kg and the lipid dose was 225 pmole/kg. Drug and lipid levels were determined as described in Chapter 2. PFVs and mitoxantrone levels are shown at 1, 4, and 24 h for PFVs containing P E G - D M P E , (clear) P E G - D P P E , (shaded) and P E G -D S P E (horizontal cross hatched). Bars represent the average ± S.E. n = 4. 105 4.2.3 Plasma Concentrations of P E G - P E and PFVs: Influence of P E G - P E Acyl Chain Lengths As has been already mentioned, the data presented in Figures 4.1, 4.2 and Table 4.1 depicted mitoxantrone-loaded PFVs, which were identical in all attributes except the acyl chain length of the PEG-lipid anchor. A l l these studies were conducted using [ 3H]-CHE label to mark the carrier lipid. In order to confirm the proposed mechanism of action of PFVs it was essential to determine the actual rate of transfer of PEG-PE. Studies were initiated in BDF-1 mice to determine the PEG-PE([ 3H]) and lipid ([ 1 4C]-CHE) levels within the plasma at various times following i.v. injection (refer to Chapter 2, section 2.9.3 for details). The initial ratio of PEG-PE to liposomal lipid was 0.1:1.0. The time courses of plasma elimination of PEG-PE over a 24 hour time-course is shown in Figure 4.3A. Very rapid loss of PEG-DMPE occurred within 1 hour after injection. Although, 70% of the injected PEG-DMPE had been eliminated from plasma after 1 hour, little exchange of PEG-lipid (< than 10%) was observed from either PEG-DPPE-PFVs or PEG-DSPE-PFVs. Comparison of the levels of PEG-DPPE and PEG-DSPE in plasma at 8 hours showed significant differences (P< 0.01). PEG-DSPE levels were 4-5 fold higher than those of PEG-DPPE at 8 hours. Furthermore, from Figure 4.3A it appears that the time taken for 50% of PEG-DSPE to be cleared from plasma was approximately 24 hours. 106 B Time (hours) Figure 4.3 Plasma elimination of PEG-PE and PFVs after i.v. administration: Influence of PEG-PE acyl composition Programmable fusogenic vesicles composed of DOPE:CHOL:DODAC:PEG-PE (30:45:15:10) were injected via the lateral tail vein into BDF-1 mice (4.5 pmole total lipid/mouse). At various times, the mice were sacrificed and plasma isolated. The marker [ 1 4C]-CHE was used to determine PFV recovery and PEG-PE levels were monitored with the corresponding tritiated PEG-PE. (A) Concentrations of PEG-PE in plasma, and (B) plasma levels of PFVs containing PEG-DMPE (•), PEG-DPPE (•), and PEG-DSPE ( A ) are shown at 0.5, 1, 2, 4, 8 and 24 hours. Values shown represent the mean ± S.E, n=4, * signifies P < 0.01 at 24 h, see Methods. 1 0 7 Plasma P F V concentrations for systems containing P E G - D M P E , P E G - D P P E or P E G -D S P E were followed using the radiolabeled cholesteroyl hexadecyl ether ( [ 1 4 C]-CHE) . As shown in Figure 4.3B, the rate of clearance of PFVs from plasma was consistent with the data shown in Figure 4.1 A . PFVs containing the longer chain phospholipid P E G conjugate (PEG-DSPE) remained in circulation longer as compared to the shorter chain PEG-PEs . At the end of 24 hours, 40% of the injected P E G - D S P E - P F V remained in circulation, whereas P E G -D M P E - P F V had already been eliminated from the blood compartment (Figure 4.3B). Interestingly, the circulation half-life of P E G - D M P E - P F V s was less than 30 minutes. 4.2.4 Plasma Elimination of P E G - P E from D S P C : C H O L Liposomes: Influence of P E G - P E Acyl Chain Length To determine whether dissociation of the P E G coating occurred from liposomes of a conventional composition (DSPC:CHOL) , plasma levels of P E G - P E were determined following an intravenous injection of conventional liposomes containing P E G - P E (5 mole%) of varying acyl chain composition. The time-course of elimination of PEG-lipids followed a pattern similar to that of P E G - P E components in PFVs. It becomes evident from Figure 4.4A that the elimination of P E G - D S P E from the plasma proceeds slower than that of P E G -D M P E . For instance, at 8 hours, the circulation levels of P E G - D S P E were 65% of the injected dose, where as less than 25% of the injected P E G - D M P E was observed in plasma. The loss of P E G - D P P E was intermediate between that of P E G - D M P E and P E G - D S P E (38% in plasma at 8 hours). It should be noted that P E G - P E was incorporated at 5 mole% in all the conventional vesicles with the exception of P E G - D P P E , which was included at 10 mole%. Apart from this, the only difference between the types of conventional vesicles studied was 108 the composition of the phospholipid anchoring the PEG to the liposome bilayer. A fourth type of conventional liposome was included in the studies to serve as a control for the radiolabeled marker used to trace the PEG-PE component (PEG-PE[3HJ). A conventional liposome containing PEG-DSPE was labeled with PEG-DPPE[ 3H] and the time taken for the loss of the PEG-PE component followed using this tracer. It is clear from Figure 4.4A that the concentrations of PEG-PE in this vesicle follow those of liposomes containing PEG-DPPE. The implications of this finding will be discussed in section 4.3. The time-course of elimination of these sterically stabilized vesicles from plasma was determined and was also found to be a function of the acyl chain composition of the PEG-PE stabilizer used (Figure 4.4B). The blood residence times of these vesicles were not different from that of PFVs containing the respective PEG-PE component, which is not unexpected given their lipid composition. The half-life of PEG-DSPE-containing conventional liposomes (PEG-DSPE-CnV) was approximately 15 hours while that of PEG-DSPE-PFV was around 17 hours. The plasma level of liposomes containing PEG-DSPE at 24 hours was 31.6% of the injected dose which is equivalent to that reported by Allen et al. (1991). They have shown that incorporation of 6.25 mole% of PEG-DSPE in DSPC:CHOL vesicles (injected at a dose of 0.5 pmole phospholipid per mouse) can result in up to 30 % of the liposomes remaining in circulation at 24 hours. 109 I 0.00 I 1 1 1 1 1 1 =»- 0 5 10 15 20 25 30 Time (hours) Figure 4.4 Elimination of PEG-PE and liposomes from plasma: influence of PEG-PE acyl chain composition Vesicles composed of D S P C : C H O L : P E G - P E (50:45:5) were injected via the lateral tail vein into BDF-1 mice (4.5 pmole total lipid/mouse). At various times, the mice were sacrificed and plasma isolated. Plasma PEG-PE([ 3 H]) and lipid ( [ 1 4 C]-CHE) levels were determined at 0.5, 1, 2, 4, 8 and 24 hours by dual label scintillation counting. (A) Plasma levels of P E G - P E , and (B) vesicles containing P E G - D M P E (•) P E G - D P P E (•) and P E G - D S P E ( A ) . The open diamond symbol represents conventional vesicles containing P E G - D S P E labeled with P E G -DPPE[ 3 H]. The data are expressed as molar ratio of injected dose to account for conventional vesicles containing P E G - D P P E (10 mole%). Results represent the mean ± S.E, n=4. 110 0 5 10 15 20 25 30 Time (hours) Figure 4.5 Loss of P E G - P E from liposomes in blood The ratio of P E G - P E (PEG-PE[ 3 H]) to liposomal lipid ( [ 1 4 C]-CHE) was determined in plasma at various time points after i.v. administration for PFVs (A) and conventional liposomes (B) containing P E G - D M P E (•), P E G - D P P E (•), and P E G - D S P E ( A ) . In Panel B , the symbol for conventional vesicles containing P E G - D S P E and labeled with PEG-DPPE[ 3 H] is an open diamond. Results represent the means of PEG-PE[ 3 H] to [ l 4 C ] - C H E ratios obtained from four mice and expressed as a percentage of (± S.E.) of the initial ratio before injection. * signifies P< 0.01 at 24 h, see Methods. I l l 4.2.5 Rate of Exchange of P E G - P E from The Carrier: Influence of Vesicle Composition On the basis of the time-course of elimination of P E G - P E and that of the carrier from the plasma (Figure 4.3 A , B and Figure 4.4A, B), it is possible to estimate the extent of P E G -P E that has exchanged out of a vesicle. Illustrated in Figure 4.5A, B are the ratios of P E G -P E lost from PFVs and conventional liposomes, respectively. The rate of transfer of P E G - P E from PFVs was determined over 24 hours and was consistent with the hypothesis that the shorter is the chain length of the P E G - P E , the more rapid was the loss of lipid. It is shown in Figure 4.5A that within 1 hour 30% of the injected P E G - D M P E had exchanged out of P E G - D M P E - P F V s and at the end of 4 hours only 10% of the P E G - D P P E had been lost from P E G - D P P E - P F V s . In contrast, even after 24 hours as much as 90% of the initial P E G - D S P E was retained within P E G - D S P E - P F V s . The relative kinetics of exchange of PEG-conjugated lipids from conventional liposomes were similar to those seen for PFVs. Within 2 hours, 45% of the P E G - D M P E had been lost; however, only 10% of P E G - D P P E had exchanged out at 1 hour. In the case of P E G - D S P E , even after 24 hours little exchange of P E G - P E had been observed (Figure 4.5B). It should be mentioned that the extent of P E G - D P P E exchange observed from either PFVs or D S P C : C H O L liposomes levels off after 8 hours. This observation was true for the P E G -D S P E containing conventional, control vesicles as well (labeled with PEG-DPPE[ 3 H]) . On the basis of the results presented so far, two important conclusions can be made. First, that the process of spontaneous exchange of PEG-lipids occurs in vivo as a function of the acyl chain composition of the phospholipid anchor, as has been demonstrated in vitro by Silvius and Zuckermann (1993) and Holland et al. (1996b). The second important inference that can 112 be made from these data is that regardless of the liposome composition, the loss of these exchangeable lipids is a function of acyl chain length. 4.2.6 In Vivo Exchange of D O D A C from Programmable Fusogenic Vesicles The next set of experiments was designed to determine whether the cationic component ( D O D A C ) of programmable fusogenic vesicles could also be lost from the bilayer. Since PFVs are primarily composed of fusogenic, non-bilayer-forming lipids which are maintained in a bilayer conformation by the presence of PEG-lipid, it was proposed that the formulation would destabilize following the exchange of PEG-conjugated lipids. Therefore, the question that remained was whether the rate of desorption of D O D A C also was influenced by the rate of exchange of the PEG-lipid. In experiments similar to those that examined exchange of P E G - P E from PFVs, D O D A C was traced using a [ 1 4 C ] - D O D A C label and [ 3 H]-CHE was used as the marker for PFVs. As shown in Figure 4.6A, the rate of D O D A C elimination from plasma was influenced by the P E G - P E species in PFVs . At 30 minutes, 10%, 30% and 90% of initial D O D A C was eliminated when D O D A C was a component of P E G - D S P E , P E G - D P P E and P E G - D M P E containing PFVs , respectively. The blood residence times of D O D A C are comparable to those of P E G - P E (shown in Figure 4.3A). At 30 minutes, 10%, 30% and 70 % of initial P E G - D S P E , P E G - D P P E and P E G -D M P E , respectively, were eliminated when these P E G - P E were a component of PFVs (Figure 4.3A). Thus, while the plasma levels of D O D A C and P E G - D P P E or P E G - D S P E were similar for P E G - D P P E - P F V and P E G - D S P E - P F V , relatively lower plasma D O D A C levels were observed as compared to P E G - D M P E for P E G - D M P E - P F V . 113 to 0.075 E -§. 0.060 0.00 0 B 10 15 20 25 30 Time (hours) Figure 4.6 Plasma elimination of the cationic lipid, DODAC from PFVs: influence of PEG-PE composition Following i.v. administration of P F V formulations differing only in P E G - P E chain length, the levels of [ 1 4 C ] - D O D A C and [ 3 H]-CHE were determined at 0.5, 1, 2, 4, 8 and 24 hours by dual label scintillation counting. Shown in panel (A) are plasma levels of D O D A C present in P E G - D M P E - P F V s (•) P E G - D P P E - P F V s (•) and P E G - D S P E - P F V s ( A ) . The rate of exchange of D O D A C from circulating P F V is illustrated in panel (B) as the molar ratio of D O D A C to PFVs ([ 1 4C]/[ 3H]). Each point represents mean ± S.E, n=4. 114 Furthermore, the kinetics of elimination of PFVs ([ 3 H]-CHE) from plasma again were very similar to those shown in Figure 4.3B, indicating that regardless of the radiolabeled marker ( [ 3 H]-CHE or [ 1 4 C]-CHE) used to trace the liposomal lipid, the results obtained were the same (data not shown). The kinetics of exchange of D O D A C from P F V can be determined from plasma levels of D O D A C and P F V , respectively. The results are illustrated in Figure 4.6B. The rate of exchange of D O D A C from circulating P F V was influenced by the P E G - P E component of P F V . Exchange of D O D A C occurred rapidly from P E G - D M P E - P F V and was slower from P E G - D S P E - P F V . At 4 hours, 70% and 20% of initial D O D A C had exchanged out of P E G -D M P E - P F V and P E G - D S P E - P F V , respectively. Rates of exchange of D O D A C from P E G -D P P E - P F V were intermediate (at 4 hours 40% of D O D A C had been lost from P E G - D P P E -PFV) between those of PFVs containing P E G - D M P E and P E G - D S P E . The apparent ratio of D O D A C to PFVs as a function of the acyl chain length of the PEG-l ipid component of PFVs was lower than the corresponding ratio of P E G - P E to PFVs. It should be noted that incorporation of 15 mole% D O D A C and 10 mole% PEG-lipids in D O P E : D O D A C : P E G - l i p i d liposomes has been demonstrated to be optimal concentrations for longer circulation lifetimes and lower distribution to the liver (Mori et al., 1998). These investigators reached these conclusions by determining plasma and liver levels of PEG-Ceramide and D O D A C over a 0-10% and 10-50% range in D O P E : D O D A C vesicles, respectively. 115 4.2.7 Distribution of PEG-PE within the Blood Compartment Determined by Fast Protein Liquid Chromatography Thus far, the results from in vivo exchange experiments confirm that P E G - P E is lost from P F V and other liposomes (Figures 4.3, 4.4 and 4.5). These studies raise an important question: To what binding site does the P E G - P E exchange? This is a relevant question considering the rationale for the mechanism of action of PFVs is based on exchange of bilayer stabilizing lipids to a hydrophobic sink. The initial sink in vivo might constitute other membranes, lipoproteins, plasma proteins and/or other hydrophobic proteins. Fast protein liquid chromatography was used to resolve the fate of PEG-lipids after they exchange out of PFVs . This technique was selected on the basis of recent results that describe the use of an F P L C procedure to separate large unilamellar vesicles from most lipoproteins and serum proteins (Choice et al., 1999). Plasma was isolated from BDF-1 mice injected with PFVs containing P E G - D M P E , or P E G - D P P E , or with conventional liposomes ( D S P C : C H O L ) containing P E G - D M P E and analyzed by F P L C (Figure 4.7). On the basis of previous studies (Mills et al., 1984; Walsh and Atkinson, 1986; Innis-Whitehouse et al., 1998), four major peaks corresponding to major lipoprotein species were identified for murine plasma. It is likely that these peaks resolved by F P L C contain other plasma components in addition to lipoproteins but for clarity they will be referred to according to the various types of lipoproteins. The first elution peak represents the very low density lipoprotein ( V L D L , fractions 45-52 ml) component of plasma and is followed by a single peak containing both low density lipoproteins (LDL) and intermediate density lipoproteins (IDL). High density lipoproteins (HDL) and the soluble protein fractions elute next. Common to all three types of vesicles ( P E G - D M P E - P F V s , P E G - D P P E - P F V s and 116 P E G - D M P E containing conventional liposomes) examined here was the observation that liposomes eluted in the early fractions (45 to 52 ml), at or near the column void volumes. At 1 hour, P E G - D M P E [ 3 H ] was observed to co-elute with D S P C : C H O L liposomes (Figure 4.7A). A second P E G - D M P E peak was observed from fractions 72 to 80 ml; this peak of P E G - D M P E coincided with the soluble-protein plasma component. After 8 hours, a reduction of 50% was observed in the first P E G - D M P E peak while the second peak in the soluble-protein fraction could no longer be observed (Figure 4.7B). The height of the peak corresponding to conventional liposomes at 1 and 8 hours was 160 and 90 nanomoles, respectively. The ratios of D M P E - P E G lipid to total P F V lipid content at 1 and 8 hours were 0.037 and 0.033, respectively. However, the ratio in the initial sample was 0.05. Therefore, data in Figure 4.7A and B indicate that a proportion of P E G - D M P E exchanges out of conventional liposomes. Data presented in Figure 4.7 panels C and D illustrate the elution profile of P E G -D M P E and PFVs containing P E G - D M P E . At 1 hour, P E G - D M P E [ 3 H ] levels detected in fractions corresponding to V L D L , L D L and H D L and albumin fractions were 6, 2.5, 2 and 2 nanomoles per plasma sample (250 pi), respectively (Figure 4.7C). Similarly, at the later time of 8 hours (Figure 4.7D), P E G - D M P E [ 3 H ] levels were detected in V L D L , L D L and H D L and albumin fractions; however, as was observed for conventional vesicles, PEG-lipid levels in the plasma fractions were lower at 8 hours. PFVs containing P E G - D M P E levels detected were 40 and 6 nanomoles per plasma sample in fractions 45-52 ml at 1 and 8 hours, respectively (Figure 4.7C, D). It should be pointed out that the V L D L peak also corresponds to these fractions; therefore, it is difficult to resolve liposomes from V L D L by F P L C . At 8 hours, a 3-fold increase in the ratio of P E G - D M P E to P F V lipid was recorded, particularly in 117 regard to the fractions where the P F V elutes (45-52 ml, Figure 4.7, panels C and D). This is contrary to what might be expected, particularly while considering earlier results of rapid desorption of P E G - D M P E from the P F V carrier over time (~ 30% in 1 hour, Figure 4.5A). However, P E G - D M P E - P F V s are eliminated very rapidly from plasma and can no longer be detected, making the assay unreliable at later time points. These results will be discussed further in section 4.3. I The elution profile of P E G - D P P E and PFVs containing P E G - D P P E is shown in Figure 4.7E and F. For the 1 hour plasma sample, P E G - D P P E predominantly corresponded with the liposome fraction with marginal concentrations in the H D L and albumin fractions (Figure 4.7E). At 8 hours, a 4-fold reduction was observed in P E G - D P P E levels co-eluting with liposomes and much lower levels of P E G - D P P E were detected along with H D L and albumin fractions (Figure 4.7 F). P E G - D P P E - P F V s eluted in the column void volume and their levels at 1 and 8 hours were 90 and 40 nanomoles per plasma sample, respectively. On the basis of the data presented in Figure 4.7E and F , the ratio of PEG-DPPE[ 3 H] to P F V [ 1 4 C ] was estimated, the ratios being 0.16 and 0.1 at 1 and 8 hours, respectively. Keeping in mind that the sample administered into mice had an initial ratio of 0.1, the F P L C data indicate that little exchange of P E G - D P P E from P E G - D P P E - P F V occurred over the 8 hour period examined, which relates well to the data presented in Figure 4.5A. 118 Figure 4.7 Separation of liposomes from plasma components using Fast Protein Liquid Chromatography: distribution of PEG-PE (following page) Plasma isolated from mice injected with conventional liposomes or PFVs was fractionated by F P L C . Plasma samples were collected at either 1 hour (panels A , C and E) or 8 hours (panels B , D and F) post-injection. Panels A and B show fractionation profiles of conventional liposomes, D S P C : C H O L : P E G - D M P E (55:45:5, molar ratio) radiolabeled with [ 1 4 C ] - C H E , (•) and P E G - D M P E (PEG-DMPE[ 3 H] , open symbols). Panels C and D show fractionation profiles of P E G - D M P E - P F V s , D O P E : C H O L : D O D A C : P E G - D M P E , (30:45:15:10, molar ratio) radiolabeled with [ 1 4 C ] - C H E , (•) and P E G - D M P E (PEG-DMPE[ 3 H]) , (open symbols). Panels E and F show P E G - D P P E - P F V s , D O P E : C H O L : D O D A C : P E G - D P P E (30:45:15:10, molar ratio) radiolabeled with [ 1 4 C ] - C H E , (•) and P E G - D M P E (PEG-DMPE[ 3 H]) , (open symbols). In each fraction, the levels of liposomal lipid radiolabeled with [ 1 4 C ] - C H E and the corresponding tritiated P E G - P E were counted by dual label scintillation counting. The lipoprotein content of each fraction was estimated by reading the absorbance of each fraction at 280 nm. In all the panels, grey diamonds show the lipoprotein profile. 119 Figure 4.7 Separation of liposomes from plasma components using Fast Protein Liquid Chromatography: distribution of PEG-PE 4.3 Discussion Sophisticated liposomal delivery systems such as PFVs, which require several components to impart specific properties, generate a need to fully characterize the stability of each component in the circulation. The results presented in this chapter illustrate that the in vivo stability of PFVs is dependent upon the acyl chain composition of the phospholipid anchor, which in turn governs the rate of exchange of PEG-lipids. In order to better understand the exchange of PEG-lipids, plasma samples were resolved by F P L C . On the basis of F P L C analysis, it was established that PEG-lipids co-elute with lipoproteins or plasma proteins. These results are discussed in the context of factors effecting the selective loss of individual vesicle components and how these impact on critical carrier properties such as circulation longevity and retention of drug. Lipid mixtures which in isolation form the non-lamellar hexagonal H n phase, can be stabilized in a bilayer organization by the addition of PEG-lipid conjugates (Holland et al., 1996a). During membrane fusion, non-lamellar structures must be formed transiently and hexagonal H n phase-forming lipids, such as phosphatidylethanolamine, play a major role in these structural intermediates (Siegel et al., 1989; Cullis et al., 1991). Destabilization of vesicles composed of non-bilayer-forming lipids plus PEG-lipid conjugates, by removal of PEG-lipid, would unmask the latent fusogenic character of these systems (Holland et al., 1996a). Although it is generally very difficult to remove one lipid component from a liposome selectively, this is possible in the case of PEG-lipids because of their relatively rapid exchange between membranes (Silvius and Zuckermann, 1993). Hydrophobic forces responsible for assembly of phospholipids in a bilayer, make it energetically unfavorable for 121 these molecules to leave the bilayer and therefore exchange of lipids between membranes, through the aqueous medium, is normally very slow. However, lipids possessing large, hydrophilic domains, such as P E G - P E , experience a much smaller energetic barrier to desorption and can therefore exchange between membranes much more rapidly. Further, this exchange rate is largely determined by the size of the hydrophobic domain "anchoring" the lipid in the bilayer and can be modulated by appropriate selection of lipid acyl composition (Holland et al., 1996a). These initial studies (in vitro) had achieved removal of P E G - P E from what were termed programmable fusogenic vesicles by incubation with "acceptor" liposomes that did not contain a PEG-lipid component. Subsequent studies have shown that a similar induction of fusion can be achieved by selective cleavage of the PEG-moiety from P F V , employing a reducible thiol linkage between the polymer chain and the phosphatidylethanolarnine (Kirpotin et al., 1996). This chemical cleavage results in loss of the steric barrier afforded by the polymer chains, and formation of a degradation product that is not capable of stabilizing the lipid mixture in a bilayer organization. Loss of PEG-lipids from PFVs would also be expected to occur in vivo as the result of exchange to cell membranes or hydrophobic binding sites on proteins. This property therefore provides a mechanism whereby the physical properties of a P F V carrier can be radically modulated in a predictable and controllable manner after systemic administration. The results presented in this chapter demonstrate that the stability of PFVs in the circulation is dependent upon the rate at which their bilayer stabilizing component, P E G - P E , is lost from the outer monolayer. Studies with MethoxyPEG 1 9 0 0-carbamate-DSPE at 4 mole% indicate that the P E G extends outward from the surface approximately 5 nm (Needham et al., 1992). Furthermore, in the absence of direct evidence, it has been speculated by Woodle and 122 Lasic (1992) that as long as the radius of the vesicle is greater than a factor of 10 relative to the length of the polymer, (varies between 6 to 8 nm for P E G 1 9 0 0 ) the distribution of P E G should be approximately equal between the two leaflets of the bilayer which is the case here (note: P E G 2 0 0 o was used in all the studies). Therefore, the loss of PEG-lipid would be expected to occur from the outer leaflet of the PFVs triggering the destabilization of the vesicle. In this regard, PEG-PEs possessing shorter acyl chains exchange out of the vesicles more rapidly and hence the vesicles are removed from the circulation more rapidly. In the case of P F V systems containing longer acyl chain P E G - P E , such as P E G - D S P E , much slower destabilization occurs and these carriers exhibit long circulation lifetimes in the blood. The "stronger anchoring properties" of longer acyl chains, in particular P E G conjugated to a ceramide anchor (C20 chain length), have recently been demonstrated for S M : C H O L vesicles and for D O P E : D O D A C formulations by Webb et al. (1998) and Mori et al. (1998) respectively. The importance of the nature of the anchor of PEG-lipids in influencing the circulation lifetime of vesicles has been recognized by Allen et al. (1991). They have compared P E G conjugated to dipalmitoylglycerol (DPG), D S P E or cholesterol lipid anchors incorporated in S M : C H O L liposomes and reported P E G - D S P E to be significantly more effective than P E G - C H O L or P E G - D P G at reducing vesicle elimination from plasma at 24 hours. However, Woodle et al. (1992) believe the lipid anchors to be equivalent. This is not surprising considering that Woodle and coworkers examined hydrogenated soy phosphatidylethanolamine (HSPE) and D S P E phospholipids conjugated to P E G , both of which are relatively longer chain anchors (HSPE, C18:C16 acyl length and D S P E represents C18). I have shown that P E G - D S P E exchanges out of the vesicle surface slowly (~ 10% at 24 hours). 123 The addition of successive methylene units and the resultant increase in hydrophobicity plays a significant role in governing the rate of spontaneous exchange of PEG-lipids. Silvius and Zuckermann (1993) have reported an approximately 6.3-fold decrease in the rate of intervesicle transfer with the extension of the acyl chain by one methylene unit. A similar influence of acyl chain lengths on the rate of transfer between vesicles has been reported for fluorescent-labeled phospholipids (Nichols, 1985; Homan and Pownall, 1988) and between vesicles and erythrocyte membranes (Ferrell et al., 1985). The contributions of two entropic factors to the free energy of desorption of PEG-lipids have been described. First, there is an increase in translational and rotational entropy associated with the exchange of a molecule from an aggregate or a macromolecular complex to a monomeric state (Janin and Chothia, 1978; Finkelstein and Janin, 1989). Second, the conformational entropy displayed by a polymer at the bilayer/water interface is lower than that of the free polymer. Silvius and Zuckermann (1993) have pointed out the contribution of an additional factor: interactions of the anchored polymer with other membrane components. Further, the exchange of amphipathic P E G derivatives from the surface of the liposome is not specific to P F V compositions-I have also shown that the acyl chain length determines the rate of exchange of PEG-PEs from conventional (stable) vesicles. While it has been reported by Connor et al. (1986) that the inclusion of cholesterol in pH-sensitive fusogenic systems causes these vesicles to become acid-insensitive, it is shown here that PFVs are stable systemically despite the inclusion of cholesterol. The exchange of D O D A C from PFVs is also influenced by the hydrophobicity of the PEG-anchor and may occur instantaneously as in the case of P E G - D M P E - P F V s . The role of PEG-PEs in forming a steric barrier (Papahadjopoulos et al., 1991; Torchilin et al., 1994a) 124 and shielding the surface charge is well known. Therefore, it follows that a factor that would facilitate the dissociation of D O D A C from PFVs is the initiation of exchange of PEG-lipid. However, it is apparent from Figure 4.6B that the exchange of D O D A C precedes that of P E G - P E from PFVs, especially in the case of P E G - D M P E - P F V s . In this regard, two points should be mentioned; first, that even in the presence of the steric barrier presented by P E G -lipids, the vesicular surface is subject to a certain degree of contact with soluble proteins. Second, on the basis of the F P L C results (Figure 4.7B and C) showing the presence of P E G -D M P E in fractions corresponding to lipoproteins and other plasma proteins, the possibility of overestimating the levels of P E G - D M P E in plasma cannot be ruled out. Therefore, it would be more appropriate to consider the rates of D O D A C exchange in PFVs containing P E G -D P P E or P E G - D S P E . The structural differences between D O D A C and other phospholipids or PEG-anchors may offer another explanation for the faster dissociation of D O D A C from P F V surface. The presence of a glycerol phosphate backbone in phospholipids enables intramolecular hydrogen bonding. In the absence of similar hydrogen bonds and a smaller headgroup, it is possible that D O D A C would be packed less firmly within the bilayer as compared to other phospholipids and thus more susceptible to dissociation from the membrane. It should also be mentioned that the loss of D O D A C would lower the probability of electrostatic interactions that promote fusion of P F V to anionic surfaces. This implies that the fusion event remains a possibility for PFVs containing the longer chain anchors such as P E G - D S P E where relatively slower exchange of both D O D A C and PEG-lipid was observed. The results on the exchange of PEG-lipids are complimented by the F P L C data resolving liposomes from lipoproteins and other serum proteins. The elution of PEG-lipids together with H D L implies that following exchange of PEG-lipid from the vesicle surface, 125 PEG-lipids bind to lipoproteins and/or plasma proteins. This is especially true in the case of PEG-DMPE which co-elutes with L D L , HDL and soluble plasma components at 1 hour as well as 8 hours. The fact that at 8 hours more PEG-DMPE than the carrier lipid is associated with the V L D L fraction raises the possibility of overestimating the plasma levels of PEG-DMPE. Therefore, the plasma elimination data for PFV containing PEG-DMPE should be interpreted with caution. In this regard, it is important to bear in mind that while all the data presented here were based on radiolabeled markers (CHE for vesicle lipid and tritiated PEG-PE), the exchange of PEG-PE is indeed represented by the tracer used and not the properties of the carrier. This is illustrated by the plasma elimination of PEG-DSPE-containing DSPC:CHOL vesicles labeled with PEG-DPPE[ 3H] being similar to PEG-DPPE-containing vesicles (Figure 4.5B). These results support the occurrence of exchange of PEG-PE from PFV and conventional vesicles in vivo. There is evidence from earlier studies for the exchange of phospholipid from phosphatidylcholine vesicles to lipoproteins (Chobanian et al., 1979; Tall and Green, 1981; Shahrokh and Nichols, 1982). Furthermore, it is expected that the desorption of cationic and PEG-lipids from the surface of PFVs should initiate membrane asymmetry, exposure of the transiently masked fusogenic lipids (DOPE) and thus destabilization of PFVs. The extent of lipid exchange thus determines the degree of vesicle destabilization. Disintegration of phosphatidylcholine liposomes in plasma as a result of interaction with high density proteins in vitro has been reported by Scherphof and coworkers (1978). In summary, programmable fusogenic vesicles are stable in the circulation. The primary factor that determines the retention of the PEG coating on the PFV is the strength of its anchorage to the bilayer by the lipid anchor. The acyl chain composition of the 126 phospholipid anchor of PEG regulates the rate of exchange of PEG-lipids from the surface of a vesicle, whether PFV or a conventional, sterically stabilized vesicle. The partitioning of the cationic component of PFVs from the PFV surface is also influenced by the chemical composition of the PEG-lipid anchor. The desorption of DODAC together with the exchange of PEG-lipids makes it possible to program the rate of transformation of the membrane characteristics of PFVs by appropriate selection of the PEG-lipid component. 127 CHAPTER 5: E V A L U A T I O N OF THE ANTICANCER PROPERTIES OF M I T O X A N T R O N E ENCAPSULATED WITHIN P R O G R A M M A B L E FUSOGENIC VESICLES 5.1 Introduction Liposomal formulations used traditionally for the delivery of anticancer agents have focused on maximizing carrier stability in the circulation, thereby enhancing the probability of their preferential accumulation at a disease site such as tumors (Proffitt et al., 1983; Mayer et al., 1990a; Allen et al., 1991a and Bally et al., 1994). However, these systems have not yielded efficacy improvements commensurate with the levels of drug accumulation observed (Mayer et al., 1994). The premise that maintenance of high concentrations of drug in the plasma compartment over prolonged periods of time would facilitate localization of the drug at a disease site has formed the basis for the design of most traditional liposomal carrier systems. A n inherent advantage of such conventional liposomes is that drug exposure to healthy tissues is minimized (Rahman et al., 1982; Olson et al., 1982). A significant disadvantage, however, is that the drug is not released efficiently from the liposome following localization at the disease site (Mayer et al., 1994). This is well substantiated by in vitro cytotoxicity studies demonstrating that as much as 100-fold higher concentrations of liposomal anticancer drug are required to produce cell toxicity equivalent to that observed with free drug. The explanation for this result is simple: encapsulated drug is not bioavailable and therefore does not contribute to biological activity. Emerging technologies such as PFVs, must be able to address this problem, while maintaining the beneficial properties of conventional liposomes. Ideally, it is important to design liposomes that: 1) minimize systemic exposure of free drug, 2) maximize delivery of liposomal drug to 128 extravascular sites and 3) undergo transformation that results in drug release following localization at the disease site. In Chapter 4, it was demonstrated that PFVs are stable following i.v. administration when appropriately selected PEG-lipids are incorporated into the formulation. Specifically, the circulation lifetime of PFVs can be controlled by altering the length of the lipid anchor of PEG. Structural modifications of PFVs, which occur as a consequence of the loss of the PEG-lipid, result in exposure of a charged surface and, eventually, destabilization of the membrane structure. The former will result in liposome elimination due to opsonization and recognition by cells of the MPS (Chapter 4), while the latter is a consequence of formation of non-bilayer lipid domains and loss of entrapped contents (Chapter 3). When considering the development of PFVs for use in the treatment of tumors, it is necessary to select formulations that exhibit attributes favoring efficient liposome and drug accumulation in the region of tumor growth. Liposome transformation would subsequently foster drug release, liposome-cell binding (mediated through electrostatic interactions) and potentially liposome-cell fusion. In order to establish the therapeutic utility of PFVs as carriers of anti-cancer agents, studies described in this chapter have focused on two tumor models, one where disease progression occurs primarily in the liver (i.v. administration of murine L1210 cells in immune competent BDF-1 mice) and the other where the disease progression is restricted to the site of cell inoculation (s.c. administration of human LSI80 cells in SCID/RAG-2 mice). These studies were initiated with three expectations in mind. First, treatment of liyer-localized disease will not be dependent on PFV circulation lifetimes, provided that plasma elimination is associated with efficient PFV accumulation in the liver. Therapy of liver-localized disease will be a consequence of drug release. Second, treatment of a s.c. tumor 129 will be dependent on both circulation lifetime and drug release. Accumulation of P F V s in the s.c. tumor will be a slow process and maximum drug delivery will only be achieved through use of PFVs that are eliminated from the plasma compartment slowly and are also able to retain drug effectively over the time-course required for optimal P F V uptake. It can be predicted on the basis of data provided in Chapter 4 (Figure 4.1) that PFVs which are eliminated rapidly following administration will be therapeutically less active then those that exhibit extended circulation lifetimes. Third, P F V formulations selected on the basis of optimal therapeutic activities will provide improved therapy when compared to conventional liposomes that have been designed to retain drug effectively following administration and which lack cell binding and membrane fusion attributes. The therapeutic studies described in this chapter used mitoxantrone as a model anticancer drug. As described in Chapter 1, mitoxantrone is a less cardiotoxic analog of doxorubicin and is also better tolerated than doxorubicin (Dukart et al., 1985; Neidhart et al., 1986; Bennett et al., 1988 and Weiss et al., 1989). Mitoxantrone is an active agent in the treatment of solid tumors (reviewed by Faulds et al., 1991). Importantly, the plasma elimination and biodistribution characteristics of programmable fusogenic vesicles (See Chapter 4) or other liposomes (Lim et al., 1997 and Chang et al., 1997) are not altered by encapsulated mitoxantrone. This is in contrast to other antineoplastic agents, such as doxorubicin and vincristine, that are known to lower the elimination rate of liposomal carriers in which they are encapsulated (Bally et al., 1990a and Daemen et al., 1995). This is particularly important for those studies evaluating liver localized disease, where drug-induced reductions in liposome accumulation in liver will affect treatment outcomes (Lim, 1999). The pharmacokinetic characteristics of encapsulated mitoxantrone and its release will 130 be determined by the elimination parameters and permeability properties of the carrier. One additional attribute of encapsulated mitoxantrone that needs to be considered in the context of these experiments concerns its retention characteristics in liposomes. When encapsulated in liposomes, the drug forms a crystalline precipitate (see Chapter 3) and is retained extremely well in vitro. Retention in vitro does not appear to be affected by temperature, lipid composition, presence or absence of a p H gradient, or the presence of serum proteins (Lim et a l , 1997). The data obtained are consistent with the anticipated results. The P F V formulations with encapsulated mitoxantrone, whether eliminated rapidly or slowly from the circulation, are significantly more active than conventional D S P C : C H O L liposomes when used to treat liver-localized disease. When therapy is evaluated in the s.c. model, mitoxantrone encapsulated in the long circulating P F V formulation (prepared with a slow exchanging P E G modified lipid, P E G - D S P E ) is more effective in delaying tumor progression than mitoxantrone encapsulated in PFVs that are rapidly eliminated following i.v. administration. Importantly, mitoxantrone encapsulated in the long circulating P F V formulation is significantly more active than mitoxantrone encapsulated in conventional liposomes, despite data which demonstrate that the latter delivers drug to the diseased site more efficiently then the PFVs . These data illustrate the benefits associated with designing liposomal carrier formulations that favor drug release. 131 5.2 Results 5.2.1 Elimination of Mitoxantrone and PFVs from plasma and their Accumulation in Solid Tumor Xenografts in SCID/RAG-2 mice Mitoxantrone and Lipid Levels in the Plasma Mitoxantrone and lipid levels were measured in the plasma of SCID/RAG-2 mice bearing established LSI80 solid tumors over a 48 hour time period following a single i.v. injection of free mitoxantrone (22.5 umole/kg), mitoxantrone encapsulated in P E G - D M P E -PFVs and P E G - D S P E - P F V s (22.5 pmole mitoxantrone/kg, 225 umole total lipid/kg) and data obtained are summarized in Figure 5.1. The plasma concentrations of mitoxantrone obtained after injection of non-encapsulated and mitoxantrone-loaded P F V systems over a 48 hour time-course are presented in Panel A . Levels of mitoxantrone observed for the P E G -D S P E - P F V formulation up to the 24 hour time point were significantly higher (e.g. 20-fold higher levels were observed at 4 hours) as compared to mitoxantrone encapsulated within P E G - D M P E - P F V s and free drug. However, little difference was seen between mitoxantrone-loaded P E G - D M P E - P F V s and free drug except at 1 hour post-injection, when the plasma concentration of mitoxantrone was 4-fold higher (in comparison to free drug) when administered encapsulated in P E G - D M P E - P F V s . In the absence of the carrier, plasma mitoxantrone levels fell to below detectable limits after one hour. Assuming that the plasma volume of a 20-22g mouse is 1 ml and an injected dose of 0.45 pmole mitoxantrone per animal, it is estimated that greater than 99% of the injected free drug was eliminated from the circulation within 1 hour after injection. In contrast, at the same time point following administration of mitoxantrone encapsulated in P E G - D M P E - P F V s or P E G - D S P E - P F V s there 132 was less then 80 and 40% drug elimination from the circulation, respectively. The plasma elimination of PFVs following i.v. administration in tumor-bearing mice is illustrated in Figure 5.IB. The PEG-DSPE-containing PFVs are consistently at higher concentrations in the blood than the P E G - D M P E - P F V s , resulting in 4- to 5-fold increases in the plasma concentrations of P E G - D S P E - P F V s at the earlier time points. The plasma P E G - D S P E - P F V levels obtained after 24 h are still significantly greater (p<0.01) than P E G - D M P E - P F V s . The differences between the two P F V preparations, however, are reduced substantially at the 48 hour time point. These elimination profiles are comparable to those obtained in animals that did not have established tumors (see Figure 4.1). Mitoxantrone and Lipid Accumulation in LSI 80 Solid Tumors The extent of drug and liposome accumulation in solid LSI80 tumors following i.v. administration was studied in SCID/RAG-2 mice bearing established LSI80 tumor xenografts and the results are shown in Figure 5.2 (panels A and B , respectively). The maximum concentration (C m a x ) of mitoxantrone achieved in tumors following administration of the P F V formulations was observed 4 hours following i.v. administration, with values of 25 and 35 nmole/g tumor for mitoxantrone encapsulated in P E G - D M P E and P E G - D S P E PFVs, respectively. In contrast, the peak mitoxantrone concentrations in tumor tissue following free drug administration (25 nmole/g) were observed at 1 hour post-injection, the earliest measured time point. Liposomal lipid levels, shown in Figure 5.2B, indicate significantly higher levels (10-fold at 24 hours and 5-fold at 48 hours) are achieved following i.v. administration of P E G - D S P E - P F V s in comparison to P E G - D M P E - P F V s , and this is consistent with the previous results suggesting that long circulation lifetimes are required for enhanced tumor accumulation. It is interesting to note that liposomal (PFV) lipid uptake occurs continuously throughout the first 24 hours following P F V administration, while drug accumulation data (Panel A ) show a decrease from the 4 to the 24 hr time point. These data support the contention that drug release does occur following administration of PFVs. Both formulations exhibit a tumor drug-to4ipid ratio at 24 hr that is at least 75% lower than that observed in the tumor one hour after administration. The data shown in Figure 5.2 were used to estimate the mean area under the lipid concentration-time curve ( A U C L ) and the mean area under the drug concentration-time curve ( A U C D , Table 5.1). For comparison, a similar analysis was completed using data obtained following administration of mitoxantrone encapsulated in conventional liposomes (prepared from D S P C : C H O L ) . There are two important points that can be made from the mean A U C analysis shown in Table 5.1. First, the mean A U C L for P E G - D M P E - P F V s in the tumor is 4-fold lower than that measured for either the P E G - D S P E -PFVs or D S P C : C H O L liposomes. Second, the mean A U C D in the tumors is greatest when the drug is administered in conventional liposomes. These data help demonstrate that the conventional liposomes retain drug better then the P F V formulations. If antitumor activity was dependent only on drug exposure then these data would suggest that the D S P C : C H O L formulation would be most active. As indicated in the following section, this was not the case. 134 A Time (hours) Figure 5.1 Plasma elimination of mitoxantrone and mitoxantrone-loaded PFVs from tumor bearing mice SCID/RAG-2 mice were injected bilaterally with 1 x 106 LSI80 cells subcutaneously. When the tumors were ~0.3 cm 3 , mice were injected with free drug (•) or mitoxantrone encapsulated within P E G - D M P E - P F V s (•) or P E G - D S P E - P F V s ( A ) at 10 mg/kg (22.5pmol/kg) via the lateral tail vein. Mice were sacrificed at 1, 4, 24 and 48 h and the plasma concentrations of lipid and drug were determined as described in Chapter 2. Panel A : Elimination of mitoxantrone from plasma, where mitoxantrone was determined using a [ 1 4 C]-radiolabelled drug as a tracer, Panel B: The liposomal lipid marker ([ 3 H]-CHE) was used to measure the rate of elimination of PFVs from plasma. Mean ± S.E, n=6. 135 o r i i i i i 0 10 20 30 40 50 Time (hours) Figure 5.2 Tumor accumulation of mitoxantrone and liposomal lipid in the human LS180 solid tumor xenograft SCID/RAG-2 mice were injected bilaterally with 1 x 106 LSI80 cells subcutaneously. When the tumors reached approximately 0.3 cm 3 , mice were injected with free drug (•) or mitoxantrone encapsulated within P E G - D M P E - P F V s (•) or P E G - D S P E - P F V s ( • ) at 10 mg/kg (22.5pmol/kg) via the lateral tail vein. Mice were terminated using C 0 2 asphyxiation and tumors were removed and processed as described in the Materials and Methods. Panel A depicts levels of drug in the tumor following i.v. administration of free or encapsulated mitoxantrone in P E G - D S P E - P F V s or P E G - D M P E - P F V s and shown in Panel B are liposomal lipid levels measured in the tumor following administration of mitoxantrone loaded P E G -D M P E - P F V (•) or P E G - D S P E - P F V (A) systems. Results shown represent an average of two separate experiments. Each experiment had a sample size of 6-8 tumors. 136 Table 5.1 Mean area under the concentration-time curves for programmable fusogenic vesicle and conventional vesicles (DSPC:CHOL) Mean area under the concentration-time curves for programmable fusogenic vesicles and conventional liposomes ( D S P C : C H O L ) obtained in tumors from 0 to 48 hours following i.v. administration of liposomes (225 pmole/kg dose) in SCID/RAG-2 mice bearing established LSI80 tumors. A U C L T U M O R 1 (pmole lipid /g tumor) hour A U C D T U M O R 2 (pmole drug /g tumor) hour D S P C : C H O L 16.7 2.2 P E G - D M P E - P F V 3.8 0.63 P E G - D S P E - P F V 16.5 1.25 1 A U C L was determined by trapezoidal integration of the mean of the lipid concentration-time. 2 A U C D was determined by trapezoidal integration of the mean of the drug concentration-time. 5.2.2 Therapeutic Activity of Mitoxantrone-Loaded PFVs Efficacy of Mitoxantrone-loaded PFVs against LSI 80 human colon carcinoma model It was important to assess how the efficacy of mitoxantrone encapsulated within PFVs would compare with that of conventional drug carrier systems used in the treatment of solid tumors. Although similar levels of liposomal lipid access the tumor, it was predicted that the P F V formulation would be more active because it favors drug release. Therefore, the properties of mitoxantrone-loaded PFVs or D S P C : C H O L vesicles were examined against a human colon carcinoma, LSI80, grown as a xenograft in immune deficient SCID/RAG-2 mice. 137 1.5 0 5 10 15 20 25 30 35 Days after Innoculation Figure 5.3 Mitoxantrone mediated LS180 solid tumor growth inhibition SCID/RAG-2 mice were injected bilaterally with 1 x 106 LSI80 cells subcutaneously. 2, 6 and 10 days after tumor cell inoculation, mice were injected with a 2.0 mg/kg dose of free mitoxantrone, (•), or mitoxantrone encapsulated in P E G - D M P E - P F V s (•), and P E G - D S P E -PFVs ( A ) , or 2.5 mg/kg dose of mitoxantrone encapsulated in D S P C : C H O L (•) via the lateral tail vein. Untreated mice served as control, (O). Data points represent the cumulative totals of tumor volumes of at least 8 tumors normalized for the number of mice. 138 In this model, LSI80 cells were inoculated subcutaneously in the flank of SCID/RAG-2 animals and tumor growth measured following i.v. administration of three injections of free or liposomal drug (dose 2.0 mg/kg) injected 2, 6 and 10 days after tumor cell inoculation. Although a number of different doses (1.5, 2.0 and 2.5 mg/kg) were studied, the therapeutic efficacy was optimal at the 2.0 mg/kg dose and the efficacy of mitoxantrone encapsulated in PFVs was evaluated against non-established tumors at this dose. Tumor volumes were calculated using the formula of Tomayko and Reynolds (1989) following measurements of tumor length and width as described in Chapter 2. For assessing tumor progression, the volumes of all the tumors in each individual treatment group were summed and the cumulative sum of tumor volumes was then divided by the number of mice. This normalization accounts for animals that had to be killed as a consequence of tumor ulceration (see Chapter 2) and is also the reason for the lack of error bars in Figure 5.3. The results of this analysis are shown in Figure 5.3. In the untreated, control group, LS180 tumors were first palpable 12 days after cell inoculation and tumor volume increased rapidly over the next 5-6 days. Mice treated with free mitoxantrone showed a 5 to 6 day delay in initiation of tumor growth, compared to the control group. By day 17, tumor volumes increased at a rate similar to tumors in untreated animals, suggesting that treatment of the s.c. tumor is primarily a consequence of delaying the time point when tumor growth is initiated. Compared to the free drug-treated group, mice treated with the same dose of mitoxantrone encapsulated in P E G - D S P E - P F V s showed a much longer delay in initiation of tumor growth (12-14 day delay). In these animals, tumors were palpable only by about day 28 (Figure 5.3). For comparison, treatment with mitoxantrone-loaded D S P C : C H O L vesicles (even though it was administered at a higher dose; 3 i.v. injections of 2.5 mg/kg) resulted in a delay in growth 139 initiation which was slightly less than that observed for free drug, but slightly longer than that measured following treatment with mitoxantrone-loaded PEG-DMPE-PFVs. Although it could have been predicted on the basis of drug delivery attributes that the PEG-DMPE-PFV formulation would be less active than the PEG-DSPE-PFV formulation, the value of the PFV technology is highlighted when results between animals treated with mitoxantrone loaded in the DSPC:CHOL and the PEG-DSPE-PFVs are compared. Efficacy of Mitoxantrone-loaded PFVs against Liver localized disease The results presented thus far stress the importance of using PFVs that are designed to exhibit long circulation lifetimes. This is particularly true when treatment is targeted against a disease that is localized in a region where extravasation (movement of the carrier from the blood compartment to spaces residing outside of the blood vessels) of the carriers is slow. The differences in the therapeutic activity of drugs encapsulated in PFV formulations should become solely dependent on drug release attributes when carrier access to the site of disease progression is not limited. To test this, therapy studies were initiated in a tumor model in which the disease is primarily restricted to the liver. On the basis of data presented in Chapter 4 (Figure 4.2), although the rate of PFV accumulation in the liver is much slower for PEG-DSPE-PFVs in comparison to PEG-DMPE-PFVs, both formulations efficiently deliver drug to the liver over a 48 hour time-course. In this study, tumor cells were inoculated intravenously, which results in seeding predominantly to the liver and spleen (Lim et al., 1997). One day after inoculation of tumor cells, animals were treated intravenously with free mitoxantrone (10 mg/kg which is equivalent to 22.5 pmole/kg) or mitoxantrone encapsulated in either PEG-DMPE-PFVs or 140 P E G - D S P E - P F V s (10 mg/kg), or mitoxantrone in conventional D S P C : C H O L liposomes (also 10 mg/kg drug dose). Control animals were injected with saline. The results of this study have been summarized in Table 5.2 and Figure 5.4. The median survival time for untreated (control) animals was 9 days and treatment with free mitoxantrone extended the median survival time to 17 days. The therapeutic activity of drug encapsulated in D S P C : C H O L liposomes was less than that observed for free drug (median survival time of 13 days) but this difference was not statistically significant. It should be noted that free mitoxantrone was given at the maximum tolerated dose, a dose at which significant weight loss was frequently observed and death due to drug toxicity could not be ruled out. However, at this dose, treatment was optimal with a median survival time of 17 days and a long-term (>60 day) survival rate of 15%. When mitoxantrone is given at the same dose but encapsulated in P E G - D M P E - or P E G - D S P E - P F V s , the therapeutic activity of the drug was increased significantly. Maximum percentage increase in life span (ILS) and median survival times could not be estimated because more than half of the treated animals survived beyond day 60 (6/8 and 5/8 survivors were observed for animals treated with mitoxantrone in the P E G - D M P E - and P E G - D S P E - P F V s , respectively, Table 5.2). For comparison, the antitumor activity of mitoxantrone encapsulated in conventional liposomes ( D S P C : C H O L ) was also evaluated. As can be seen from the data in Table 5.2 and Figure 5.4, the D S P C : C H O L formulation is significantly less effective in treating this liver-localized tumor when compared to the P F V formulations. It should be noted that all long-term (> 60 day) survivors were re-inoculated with L1210 cells at the end of the experiment to insure that survival was not due to the animals acquiring an immune response to the tumor cells. A l l of the re-inoculated animals developed tumors in the expected time frame (7-9 days). The L1210 141 antitumor studies summarized in Table 5.2 and Figure 5.4 clearly demonstrate that the two P F V formulations were more therapeutically active than free drug or drug encapsulated in D S P C : C H O L liposomes. However, differences in efficacy of P E G - D M P E - P F V and P E G -D S P E - P F V formulations were not statistically significant. 142 0 10 20 30 40 50 60 Time (days) Figure 5.4 Therapeutic activity of mitoxantrone-loaded PFVs against a murine L1210 leukemia model. Survival curves of BDF-1 mice inoculated with L1210 cells intravenously and treated with free mitoxantrone (•), mitoxantrone in D S P C : C H O L liposomes (•), mitoxantrone in P E G -D M P E - P F V s (•), or mitoxantrone in P E G - D S P E - P F V s (•) , 24 hours after tumor cell inoculation. Untreated animals served as control (O). Data represent treatment groups of at least 8 mice. 143 u a to Q a CO > to PH 'O ii TS « jo • a a © c O a -a a CQ 4> a> o w o a s c o 1—1 (N 1—I >r, u 3 es H CD CO CD bB cd "S CD O (-1 CD > | 3 X3 CD CD on |S CD c o o o I <-l O < O N OO CO ro ro ro IT) CO Q bO bD ro ro ro ro a o l o bO CD CD i «-> ^1-O N o ON I—1 o o o PH . „ Q Q oo vo Q 00 > fe PH I W PH Q 1 O w PH Q oo in Q 00 > fe PH W PH C O Q 6 PH o o o T3 CD •s CD H T3 CD t d <D U o co CD a CD s £H CD & CD o cd ninei was i CD _c C O CD CD o CD M were o x CD I /O cn were J3 cd were +H CO u CO <D % valu PO • i-H valu VO c3 CO +-» en C O CD S ,w M-H o eri in Li ved n :e exp CD *c cd CO cd b CD CO CM CD CO o CO fl h H ^ mal ble. VO p e ILS ani lica c O cfcl e ILS he CM CH cd bo « cd^ cd cd -»-» h H -4-» 7 3 C o T 3 CD CD ere up: Pool PH o >- Pool to 00 5.3 Discussion The results presented illustrate the therapeutic benefits that can arise when using PFVs for systemic delivery of an anticancer drug, such as mitoxantrone. In particular, it is demonstrated in the treatment of human xenografts that liposomal formulations displaying comparable circulation lifetimes, comparable accumulation rates in tumors and comparable drug delivery attributes can exhibit remarkably different antitumor activities. It is suggested that the differences observed in therapeutic activity are a consequence of differential drug release and improvements in drug bioavailability, features that are specifically designed into P F V carrier formulations. Differences between P F V and conventional formulations that may have direct impact on the antitumor activity of an encapsulated anticancer drug are discussed. In order for an encapsulated drug to be active it must first be released from the carrier. This problem is illustrated in the present research (compare the therapeutic activity of mitoxantrone encapsulated in D S P C : C H O L liposomes in comparison to P E G - D S P E - P F V s ) . This problem, however, has been defined in earlier studies that examined the dependency between the stability or "leakiness" of conventional liposomes and the anticancer activity of mitoxantrone (Lim et al., 1997). These studies and others confirm the importance of bioavailability in the design of drug carrier systems (Mayer et al., 1989; Mayer et al., 1994). Until now, however, the properties of a liposomal carrier needed to maximize drug retention in the circulation and carrier accumulation at a tumor site have not been balanced with the requirements to ensure drug bioavailability to the tumor cells. Specifically, a simple (conventional) liposomal formulation can be defined to achieve improved therapeutic effects based on increased drug permeability across the liposomal membrane. However, drug 145 leakage is initiated immediately following drug administration. These liposomes can accumulate within the disease site, but i f this process is slow, as is the case when the carrier must cross blood vessels in solid tumors, the liposomes that accumulate have lost a good portion of their encapsulated drug. Many investigators believe that the pool of drug that localizes in the region of tumor growth (Gabizon and Papahadjopoulos, 1988; Wu et al., 1993), in contrast to drug released from carriers residing in the plasma compartment, is what dictates therapeutic activity. The development of PFV technology, therefore, is focused on delivering maximal levels of drug to tumors by optimal accumulation of drug at the tumor site followed by increased rates of drug release. In the case of the formulations described here, exchange of PEG-lipids from the liposome helps to destabilize the carrier's lipid bilayer, resulting in increased drug leakage and/or total loss of entrapped contents. Once sequestered at the disease site the factors that determine release from a vesicle vary markedly between PFVs and conventional liposomes. In general, release of encapsulated drug from a conventional liposome, which is primarily made of bilayer-forming phospholipids, occurs by way of passive diffusion. On the other hand, two factors responsible for determining the release of PFV contents are: (a) the loss of PEG-lipids from the surface of PFV, and (b) the lipid composition of PFVs. As has been stated before, the principal component of PFVs is a fusogenic, non-bilayer-forming lipid, DOPE. There is evidence in vitro that the presence of PEG-lipids regulates a bilayer conformation as well as fusogenicity of PFVs (Holland et al., 1996a and 1996b). This would suggest that the loss of PEG-conjugates would trigger the loss of membrane stability and the presence of DOPE would further advance PFV destabilization. Thus, in contrast to conventional liposomes, transformations in PFV membrane attributes are expected to promote drug release. 146 Preliminary efficacy studies shown in this chapter are consistent with the difference in behavior predicted between PFV and conventional systems. Therapeutic activity may result from more than one drug pool, for example free drug or liposomal drug, each of which is localized in the blood compartment as well as within various tissues, including tumors. Sources of drug within the tumor include free drug released from liposomes that have extravasated into the site and drug released from sites that are distant from the tumor (including the blood). Antitumor effects have been attributed to drug released from liposomes localized within the tumor with minimal contributions from systemic release of drug (Mayer et al., 1994). The source of drug responsible for therapeutic activity, therefore, will be determined by the rate and extent of liposome accumulation and the rate of drug release into tumor from extravasated carriers, while ignoring drug from sources other than extravasated vesicles. Therefore, in order to have a therapeutically active formulation, three criteria would have to be met: (1) access to the target site, (2) drug delivery and (3) drug bioavailibility. In the absence of differences in carrier accumulation within the tumor between PEG-DSPE-PFV and conventional vesicles (Table 5.1) and on the basis of data evaluating efficacy against solid tumors (Figure 5.3), it appears that PEG-DSPE-PFV are able to fulfill all the three aforementioned pre-requisites. Interestingly, mitoxantrone administered in PEG-DMPE-PFV showed less therapeutic activity than free drug against the solid tumors. The tumor drug accumulation data indicate that similar levels of mitoxantrone were delivered when the drug was administered in free form or encapsulated within PEG-DMPE-PFVs (Figure 5.2). Based on arguments given above, it must be assumed that a portion of the drug in the tumor xenograft is not bioavailable and perhaps is still retained by liposomes that have accumulated within this site. 147 This explanation is not unreasonable, considering the potential that PEG-modified lipid exchange and associated changes in membrane surface features and barrier properties may result in formation of new liposome sub-populations. Those liposomes that are retained in the plasma compartment and have the opportunity to localize within the disease site may have lost their ability to undergo changes that favor drug release. With respect to the enhanced therapeutic efficacy observed with mitoxantrone-loaded P E G - D S P E - P F V compared to D S P C : C H O L liposomes against liver-localized tumor (Figure 5.4 and Table 5.2), the answer again lies in differences in the release characteristics of mitoxantrone from the carrier. It is interesting to note that in this tumor model both P F V formulations exhibited similar therapeutic activity (not statistically different) despite the very different pharmacokinetic properties illustrated in Figure 4.1 (Chapter 4). The explanation for this effect can be found by evaluating the data shown in Figure 4.2 (Chapter 4). Although rapid loss of P E G - D M P E occurs from PFVs containing this short acyl chain lipid, this results in carrier accumulation in M P S organs such as the liver and spleen. As noted above, L1210 tumor cells inoculated i.v. seed to these same organs and therefore are exposed to high mitoxantrone concentrations. Similar levels of drug are achieved when using the P E G -D S P E - P F V formulation; however, it requires a longer time period in which to achieve delivery. As with the solid tumor, provided that the PFVs still retained drug while in the circulation, there will be efficient delivery to the liver. Two important points should be made concerning the present research. First, the animal studies described here were designed primarily to illustrate the principle of how programmable fusogenic vesicles can enhance drug bioavailabilty as measured through assays that evaluate tumor growth rates and/or enhanced survival in tumor-bearing animals. 148 In particular, the intention was to demonstrate how differences in the pharmacokinetic behavior of PFVs , which in turn are governed by P E G - P E acyl composition and exchange rate, translate into differences in drug efficacy. The P F V systems described have not been optimized as drug delivery carriers and further improvements in therapeutic properties are likely. For example, by using bilayer stabilizing lipids which exchange out of the carrier at an even slower rate than P E G - D S P E and, possibly, by incorporation of targeting elements. Second, it is not possible at present to identify whether mitoxantrone delivered to the tumor site by PFVs is delivered specifically into neoplastic cells. As illustrated in Chapter 1 (Figure 1.10), destabilization of PFVs that have accumulated within tumors could trigger vesicle fusion with adjacent cells, thereby directly introducing encapsulated mitoxantrone into the cell cytoplasm. Alternatively, destabilized PFVs may become leaky allowing drug release and subsequent uptake of free mitoxantrone by the tumor cells. Either mechanism will ensure good drug bioavailability for small molecules, such as mitoxantrone, which are readily able to cross the plasma membrane. In summary, the programmed release of mitoxantrone can be tailored by encapsulating it in PFVs and the therapeutic properties of the encapsulated drug are improved when compared to conventional liposome formulations. The loss of PEG-modified lipids, the presence of a cationic surface charge and destabilization of the membrane bilayer into a fusion competent structure, are all critical factors to consider when engineering the rate of transformation of PFVs from circulation-stable vesicles into destabilized ones, capable of releasing their contents. Recent studies have demonstrated fusion between PFVs and eukaryotic cells in culture and shown that this can allow introduction of vesicle contents to 149 the cell cytoplasm. Studies to extend this observation to the in vivo situation are presently ongoing. 150 C H A P T E R 6: DISCUSSION and I M P L I C A T I O N S 6.1 Significance of Results In Chapter 1, it was proposed that the use of programmable fusogenic vesicles as a carrier for mitoxantrone would enhance the bioavailability of mitoxantrone to tumor tissue. The results presented in this thesis support this hypothesis. Essentially, three areas of investigation were explored in this thesis. The first included defining the in vitro characteristics of PFVs with respect to morphology, ability to encapsulate mitoxantrone and further to demonstrate controlled destabilization. Second, the properties of PFVs were examined in vivo as a function of the PEG-l ipid component. The final study examined the efficacy of mitoxantrone-loaded PFVs against animal models of cancer. In characterizing programmable fusogenic vesicles in vitro, I show that PFVs can be formulated and that P E G conjugates are essential for the structural integrity of PFVs . This was anticipated because there is evidence to show that PEG-lipids stabilize non-bilayer-forming lipids into a bilayer conformation (Cullis et al., 1991; Holland et al., 1996a). Given the composition of PFVs, the observation that mitoxantrone could be stably encapsulated into PFVs using a transmembrane p H gradient was somewhat more surprising. Central to testing the hypothesis was determining that the mechanism by which PFVs acted was indeed as proposed in section Towards this end it was established in vitro that PEG-lipids play a critical role in determining the characteristics of release of aqueous contents of P F V (Chapter 3). This was a significant finding from the standpoint of enhancing the bioavailability of a drug encapsulated in a liposome. The extension of these results in vivo 151 further amplifies the significance of these results. The kinetics of mitoxantrone release from P F V is controlled by the rate of exchange of PEG-lipids from the surface of P F V . The phenomenon of exchange of PEG-lipids is shown to be common to vesicles of different lipid composition (PFV and conventional liposomes; Chapter 4). However, PFVs have an inherent advantage over conventional liposomes in being transiently stable and composed of primarily fusogenic lipids. The demonstration that controlled destabilization of P F V translates into a higher efficacy of mitoxantrone when loaded in P E G - D S P E - P F V confirms that it is possible for a liposome to have both prolonged blood residence time as well as triggered release of contents (Chapter 5). This is a meaningful advance in the context of attributes desirable in a liposome delivery system, in particular, long circulation lifetimes, accumulation at the disease site and controlled release of encapsulated drug. A n important conclusion of this work is that P F V technology relies on component lipids and membrane-specific structural transformations to play an active role in an associated drug's biological activity. 6.2 Implications The results presented here have far reaching implications for the role of intravenously injected carriers in intracellular delivery. I here characterized a multi-functional, transformable liposome on the basis of the exchangeability of the P E G coating. It is demonstrated that small, conventional drugs like mitoxantrone can be stably encapsulated within such meta-stable carriers (Chapter 3) and drug release can be programmed by the timed loss of PEG-lipid component (Chapter 4 and 5). Furthermore, the contrast between the therapeutic activity of PFVs and conventional liposomes in terms of enhancing the drug 152 bioavailibility was highlighted (Chapter 5). This was of particular significance as most liposomal drug carrier work has been carried out with an emphasis on enhancing circulation longevity. However, in achieving prolonged blood residence of the carrier another attribute of liposomes was being overlooked - release of drug from the liposome. This was the fundamental contradiction presented in Chapter 1. It was predicted that a carrier whose properties could be differentiated on the basis of where it resided in the body would be able to resolve this contradiction. The use of PEG-DSPE-containing PFV shows that an increase in blood residence times, delayed destabilization and release of entrapped mitoxantrone at the disease site can be directly translated into a more efficacious drug-loaded formulation. This result presents a resolution of the aforementioned contradiction. From a mechanistic point of view, the studies presented here illustrate three steps in the functioning of circulating PFVs: 1) exchange of PEG-lipids, 2) the resultant structural transformation of PFVs, and finally 3) the release of encapsulated contents. However, the question that remains in elucidating the complete mechanism of action of programmable fusogenic vesicles (as their name suggests) requires the demonstration of either drug leakage in the vicinity of tumor cells or actual fusion with such cells. This opens up an interesting avenue for further investigation: demonstrating fusion of PFVs with target cells. The sequence of events demonstrated to be essential for fusion of DOPE:DODAC liposomes with negatively charged target vesicles in vitro include the close apposition of the two membrane surfaces promoted by electrostatic attraction of opposite charges; surface dehydration by neutralization of mutual surface charge and promotion of non-bilayer lipid structures by charge reduction and the presence of the fusogenic lipid, DOPE (Bailey and Cullis, 1997). It is known that PEG-lipid conjugates inhibit the close 153 apposition of bilayers (Holland et al., 1996a); therefore, it follows that their loss should promote membrane contact. Further Mori et al. (1998) have shown fusion of DOPE:DODAC:PEG-Ceramide-containing liposomes with erythrocyte ghosts. Taken together with the information gained from the present studies of various steps in the mechanism of action of PFVs , it may be possible to demonstrate fusion of PFVs with tumor cells in vivo. The present research was focused on demonstrating the principle of programmable fusogenic vesicles using mitoxantrone as a model anticancer drug. In addition to their application as delivery vehicles for conventional drugs, they have exciting potential for the intracellular delivery of large polar molecules such as proteins, oligonucleotides and plasmids. In the areas of antisense technology and gene therapy, for example, the primary limitation to efficacy derives from the difficulty of introducing these new classes of therapeutic agents to their intracellular sites of action. Uptake of antisense oligonucleotides and "naked" D N A into cells is generally an inefficient process. In the case of plasmids for gene therapy, this has necessitated the use of viral or non-viral (cationic lipid) vectors but these suffer from problems associated with immune response (viral vectors) or poor systemic stability (non-viral carriers, Crystal, 1995). 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