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

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BIOPHYSICAL AND ANTICANCER PROPERTIES OF MITOXANTRONE IN P R O G R A M M A B L E FUSOGENIC VESICLES by GITANJALI  ADLAKHA-HUTCHEON  B.Sc., Delhi University,  1988  M . Sc., G . B . Pant University of Agriculture and Technology,  1991  A T H E S I S 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 THE REQUIREMENT FORTHE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF P H A R M A C O L O G Y A N D THERAPEUTICS 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  degree freely  this  at the available  copying  of  department publication  of  in  University  of  for reference  this or  thesis  thesis by  for  his  this thesis  or  partial  fulfilment  of  the  requirements  British Columbia, I agree that the and  study.  scholarly her  I further agree that  purposes  may  representatives.  be  It  for financial gain shall not  is be  for  Library  granted  by  understood  of  P H A R M A C - Q LO^Y  The University of British C o l u m b i a Vancouver, Canada  2b* 3»*n 4  Date  DE-6 (2/88)  OAXJ  Trf £ R A  the that  allowed without  PfTUT/CS  advanced  shall make  permission for  permission.  Department  an  it  extensive  head  of  my  copying  or  my  written  ABSTRACT This thesis characterizes programmable fusogenic vesicles (PFVs) and examines their usefulness as carriers for an anticancer drug. exhibit time-dependent destabilization.  P F V s are cationic liposomes designed to  They consist of non-bilayer-forming lipids that are  stabilized into a bilayer by exchangeable poly(ethylene lipids).  glycol)-conjugated  lipids ( P E G -  The rate at which the vesicles destabilize is determined by the rate at which the  bilayer stabilizing component, PEG-phosphatidylethanolamine ( P E G - P E ) , exchanges out o f 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 P F V s was examined as a function of lipid composition using cryo-transmission electron microscopy (cryo-TEM).  While predominantly unilamellar,  P F V s exhibit a variety o f morphologies, including spherical, discoid and invaginated shapes. In the absence of P E G - l i p i d , P F V s 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 P F V s , these vesicles can efficiently accumulate the anticancer drug mitoxantrone in response to an imposed transmembrane proton gradient. Release o f intravesicular contents o f P F V s was demonstrated using radiolabeled sucrose. The rates o f plasma elimination and biodistribution of P F V s and mitoxantrone-loaded P F V s were characterized as functions o f P E G - P E acyl chain length. The presence  of  mitoxantrone did not alter the time-course of elimination o f P F V s from the circulation. The rate o f elimination of P F V s from the circulation depended on the chain length of P E G - l i p i d  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 P F V s . For instance, P F V s containing P E G - 1 , 2 d i s t e a r o y l - s « - p h o s p h o e t h a n o l a m i n e (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 P F V s were examined using a human colon carcinoma (LSI80) xenograft in severe combined immune deficient (SCID) mice.  P F V s prepared with a slow-exchanging P E G - l i p i d delayed tumor  progression more effectively than either P F V s that are rapidly eliminated from the circulation or conventional liposomes.  Efficacy of mitoxantrone-loaded P F V s was also evaluated  against a murine tumor model in which disease progression occurs primarily in the liver. A l l PFV  formulations were significantly more active against this tumor model than free  mitoxantrone or mitoxantrone-loaded conventional liposomes  suggesting that P F V s are  capable of releasing drug more efficiently than conventional formulations.  iii  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E O F 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 1.2 Lipids: Building Blocks of Liposomes 1.2.1 Chemistry and Physics of Lipids  1 3 3  1.2.1.1 Phospholipids 1.2.1.2 Sphingolipids 1.2.1.3 Cholesterol 1.2.2 Lipids: Structure and Behavior  3 5 5 6  1.2.2.1 Lipid Polymorphism 1.3 Types of Liposomes 1.3.1 Liposome Classification: Based on Size  6 9 10  1.3.1.1 Multilamellar Vesicles (MLVs) 1.3.1.2 Large Unilamellar Vesicles (LUVs) 1.3.1.3 Small Unilamellar Vesicles (SUVs) 1.3.2 Liposome Classification: Based on Lipid Composition  10 12 13 13  1.3.2.1 pH-sensitive Liposomes 1.3.2.2 Target-sensitive Immuno-Liposomes 1.3.2.3 Temperature-sensitive Liposomes 1.3.3 Surface Modifying Lipids  14 14 15 15  1.3.3.1 Poly(ethylene glycol) Lipids 1.3.3.2 Cationic Lipids 1.4 Liposomal Drug Loading 1.4.1 Hydrophobic Association 1.4.2 Passive Encapsulation 1.4.3 Active Loading 1.5 Mitoxantrone - An Anthracenedione Derivative 1.5.1 Structure 1.5.2 Biological Effects  21 23 23 24 24 27 27 27  1.5.2.1 Pharmacodynamics 1.5.2.2 Pharmacokinetics • 1.5.3 Liposomal Mitoxantrone 1.6 Liposomes in a Biological Milieu 1.6.1 Liposome-Macromolecule and/ or Cell Interactions 1.6.2 Fate of Intravenously Injected Liposomes  1.6.2.1 Factors Influencing the Blood Residence Times ofLiposomes  28 31 32 33 33 36  15  1.6.2.2 Mononuclear Phagocyte System 1.6.2.3 Passive and Active Targeting 1.6.3 Extravasation of Liposomes from Blood  40 41 42  1.6.3.1 Extravasation: From the Blood to the Interstitium 1.7 The Ideal Liposome - An Enigma 1.7.1 The Conceptual Dilemma 1.8 Novel Liposome Design 1.8.1 Programmable Fusogenic Vesicles  42 45 45 46 47  1.8.1.1 Composition 47 1.8.1.2 Mechanism ofAction ofProgrammable Fusogenic Vesicles: A Hypothesis 1.10 Research Hypothesis  50  CHAPTER 2: MATERIALS and METHODS 2.1 Materials 2.2 Cell lines and Animals 2.3 Preparation of Programmable Fusogenic Vesicles 2.3.1 Establishment of pH Gradient 2.3.2 Establishment of an Osmotic Gradient 2.4 Determination of Trapped Volume of PFVs 2.5 Size Analysis of PFVs 2.6 Cryogenic-Transmission Electron Microscopy (cryo-TEM) 2.6.1 Interpretation of Cryo-Transmission Electron Micrographs 2.7 Encapsulation of Mitoxantrone 2.8 Release of PFV Contents 2.8.1 Release of Mitoxantrone from PFVs 2.8.2 Release of [ C]-SucrosefromPFVs 2.9 Animal Studies of Mitoxantrone-loaded PFVs 2.9.1 Plasma Elimination Studies 2.9.2 Tissue Biodistribution 2.9.3 Studies on Exchange of PEG-lipidsfromPFVs or Conventional Vesicles in vivo 2.9.4 Plasma Elimination Studies of Exchange of DODAC from PFVs in vivo 2.9.5 Analysis of Plasma Samples by Fast Protein Liquid Chromatography 2.9.6 Tumor Accumulation and Plasma Elimination Studies of Mitoxantrone-loaded PFVs 2.9.7 Establishment of Maximum Tolerated Dose 2.9.8 Antitumor Activity against Human Colon Carcinoma Xenografts 2.9.9 Antitumor Efficacy against Murine L1210 Leukemia 2.10 Statistical Analysis  52 53 53 54 54 54 55 56 56 57 59 59 59 60 60 60 61 61 62 63 63 64 65 65  14  CHAPTER 3: CHARACTERIZATION OF P R O G R A M M A B L E FUSOGENIC VESICLES IN VITRO 3.1 Introduction 67 3.2 Results 69 3.2.1 Morphology of PFV 69  3.2.1.1 Morphology ofPFV: Influence of PEG-PE 3.2.1.2 Morphology ofPFVs: Influence of Cationic Component, DODAC 3.2.2 Uptake of Mitoxantrone in PFVs  69  76  3.2.2.1 Uptake ofMitoxantrone in PFVs: Influence of PEG-lipid Species 3.2.2.2 Effect ofDrug to Lipid Ratio on Uptake of Mitoxantrone in PEG-Ceramide Cont 3.2.2.3 Stability of Mitoxantrone Uptake in PFVs Containing PEG-Ceramide 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 T H E 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 T H E ANTICANCER PROPERTIES OF MITOXANTRONE ENCAPSULATED WITHIN P R O G R A M M A B L E 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  5.2.1.1 Mitoxantrone and Lipid levels in the plasma 5.2.1.2 Mitoxantrone and Lipid Accumulation in LSI 80 Solid Tumors 5.2.2 Therapeutic Activity of Mitoxantrone-Loaded PFVs  132 13 137  5.2.2.1 Efficacy of Mitoxantrone-loaded PFVs against LSI 80 human colon carcinoma mo 5.2.2.2 Efficacy of Mitoxantrone-loaded PFVs against Liver localized disease 5.3 Discussion  145  CHAPTER 6: DISCUSSION and IMPLICATIONS 6.1 Significance of Results 6.2 Implications  151 152  REFERENCES  155  vi  LIST O F FIGURES Figure 1.1 Structures o f Common Lipids  4  Figure 1.2 L i p i d Polymorphism  7  Figure 1.3 Types of Liposome  11  Figure 1.4 Structures o f Surface-modifying Lipids  19  Figure 1.5 Active Drug Encapsulation in Liposomes  26  Figure 1.6 Structure o f 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 P F V s in the absence of P E G - P E . . . 73 Figure 3.2 Cryo-transmission electron micrographs of P F V o f different compositions....  75  Figure 3.3 Uptake o f mitoxantrone in P F V s : Influence o f P E G - l i p i d species  78  Figure 3.4 Uptake of mitoxantrone by P F V s : Influence o f drug to lipid ratio on uptake o f mitoxantrone in P F V s containing PEG-Ceramide  79  Figure 3.5 In vitro release of mitoxantrone from PEG-Ceramide-PFVs: effect o f drug to lipid ratio Figure 3.6  80 Cryo-transmission electron micrograph o f "empty" programmable fusogenic  vesicles and mitoxantrone-loaded P F V s  82  Figure 3.7 In vitro release of aqueous contents from P F V s : Influence of concentration and acyl composition of P E G - P E  88  vii  Figure 4.1  Plasma elimination of mitoxantrone-loaded P F V s after i.v. administration:  Influence of P E G - P E acyl composition  103  Figure 4.2 Biodistribution of P F V s and mitoxantrone in the liver of B D F - 1 mice  105  Figure 4.3  Plasma elimination of P E G - P E and P F V s after i.v. administration: Influence of  P E G - P E acyl composition Figure 4.4  107  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  Ill  Figure 4.6  Plasma elimination of the cationic lipid, D O D A C from P F V s : influence of P E G -  P E composition Figure 4.7  Separation of liposomes from plasma components using Fast Protein Liquid  Chromatography: distribution of P E G - P E Figure 5.1  120  Plasma elimination of mitoxantrone and mitoxantrone-loaded P F V s from tumor  bearing mice Figure 5.2  114  135  Tumor accumulation of mitoxantrone and liposomal lipid in the human L S I 8 0  solid tumor xenograft  136  Figure 5.3 Mitoxantrone mediated LS180 solid tumor growth inhibition  138  Figure 5.4  Therapeutic activity of mitoxantrone-loaded P F V s against a murine L1210  leukemia model  143  viii  LIST O F T A B L E S Table 1. Types o f 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 o f release of the aqueous contents o f P F V s as a function of concentration  and  PEG-PE  chain  length  on  day  2  in  the  presence  of  sink  85  Table 4. A comparison o f release of the aqueous contents o f P F V s concentration  10-fold  and  PEG-PE  chain  length  sink  on  day  7  in  the  as a function o f  presence  of  30-fold 87  Table 5. Mean area under the curve ( A U C ) 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 P F V s in B D F - 1 mice. 144  ix  ABREVIATIONS AUC  area under the curve  CHOL  cholesterol  CHE  cholesteryl hexadecyl ether  CnV c  conventional liposomes tumor concentration levels  cryo-TEM  cryogenic transmission electron microscopy  DDAB  N, Af-dimethyl-A , A^-dioctadecylammonium bromide  DODAC  N, TV-dioleyl-Af A^-dimemylammonium chloride  DOPC  1,2-dioleoyl-.s«-glycero-3 -phosphatidy choline  DOPE  1,2-dioleoyl-5«-glycero-3 -phosphatidy lethanolamine  ^ m a x  7  DSPC  1,2-distearoyl-5/7-glycero-3 -phosphatidylcholine  EDTA  ethylenediaminetetraacetic acid  EPC  egg phosphatidylcholine  FATMLV  frozen and thawed L U V s  FPLC  fast protein liquid chromatography  G i  monosialoganglioside G  H HBS  hexagonal phase  HDL  high density lipoprotein  HEPES  A^-2-hydroxyethylpiperazine-7V-2-ethane-sulphonic acid  i.p.  intraperitoneal  i.v.  intravenous  LDL  low density lipoproteins  LUVs  large unilamellar vesicles  MePEG  monomethoxypoly(ethylene  MLVs  multilamellar vesicles  MPS  mononuclear phagocyte system  MTD  maximum tolerated dose  PA  phosphatidic acid  PC  phosphatidylcholine  PE  phosphatidylethanolarnine  PEG  poly(ethylene glycol)  PEG-Cer  poly(ethylene glycol)  PEG-CerC20  1 -0-(2' -(w-methoxypolyethyleneglycol  PEG-PE  poly(ethylene glycol)  PEG-DMPE  poly(ethylene glycol)  (2000)  -dimyristoylphosphatidylethanolamine  PEG-DPPE  poly(ethylene glycol)  (2000)  -dipalmitoylphosphatidylethanolamine  PEG-DSPE  poly(ethylene glycol)  PEG-DMPE-PFV  P F V prepared with P E G - D M P E  PEG-DPPE-PFV  P F V prepared with P E G - D P P E  M  n  M 1  HEPES-buffered saline  (2000)  glycol)  -modified ceramide succinoyl)-2-A /  (2000)  arachidoylsphingosine (2000)  -modified phosphatidylethanolarnine  (2000)  -disteroylphosphatidylethanolamine  PEG-DSPE-PFV  P F V prepared with P E G - D S P E  PFV  programmable fusogenic vesicle  PG  phosphatidylglycerol  PI  phosphatidylinositol  POPS  1 -palmitoyl, 2 - o l e o y l - i « - g l y c e r o - 3 - p h o s p h a t i d y l c h o l i n e  QELS  quasielastic light scattering  RES  reticulo-endothelial system  ,  s.c.  subcutaneous  SCID  severe combined immune deficient mice  SM  sphingomyelin  SSL  sterically stabilized liposomes  SUVs  small unilamellar vesicles  T  transition temperature  VLDL  very low density lipoproteins  * c  ACKNOWLEDGMENTS " 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 G o d and the powers that be, for giving me the perseverance to do as I dreamt. I am grateful to Dr. T o m Madden, for giving me a chance just when I almost quit. I would also like to thank T o m 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 o f the FPLC.  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 o f British Columbia and University Graduate Fellowship from The University o f British Columbia for financial support. I am hard pressed for words to acknowledge my family members who believed in me unconditionally.  T o 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, A m i , 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  DEDICATED T O my husband, my second conscience BRUCE  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.  A n 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 L i p i d s  1.2.1.1 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, P G or PI.  3  R-CH CH N(CH )  Phosphatidylcholine (PC)  2  2  Phosphatidylethanolarnine (PE)  >  3  +  R-CH CH NH 2  C Z z j ^ > Headgroup  2  3  R-H  Phosphatidic acid (PA)  Glycerol Backbone  3  H + RCH—C-NH  Phosphatidylserine (PS)  3  nun.-— _  ioo-  Phosphatidylglycerol (PG)  R-CH CH(OH)CH OH 2  2  Phosphatidylinositol (PI)  Some naturally occurring fatty acids  C=£>  Acyl chain  Laurie (12:0)  CH,(CH,), COOH 0  Myristic(14:0)  CrycH^coOH  Palmitic (16:0)  CR,(CH,)„COOH  Stearic (18:0)  CH,(CH,)„COOH  Palmitoleic (16:1,A9) Oleic (18:1. A ) 9  CH,(CH ) CH=CH(CH,) COOH J  5  7  CH,(CH3) CH=CH(CHj) C00H 7  7  Linoleic (18:2, A 9 ' 1 2 ) CH (CH,) CH=CHCH CH=CH(CH ) C00H J  4  2  J  7  Unolenic(18:3,A ' ' ) 9 12 15  CHjCHjCH-CHCHjCHoCHCHjCH'CHfCHjJjCOOH  B Sphingolipid  Cholesterol (Choi)  R-NH OR"  R = H; R" = H  Sphingosine  R = COR"; R' = H  Ceramide (Cer)  R = COR"; R' = phosphocholine  Sphingomyelin (SM)  (R" = hydrocarbon)  Figure 1.1 Structures of Common Lipids  4  1.2.1.2 Sphingolipids L i p i d 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 LIB).  One of the  most common membrane  sphingolipids  is  sphingomyelin (SM).  Sphingomyelin has a phosphocholine group esterified to the 1-hydroxyl group of a ceramide.  1.2.1.3 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 1.2.2.1) above their transition temperature (T , Oldfield and Chapman, 1972; Demel and de c  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). A s 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  L i p i d s : 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 and isotropic n  phases) have also been characterized.  1.2.2.1 Lipid Polymorphism Lipid polymorphism is defined as the ability of lipids to adopt different phases such as the lamellar or bilayer, the hexagonal, H phase and other non-bilayer phases grouped n  together as the "cubic" phases (Cullis et a l , 1986).  B LIPIDS  PHASE  Lysophospholipids  Detergents  • Micellar  Phosphatidylcholine Sphingomyelin Phosphatidylserine Phosphatidylinositol Phosphatidylglycerol Phosphatidic Acid Cardiolipin Digalactosyldiacylglycerol  MOLECULAR SHAPE  Inverted Cone  If i l 11 111 Bilayer  Cylindrical  Hexagonal (H||)  Cone  Phosphatidylethanojamine Cardiolipin - Ca Phosphatidic Acid - Ca Phosphatidic Acid (pH<3.0) Phosphatidylserine (pH<4.0) Monogalactosyldiacylglycerol  Figure 1.2 L i p i d 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 o f 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 threedimensional 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 ( C L ) 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 o f 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 o f factors such as p H , temperature and addition of counter-ions allows alteration o f the preferred phase of a given lipid. The addition of micelle-forming species like detergents (Madden and Cullis, 1982) or 20-50 moi % o f 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 phase is shown in Figure 1.2A. n  O n the basis of their phase transition temperature (T ), lipids can adopt either a c  'frozen' gel, solid ( L ) phase or a liquid crystalline ( L J phase. T is principally dependent on p  the  length  c  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 , the more fluid liquidc  crystalline state exists, exhibiting decreased order and an increased movement  of the  membrane components: lateral diffusion, rotation around the long molecular axis and transgauche isomerization occurs.  In addition, membranes are more permeable to a variety of  solutes and solvents at or above T than below (Bittman and Blau, 1972). c  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). small size.  Further processing of M L V s can produce liposomes that result in uniform and 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  Lipid Composition  Function  MLVs (1 pm - 100 pm)  LUVs (0.05 pm - 1.0 pm)  SUVs (< 0.05 nm)  Conventional: base carrier  Second generation: "Stealth", incorporation of PEG-lipids  Third generation: surface-associated targeting information (antibodies)  pH-sensitive liposomes  Target-sensitive immuno-liposome (antibodies attached)  Temperaturesensitive 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.  1.3.1.1 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) L U V s , 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 ( F A T M L V s , 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).  1.3.1.2 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 L U V s 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). L U V s 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 L U V s , 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.  1.3.1.3 Small Unilamellar  Vesicles (SUVs)  Unilamellar vesicles possessing the smallest diameter (typically < 50 nm) fall into the class of small unilamellar vesicles (SUVs). S U V s 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 S U V s 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,  al.,  1969;  Barenholz et  1979)  and lipoprotein-induced  leakage  of  contents  (Scherphof and Morselt, 1984), all of which make S U V s 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.  changes in liposomal lipid components were intended to optimize a specific  Most  liposomal  13  attribute.  F o r instance, attempts to formulate fusogenic l i p o s o m e s s u c h as p H - s e n s i t i v e and  target-sensitive i m m u n o - l i p o s o m e s were intended for the c y t o p l a s m i c d e l i v e r y o f entrapped contents.  V e r y often the p r i m a r y l i p i d c o m p o n e n t o f these fusogenic l i p o s o m e s w a s a l i p i d ,  l,2-dioleoyl-.yH-grycero-3 -phosphatidylethanolarnine ( D O P E ) , w h i c h is n o n - b i l a y e r f o r m i n g and fusogenic under p h y s i o l o g i c a l c o n d i t i o n s . result  o f l i p o s o m e designs  Although  pH-sensitive,  Temperature-sensitive  a i m e d at c o n t r o l l i n g the  target-sensitive  and  l i p o s o m e s were the  release o f encapsulated  temperature-sensitive  contents.  liposomes  were  c o n c e p t u a l l y a d v a n c e d o v e r c o n v e n t i o n a l l i p o s o m e s , their a p p l i c a b i l i t y w a s l i m i t e d in vivo. S o m e o f these l i m i t a t i o n s are detailed b e l o w .  Surface properties  o f l i p o s o m e s c a n be  m o d i f i e d b y the i n c l u s i o n o f p o l y m e r s s u c h as p o l y ( e t h y l e n e g l y c o l ) and/or c a t i o n i c l i p i d s . These l i p i d s w i l l be c o v e r e d i n greater depth i n section 1.3.3.  1.3.2.1 pH-sensitive Liposomes p H - s e n s i t i v e l i p o s o m e s t y p i c a l l y consist o f D O P E s t a b i l i z e d b y a p H - s e n s i t i v e fatty a c y l a m i n o a c i d or fatty a c i d s u c h as o l e i c a c i d . P r i m a r i l y these carriers e m p l o y a v a r i e t y o f protonable l i p i d s to trigger v e s i c l e d e s t a b i l i z a t i o n under a c i d i c c o n d i t i o n s t a k i n g advantage o f e n d o s o m a l a c i d i f i c a t i o n ( Y a t v i n , 1980; C o n n o r and H u a n g , 1985; C o n n o r et a l . , 1986). H o w e v e r , the disadvantage o f s u c h p H - s e n s i t i v e l i p o s o m e s stems f r o m their tendency  to  aggregate a n d b e c o m e l e a k y i n the presence o f serum ( C o n n o r et a l . , 1986).  1.3.2.2 Target-sensitive Immuno-Liposomes Target-sensitive i m m u n o - l i p o s o m e s are s i m i l a r to the class o f p H - s e n s i t i v e , D O P E c o n t a i n i n g l i p o s o m e s but differ i n that these are s t a b i l i z e d d i r e c t l y b y a n a c y l a t e d m o n o c l o n a l  14  antibody (Huang et al., 1982; H o et a l , 1986). confer target cell specificity.  The monoclonal antibody was included to  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).  1.3.2.3 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 L i p i d s  1.3.3.1 Poly (ethylene glycol) Lipids There is a wealth of data available on polymer-coated nanoparticles and other polymer-coated  spheres that persist  (reviewed by Brannon-Peppas, 1995).  in the circulation longer than uncoated  particles  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) P E G 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 -CH ] -) (Figure 1.4). This is primarily because PEG 2  2  n  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, P E G 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 % PEG o-PE (Needham et 200  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% PEG o-PE or 0-6 mole % PEG -PE) of 200  5000  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 D a (approximately 46 monomeric residues) at 5 mole %. Liposomes containing PEG4ipids in the bilayer have often been referred to as "sterically stabilized liposomes" (SSL). circulation longevity  Although the mechanism underlying liposome  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; M o r i 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: D O D A C and D D A B  19  It is of interest, however, that little attention has been paid to the extent to which the P E G 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 PEGlipids include the length and degree of saturation of the lipid fatty acyl chains and the size of the P E G moiety (Silvius and Zuckermann, 1993). Consequently, liposomes composed of shorter or unsaturated acyl chains (POPE-PEG  2000  ) were found to have a shorter circulation  life in vivo than the longer, saturated P E G derivatives (DSPE-PEG  2000  ; 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.  1.3.3.2 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. T o date, two hypotheses for the mechanism of entry of lipid-based transfection agents into transfecting cells have been put forward. route is via membrane fusion, while the second is by way of endocytosis.  The first  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 i p i d - D N A 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,Ndimethyl-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  H y d r o p h o b i c 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 o f drugs subsequent to passive encapsulation is frequent and occurs rapidly.  For  example,  egg  between 20  - 60% of passively  loaded doxorubicin is released  from  phosphatidylcholine (EPC): cholesterol vesicles by 1 hour (Gabizon et al., 1982). In general, the retention rates can be improved significantly with the incorporation o f 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  (CHOL)  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  K  Inside pH 4.0  K — [B],[H ],  = [B] [H ] [BH ] +  0  +  +  0  [BH ], +  0  At equillibrium, if: Then:  [BL = [BH ], +  =  [BH ] +  0  P I  [H], [H ] +  0  Figure 1.5 Active Drug Encapsulation in Liposomes R e d i s t r i b u t i o n o f a w e a k base ( l i p o p h i l i c amine) i n response to a transmembrane p H gradient ( A p H ) , across the l i p o s o m a l bilayer. O n l y the neutral, u n c h a r g e d f o r m o f the base is capable o f c r o s s i n g the l i p i d bilayer. (Figure t a k e n f r o m 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-9,10anthracycenedione 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; L a i 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.  1.5.2.1 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; L o w n et al., 1984).  Finally, the  oxidative  non-protein-  associated  activation of mitoxantrone  by free  radical  D N A strand breaks (Fisher and Patterson,  generation 1989).  induces  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.  A s 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 vs. 9 doses of doxorubicin at 60 m g / m (Dukart and Barone, 2  1984) .  2  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 nonprotein-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[ CJ). The asterisk 14  denotes the position o f 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). observations  point towards  a multifactorial mechanism  mitoxantrone as with most anticancer drugs.  Taken together, these  of resistance  in response  to  Tumor cell lines exhibiting resistance  to  mitoxantrone in vitro display an atypical resistance profile, showing only partial crossresistance 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 nontransport-mediated drug efflux.  1.5.2.2 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 , suggesting that much of the drug is sequestered in 2  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 lipidmitoxantrone 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  Figure 1.7  Lipid Exchange  Adsorption  Fusion  Endocytosis  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 L i n , 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 (HermandezCaselles 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 , S M ) 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. containing negatively  Liposomes  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 A p o A I , 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) Morselt,  and the complete destruction of liposome  1984). It was  structure (Scherphof and  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 compositional  differences  including different phospholipids  in vivo are gross  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).  1.6.2.1 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 i f 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 V a n 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 ( S U V s ) too are cleared very quickly from circulation (Roerdink et al., 1981). Higher circulation levels have also been attributed to higher administered doses. A t 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  1.6.2.2 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.  1.6.2.3 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. fenestrations  It takes advantage of the  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 antibodycoated 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 1.6.3.1 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  43  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 villi 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 1.3.3.1), 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.  1.8.1.1 Composition In the present studies, P F V systems contain the non-bilayer-forming lipids 1,2dioleoyl-^«-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  glycol o) 200  (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.  1.8.1.2 Mechanism ofAction 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 o f P E G - l i p i d leading to P F V destabilization. This critical step would be followed by one of three outcomes: (A) the elimination o f the destabilized P F V s via the reticuloendothelial cells of the body (B) the release of the contents o f P F V s in the vicinity of the target cell or (C) the fusion of the destabilized P F V s 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  C H A P T E R 2: M A T E R I A L S and METHODS  2.1 Materials A l l phospholipids including PEG-PE conjugates were purchased from Avanti Polar Lipids, (Alabaster, A L ) .  D O D A C was a generous gift from Inex Pharmaceuticals  Corporation, Vancouver, B.C., Canada. [^CJ-A^TV-dioleyl-A^A^-dimethylammonium chloride ([ C]-DODAC) was custom synthesized by Stephen Ansell of Inex Pharmaceuticals 14  (Vancouver, B.C).  [ H]-Cholesteryl Hexadecyl Ether ([ H]-CHE) was purchased from 3  3  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 [ C] mitoxantrone used as a tracer was generously donated by Wyeth 14  Ayerst (Montreal, PQ). N-[poly(ethylene  1,2-dimyristoyl (9',10'-di- H)-^«-glycero-3-phosphoethanolamine-  glycol)  3  2000  ]  (PEG  -DMPE[ H]); 3  2000  1,2-dipalmitoyl  glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) distearoyl (PEG  2000  ] (PEG  (9',10'-di- H)-5«3  -DPPE[ H]) and 1,23  2000  (9', 10' -di- H)-5«-glycero-3 -phosphoethanolamine-N- [poly(ethy lene 3  glycol)  2000  ]  -DSPE[ H]) were custom synthesized and radiolabeled by Xue M i n Zhou of 3  2000  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, L S I 8 0 , 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 B D F - 1 mice. Female B D F - 1 mice (8-9 weeks old, 18.0-23.0 g) were purchased from Charles River Laboratories (Ontario, Canada).  Severe combined immune  deficient mice, S C I D / R A G - 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 L i p i d components (DOPE:Cholesterol:DODAC:PEG-lipid, 30:45:15:10 molar ratio) containing the non-exchangeable, non-metabolizable lipid label (Stein et al., 1980) [ H]3  Cholesteryl Hexadecyl Ether ([ H]-CHE) or [ C ] - C H E were solubilized in 1-2 3  14  ml of  benzene:methanol (95:5, v/v) and then lyophilized for a minimum of 5 h at a pressure o f <60 m Torr using a Virtis lyophilizer equipped with a liquid nitrogen (N ) trap. 2  prepared for  For P F V s  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).  T o assist lipid hydration, the suspension was  vortexed and then frozen (liquid N ) and thawed ( 3 7 ° C ) five times (Mayer et al., 1985a). The 2  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 P F V 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 m M HEPES buffer; M u i 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 [ H]-CHE) was carried out in HEPES-buffered saline (pH 7.4) containing 3  1 m M sucrose, radiolabeled with [ C]-sucrose, as the impermeable trapped volume marker 14  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 [ C]-sucrose and [ H]-CHE lipid 14  3  counts. From the rest of the extruded vesicle preparation, the unentrapped [ C]-sucrose was 14  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 N a C l 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 o f the mini columns was then assayed by dual label scintillation counting for [ C]-sucrose 14  and [ H ] - C H E lipid levels. 3  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 [ C]-sucrose counts in the initial liposome sample (100 pi). 14  L i p i d 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 o f the [ H]-liposomes 3  used (Hope et al., 1985).  Entrapped volume was expressed as pi o f trapped [ C]-sucrose/ 14  pmole o f 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 ( Q E L S ) using a Nicomp 270 submicron particle sizer (Nicomp Instruments, Goleta, C A ) operating at 632.8 nm and 5 m W , 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 o f 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 c r y o - T E M 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  Dubochet et al., 1988 and Bellare et al., 1988.  has  been  described  in  detail  by  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 k V . 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 c r y o - T E M pictures, it is important to realize how the twodimensional representation of a three-dimensional bilayer aggregate depends on the projected thickness of the depicted aggregate.  A s 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 P F V s 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 /pH 7.4 ) were added to mitoxantrone (2.0 in  0Ut  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 P F V s 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 m l 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 o f a spherical liposome (A), a flattened liposome (B), and a bilayer disc (C) all appear as circular objects in the  cryo-TEM  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 o f a three dimensional volume as a two dimensional image.  58  2.8 Release of PFV Contents 2.8.1 Release of Mitoxantrone from P F V s The kinetics of mitoxantrone releasefromPFVs 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 [ C]-Sucrose from P F V s ,4  The initial volume of [ C]-sucrose entrapped within PFVs was measured as described 14  in Section 2.4 as a function of the acyl composition of the PEG-PE component of PFVs. The kinetics of [ C]-sucrose release from PFVs was monitored by assaying the change in the 14  contents of trapped [ C]-sucrose under various conditions. PFVs differing only in the PEGl4  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 [ C]-sucrose content was measured. The release of [ C]-sucrose from PFVs was 14  14  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  intravenous doses via the lateral tail vein at 10 mg/kg mitoxantrone (22.5  single  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 drugloaded P F V s .  A t 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 50°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 0 2  (200 pi) and 10 N HC1 (25 pi). Samples were  2  incubated at room temperature for 1 hour.  Following the addition of scintillation cocktail  (Ultima Gold, Packard, Meriden, C T ) , 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 P F V s  or conventional  vesicles  was  determined in female B D F - 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 P F V s and conventional liposomes, respectively.  A t 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 ( [ C ] - C H E ) and P E G - P E [ H ] levels in plasma were determined by dual 14  3  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, [ C ] - D O D A C was 1 4  incorporated in P F V s labeled with [ H ] - C H E as the vesicle marker. 3  Plasma elimination  studies to determine the rate of loss of D O D A C 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 PEGDMPE[ H] or PEG-DPPE[ H] or D S P C C H O L liposomes containing PEG-DMPE[ H] after 1 3  3  3  and 8 hours, respectively. Radiolabeled Cholesteryl Hexadecyl Ether ([ C]-CHE) was used 14  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 F P L C 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 m M NaCl, 10 m M Tris, 0.03 % sodium azide (NaN ) at pH 7.4. The absorbance of each fraction was read at 280 nm and the lipids tracked 3  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 ( S C I D / R A G - 2 , 18-22g, female mice, 4 per group, B . C . Cancer Agency) were inoculated bilaterally with 1 x 10 human colon carcinoma, 6  L S I 8 0 cells subcutaneously on the hind regions of the back. Mice were injected with a 10 mg/kg dose o f free mitoxantrone or mitoxantrone-loaded P E G - D M P E - P F V s or P E G - D S P E P F V s when the tumors had reached a size measurable by calipers. A t 1,4, 24 and 48 hours, animals were terminated by C 0 asphyxiation and whole blood collected via cardiac puncture 2  and placed into EDTA-coated tubes. Plasma was isolated following centrifugation o f whole blood at 500 x g for 10 min.  Aliquoted plasma samples (100 pi) were mixed with 5 m l  scintillation fluid (Packard, Meriden, C T ) and counted for [ H] and [ C ] . 3  l4  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 L S I 8 0 tumor model were  determine the maximum tolerated dose ( M T D ) of mitoxantrone.  performed to  These studies were  completed using protocols approved by the University o f British Columbia Animal Care Committee and were done in accordance with the guidelines o f the Canadian Council on Animal Care ( C C A C ) .  It should be noted that only two of 25 animals ( S C I D / R A G - 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 or L D 0  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 30day period, animals were sacrificed by C 0 asphyxiation and necropsies were completed to 2  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 10 human colon carcinoma, LSI80 cells in a volume 6  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 P F V 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 (cm ) = 7tab /6 3  2  The volumes of all the tumors in each individual treatment group were summed. cumulative sum of tumor volumes was then divided by the number of mice.  The  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 cm ) and tumor size on the 3  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 o f 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 P F V s was determined using the murine L1210 leukemia model.  B D F - 1 mice (8 per group) were  inoculated intravenously with 10 L1210 murine tumor cells derived from the ascites fluid of 4  a previously infected B D F - 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 o f 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). A N O V A , a post  If a significant difference between means was detected by  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 o f variance followed by Scheffe's test was also used to evaluate differences between groups for studies on exchange of PEG-lipids from P F V s or conventional liposomes.  For the LI210 efficacy  studies,  survival times (in days) were ranked and statistically analyzed using a Cox's F test.  65  C o m p a r i s o n s i n d i c a t e d as h a v i n g statistical significance h a d P values f r o m the C o x ' s F test o f < 0.01.  M e a n area under the curve analysis was p e r f o r m e d u s i n g t r a p e z o i d a l integration.  66  C H A P T E R 3: C H A R A C T E R I Z A T I O N 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 multicomponent 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 C H O L and the non-bilayerforming lipid DOPE were stabilized in a lamellar organization by inclusion of PEG-PE or PEG-Ceramide. In addition, a cationic lipid, D O D A C , 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  67  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 o f 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 o f 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 P F V s 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 P F V s to sink would sufficiently destabilize P F V s to expose D O D A C and subsequently trigger electrostatic interactions with negatively charged vesicles.  Therefore,  the release o f 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 o f P F V s 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 (cryoTEM) was utilized to delineate P F V 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 [ C]-sucrose as a marker for PFV contents. 14  3.2 Results 3.2.1  M o r p h o l o g y of P F V  3.2.1.1 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 D O P E : C H O L : D O D A C 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 ) phase n  similar to those observed by Gustafsson et al. (1995). In contrast to systems lacking PEG-lipid, P F V 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 et.al., 1997). Average vesicle diameter, as determined from c r y o - T E M  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 P F V s 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. direct measurement  from c r y o - T E M  In comparing vesicle diameters determined by  electron  micrographs with  sizes determined  by  quasielastic light scattering ( Q E L S ) , 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)  Mean Diameter (nm)  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)  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  b  c  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  3.2.1.2 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 D O D A C 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 P F V s 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 ) phase. The bar represents 100 nm. n  73  Figure 3.2 Cryo-transmission electron micrographs of PFV of different compositions (following page) C r y o - t r a n s m i s s i o n electron m i c r o g r a p h s o f (a) P F V s c o m p o s e d o f D O P E : C H O L : D O D A C : P E G - P E , ( 3 0 : 4 5 : 1 5 : 1 0 , m o l a r 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 c o m p o s e d 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, m o l a r ratio).  The  arrows denoted E and F p o i n t out b i l a y e r discs as v i e w e d edge-on and face-on, r e s p e c t i v e l y . (c) P F V s i n the absence o f the c a t i o n i c l i p i d component, D O D A C  (DOPE:CHOL:PEG-  D S P E , 4 5 : 4 5 : 1 0 , m o l a r ratio), m a d e 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). T h e arrows denote i n v a g i n a t e d structures v i e w e d face-on. (d) P F V s i n the absence o f the c a t i o n i c l i p i d component, D O D A C , d i l u t e d 1:1 i n h y p o o s m o t i c (25 m M N a C l , 25 m M H E P E S , p H 7.4) s o l u t i o n . (e)  PFVs  (DOPE:CHOL:DODAC:PEG-PE,  30:45:15:10,  m o l a r ratio)  diluted i n hypo-  o s m o t i c buffer ( 25 m M N a C l , 25 m M H E P E S , p H 7.4). T h e bar represents 100 n m .  74  3.2.2 Uptake of Mitoxantrone in P F V s  3.2.2.1 Uptake ofMitoxantrone 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 P F V s 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. A s 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 P F V s containing PEG-Ceramides with N-palmityl (C16, P E G - C e r C 1 6 ) 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. P E (Figure 3.3B).  Similar drug loading efficiencies are observed for P F V s containing P E G Mitoxantrone encapsulation was rapid with essentially complete uptake  seen by 30 minutes on incubation at 4 5 ° C . Again, P F V formulations containing either P E G DMPE,  (dimyristoylphosphatidylethanolamine)  or  PEG-DSPE  (distearoylphosphatidylethanolamine) exhibited similar loading properties.  76  3.2.2.2 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 P F V s ( 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). A t 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 o f loading are slower at higher ratios (Figure 3.4).  3.2.2.3 Stability of Mitoxantrone Uptake in PFVs Containing PEG-Ceramide Having demonstrated that mitoxantrone can be efficiently encapsulated within P F V s , the length o f time for which mitoxantrone was stably retained within P F V s was determined next.  Release of mitoxantrone from P F V s ( 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 3 7 ° C was monitored for vesicles of varying drug:lipid molar ratio (Figure 3.5). In the case o f vesicles loaded at 0.1:1 dragdipid ratio, no mitoxantrone leakage was observed over 24 hours. A t 0.2:1 molar ratio, slow release was observed with approximately 85% of the encapsulated drug retained at 24 hours. A t 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  120  o '•+-« CO  l_  100  •g  r-  Q.  76  CD  (0 -I—<  Q.  3  B  o ro •g CD  "D  60  ^  40  ro -»—'  Q.  3  20 -  oi o  90  60  30  120  Time (minutes)  Figure 3.3 Uptake of mitoxantrone in P F V s : Influence of P E G - l i p i d specie Mitoxantrone was loaded at a drug to total lipid molar ratio of 0.1:1.0 into P F V s exhibiting a transmembrane  p H gradient  CeramideC16 (•) D M P E (•),  (pH 4.0 /pH  0Ut  or PEG-CeramideC20 (•)  or P E G - D S P E (•,)  deviation, n = 3.  7.4 ).  in  at 45 ° C .  Panel  A : PFVs  containing P E G -  at 37 ° C . Panel B : P F V s containing P E G The data points represent a mean ± standard  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 P F V s 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 P F V s 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). T o ascertain the suitability of P F V s 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 P F V s containing either P E G - D S P E or P E G - C e r C 2 0 as their bilayer-stabilizing lipid were examined by c r y o - T E M . Electron micrographs shown in Figure 3.6 demonstrate that both empty and mitoxantroneloaded P F V s exhibit vesicle shapes ranging from spherical to discoid or tubular (average size approximately 100 nm, see Table 3.1).  In contrast to empty P F V s , 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  P F V s 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 P F V s containing either P E G - D S P E or P E G - C e r C 2 0 , for both empty and mitoxantrone-loaded systems.  In general, P F V s containing PEG-Ceramide appear more spherical compared to  P F V s containing P E G - D S P E which show a preponderance of tubular structures, possibly reflecting differences in overall net charge on the P F V s .  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" fusogenic vesicles and mitoxantrone-loaded PFVs  programmable  P F V s were made o f 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 P F V s 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 P F V s 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 P F V s .  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 P F V s  in vitro, following destabilization. P F V s 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 [ C]-sucrose as a marker for the intravesicular contents (Chapter 2). 14  It should  also be mentioned that the initial volume trapped within P F V s 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 P F V s 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 P F V s 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 P F V s .  In addition, the loss of P E G - P E would unmask the fusogenic lipids and in  doing so sufficiently destabilize the P F V s to trigger release of P F V contents. this hypothesis  In order to test  in vitro, changes in [ C]-sucrose content within P F V s were investigated as a 14  83  function o f the concentration of PEG-lipids (5 or 10 mole%) and the acyl composition of PEG-lipids  ( P E G - D M P E and P E G - D S P E ) .  Experiments involving two-step incubations  were conducted to record the variation in the aqueous contents of P F V s . Vesicles were first incubated with a concentration gradient of sink (first step) with the rationale o f promoting loss of PEG-lipids. Negatively charged vesicles containing the phospholipid, 1-palmitoyl, 2o l e o y l - ^ « - g l y c e r o - 3 - p h o s p h o s e r i n e (POPS) were then added for 1 day (second step) to trigger electrostatic interactions with the positively charged component of P F V s .  Two sets o f these  experiments were carried out having different durations for the first step o f incubation. In the first set of experiments, release o f sucrose from P F V s 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). A s shown in Table 3.2, the loss of sucrose content from P F V s containing 5 mole% P E G - D M P E in the presence o f sink was equivalent to 23.6% of the initial trapped sucrose. The extent o f sucrose release increased to 59% upon incubation o f the same P F V s with negatively charged vesicles.  Upon incubating P F V s containing P E G - D S P E (5 and 10  mole%) with negatively charged vesicles no further increase in release o f sucrose was observed over that of sink vesicles (Table 3.2).  A s a control for the second incubation step  with anionic vesicles, P F V s already exposed to sink were incubated with a solution o f 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.  C/2  +  •a  I5D  OX  a  *CN >>  Qa  <D  vesicl  PS (-  >  s  in  o o  +1  m  c/3  s  o  ©  © ©  ©  ON  +1  +1  ro  o  CN  OO CN  CU  o  >  +1  <D CJ)  ON  o  *~'  cj >  St  u  U  PM I  +1  - f i  c  PM  -g  -O fl «  c/3 X o  fl  *CN  >,  ca Q  o o  in o  ON  O  in O  +1  +1  +1  +1  <N  O NO  NO  CN  in  CN  +1  +1  +1  ON  ro  CN  O  o  ©  o  ON  ro  o o  ©  o o  ©  V  OH fl"  13  O  m  «t  s-  fl U fl  o o fl  <s .2 +H  (N 5H  •S §  m  « at ^ a> cs  PH  ©  fl fl  o u Mi  s  o s  Q  o o  oo in  © ©'  © ©  o ©  +1  t--  + Q C/3  c  ,3 o  >  CO %-» 'fl  +1  O ' f t  "o  O  +1  +1  rO 00  •st  in  cr-  vo  o  +1  00  r-;  r-  o ©  +1  CN ON  fl  .3.  «  H-<  "3  fl o SB  « a S o u  in  >  >  MPE-PF  «  O  SPE- PFV(  V  Q  P  Q  PH  PH  6 6 6 W w w Ui  /•—s  O  o  ;G-DSPE-PFV  o  MPE-PF  •  PH  •=tt  s  85  In the second set of experiments, the loss of aqueous contents o f P F V s  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 o f the PEG-lipids.  With respect to initial trapped sucrose, release o f sucrose  was observed from P F V s 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 P F V ) .  P F V s formulated with 10 mole% of  P E G - l i p i d 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.  U p o n the subsequent incubation of P F V s 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 o f 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 o f P E G - P E concentration in P F V s became apparent when sucrose release from P F V s containing different mole % P E G - P E was compared. A s 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 P F V s (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 P F V s , respectively) was lower than in the presence of sink alone.  86  CO  CD  + Ml  OX  a  cu  ay 7  * Q  CO  vesicl  PS (-  >  s  ro O ©  CN O ©  ipn  o  ©  +1  +1  +1  +1  in  00 ©  ©  ©  S  CO  S  t--  >  ©  -a  o  m  .g '3  >  JS  u  U fl  w I O w  XSii  -a  PH  T3  O (3 a o  o  * >? a Q  — 'o +1 1  o ©  +1  © ©  +1  Os 0\  o  ro  ro p ©  VO p ©  CN  o  o\  oo  o  ©  +1  ,—1  I.  a  o  .2  Q  u  +1  +1  i—|  ©  +1 vq  o p ©  +1  r-  vq  09 CO >  «n  o  a -**  S  O  CU 3  O"  cu  cu  *—<  ©  ©  +1 vq  i—<  CO  a  SPE- PFV(  ce  Q  Q  6  6 pa  W  CH CO  o  +1 00  +1  »—<  O in  MPE -PFV  o  o  ©  .2  c  Os  in .a  in  C  o  ro oo  CN  o  PH  ^—'  (%0  cu "a*  a  "o c c  +1  ro  ^°  CA  rt  iT 'a,  ©  SPE- PFV(  cn  O  MPE -PFV  n  CJ  U  CU  +i  a o  nitial Tr ap Vi  ~  &  m  S.D  rffei  m  a o w o a  Q  Q  6 w  6  PH  PJ PH =tfc  • =*fc  CJ  87  •g so. 2.0  X  ro -i—>  o-> + o E  1.5 1.0  0)  E O o 0.5 > Q.  ro  0.0 PEG-DMPE (5%) PEG-DMPE (10%) B  •g S 2.0 -*ro —•  o  -<—•  0)  o E ^  1.5 1.0  E O o 0.5 > tiro  0.0 PEG-DSPE (5%) PEG-DSPE (10%) Figure 3.7 In vitro release of aqueous contents from P F V s : Influence of concentration and acyl composition of P E G - P E The volume of [ C]-sucrose-labeled buffer remaining within P F V s (pl/pmole lipid) ± S.D 14  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  (DOPE:CHOL:POPS,  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 P F V s 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 P F V s 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 i s c u s s i o n O n 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) P F V s readily accumulate and retain mitoxantrone, and (c) the release of aqueous contents of P F V s occurs as a function of P E G - P E acyl composition in the presence of sink and negatively charged vesicles. When morphologies.  PFVs  are  prepared in  H B S , they  adopt  predominantly  non-spherical  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 non89  spherical ( M u i et al., 1993; M u i 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 P F V s is therefore not unique but has been suggested by previous studies of extruded vesicles. While many non-spherical vesicles can be the result o f the extrusion process, the lipid composition of P F V s 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 o f elongated vesicles (Edwards et a l , 1997).  In this regard, the presence o f 45  mole% cholesterol in P F V s , together with other non-bilayer-forming lipids, may promote non-spherical morphologies. Vesicles prepared in the absence o f 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 P F V s , such area imbalances could result from loss of P E G - l i p i d from the outer monolayer. A s noted in Chapter 1, P F V s 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 o f P F V s in the presence of "acceptor" liposomes (Holland et al., 1996b), and will readily occur  in vivo through binding o f PEG-lipids to  lipoproteins or cell membranes. Although "acceptor" systems were not present for the cryoT E M studies, it is possible that some PEG-lipid desorption occurs by the formation o f 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. A t mole ratios lower than 5 mole%, the polymer chains are able to occupy a larger surface area and sweep out a "mushroom-shaped" volume. A t 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 capacity  o f the  bilayer to  accommodate  2 0 0 0  - P E ranging from 0-10 mole%. When the  PEG-PE  is  exceeded,  however,  membrane  destabilization is triggered with the formation of P E G - l i p i d 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 o f D S P C : C H O L . They attributed these structures to the presence o f 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 o f the fact that P F V s contain predominantly unsaturated lipids ( D O P E 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 P F V s could be prepared with 10 mole% PEG-lipid but, in agreement with Edwards et al. (1997), open bilayer discs were also observed when P F V s were prepared with 20 mole % P E G - D S P E . A s 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 o f 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 c r y o - T E M 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.  A s a result o f the  influence o f 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 o f an  92  accumulated drug.  A s the current research demonstrates,  this  is not the  case and  mitoxantrone can be efficiently and stably loaded into P F V s employing a proton gradient. The physical state of anticancer drugs loaded within liposomes using this technique has been the subject o f 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  intravesicular core (Madden et al., 1990b).  might  undergo  precipitation  within  the  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 o f 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 c r y o - E M and X-ray diffraction to characterize such systems. This study demonstrates that doxorubicin forms bundles o f fibers in the aqueous core with no significant perturbation of the vesicle bilayer ( L i et al., 1998). In the current study, similar electron opaque structures were observed within mitoxantroneloaded P F V s .  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 o f 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 o f 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 o f contents was observed from P F V s 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 P F V s 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 P E G - l i p i d exchange has been reported (Silvius and Zuckermann, 1993). In addition, the greater loss o f vesicular contents of P F V s 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 o f acceptor vesicles may reflect exchange of a higher proportion of the initial PEG-lipids.  The observation that  incubation o f P F V s 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 o f anionic vesicles is mediated by a fusion event and hence is not seen in the presence o f an anion such as aspartic acid. It was recognized that the use of [ C]-sucrose would serve as a surrogate marker for 14  encapsulated drug but was used in order to demonstrate the mechanism o f action o f P F V s . 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 o f 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 [ C]-sucrose could occur, possibly by diffusion (less than 10 % 14  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 PEGDMPE 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  C H A P T E R 4: C H A R A C T E R I Z A T I O N OF T H E SYSTEMIC PROPERTIES OF PFVs: I N F L U E N C E OF P O L Y ( E T H Y L E N E G L Y C O L ) - P H O S P H A T I D Y L E T H A N O L A M I N E 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; L i u 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  biologically relevant time scales.  to phospholipid anchors  occurs on  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 P E G conjugated lipid.  The rate of loss of the cationic component of PFVs, D O D A C , 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 P F V 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 6and 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 A U C s 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  DSPC:CHOL:PEG-DSPE liposomes.  estimated  for  conventional  DSPC:CHOL  or  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 ( A U C ) / mean liposomal lipid A U C (AUC ). Values of 0.016, 0.037 and 0.068 were D  L  obtained for PFVs containing PEG-DMPE, PEG-DPPE and PEG-DSPE, respectively. should be noted that the  A U C D / A U C  l  It  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 P E G - D M P E 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 P F V 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-DSPEcontaining 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 A U C s 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 A U C s 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 A U C s for spleen, lung and kidney. A comparison of mean liposomal lipid A U C s 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 [ H]-CHE was used to determine P F V recovery and mitoxantrone levels were monitored with [ C]-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. 3  14  102  Time (hours)  Q  <u o  -fl  °.3  Vi  S  Vi Vt  bO  CU CU  o  o a  s  A.  6 -o cu  -O  cu cd _ cj ~ -fl fl H  bO  o cu  fl «  a VI  St  "Si 'a  ta PH  -B fl « « fl O )H  fl « o H->  a  s.o U  < CU  s  ~a  fl s «  CU  s« fl  mol/g/h)  bO  «« .2 fl. o fi -S rt .2 2 in OH 3 g CN X CN O 3. o -fl T 3 OH CU 2 § & OH  PH  O  60 fl  cu  CU  bO fi bo o  cd  T3 fl cu  rt o fl o £ t3 rt •1=! cd « ~ CO CU  cu  rt  a ocu o CO  o OH  -fl  CU  -a * co  CU  N  <-  rfl ta -O  PH  >r> cd  -3 -2 cu  cu  g  s S bo  a>"bb ^ a cu  R  ° rt o  s fl co m CN <N  ^  S g  .2"  cu -fi  co  o 2 3 fi W TIOH O H C^U CU P H I flo o pq o o  PH  C+H  H  OO  CN o CN  Q  u  ON  o o  o  ON VO  in r--  O  o  oo CN  o  vq  in  r-CN  vo in CN  ON  00 ro  u  <  fl  vo  OS  oo CN  ON C-O  co cd  VO  in  td i-l  a O  o OH  i  o  ro vo  VO  00  u  ro oo  rt  in  <  ON  in  CN in  <  o  CN  c> ro  00 m  CN ro ro  r--  m  ON ON  o  fl.  fl cu  rt "El  00  -s? fl fl  <  r-  oo  m  o  oo  00  cu  >  a CO  ta  o  00  fl. o  fl.  00  CN  Q  u  <  as ON  vo CN  CN  ro  vo  CN  O  m  in  oo  o o  O  o  O  u  ON  in  <  r--  vo  ON  as C3  VO  VO VO  ro  ON  rON ON  CN  vo in  cd CN  rt  00  CN  H-> cu  bO  VO  CN  CN  CN  VO  -fl U a o cc B  ta °  w OH 6 w  Ui  UH  <&  >  O  o  o u  X CO <D  •a  PH  * -g  o fl o sg .o£ s s cu a> c & OH fl X X o o u S ' s l  o  O  fl *  cu  o  o  u  co O 5 2  cd fl  o  CU  1  ^  O  ro CN O  A.  •5 o  u <u pfl S-  00  Tt  <  Kid  o  CD  nol/g/h)  H-»  S rt  ON  Ti-  VO  nol/ml/h)  Vj U <U  OH  -a ^  o  > cu  "3  o  s  o —  o  >  >  PH 1  PH I  PH I  o  w  p<  w  PH  Q 1  u Ui  Q *  Q *  a w PH  w PH Q I  a w PH  PH Ui  Q I  a PH  -+-» S3  -euoe  OH  cu kH  cd td  Q  104  CD >  60  CD 00  o Q  40  •g CL  "D CD  SO  20  CD  0 CD >  B  60  CD CO  o Q CD c o  40  i—  -4—" c cc X  |o  20  TJ CD O CD *S>  0 1 Hour  4 Hour  2 4 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. P F V s and mitoxantrone levels are shown at 1, 4, and 24 h for P F V s 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 [ H]-CHE 3  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([ H]) and lipid ([ C]-CHE) levels within 3  14  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 P E G - D M P E occurred within 1 hour after injection. Although, 70% of the injected P E G - D M P E had been eliminated from plasma after 1 hour, little exchange of PEGlipid (< 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 PEGDPPE 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 [ C]-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. 14  107  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 ( [ C ] - C H E ) . 14  As  shown in Figure 4.3B, the rate of clearance of P F V s from plasma was consistent with the data shown in Figure 4.1 A . P F V s containing the longer chain phospholipid P E G conjugate ( P E G D S P E ) remained in circulation longer as compared to the shorter chain P E G - P E s .  A t 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 DMPE-PFV  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 ( D S P C : C H O L ) ,  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 P F V s . 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 DMPE.  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 P E G 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[ HJ). 3  A conventional  liposome containing PEG-DSPE was labeled with PEG-DPPE[ H] and the time taken for the 3  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 P E G 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 B D F - 1 mice (4.5 pmole total lipid/mouse). A t various times, the mice were sacrificed and plasma isolated. Plasma P E G - P E ( [ H ] ) and lipid ( [ C ] - C H E ) levels were determined at 3  14  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 D P P E [ H ] . The data are expressed as molar ratio of injected dose to account for conventional 3  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[ H]) to liposomal lipid ( [ C ] - C H E ) was determined in plasma 3  14  at various time points after i.v. administration for P F V s (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 P E G - D P P E [ H ] is an open 3  diamond.  Results represent the means of P E G - P E [ H ] to [ C ] - C H E ratios obtained from 3  l 4  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.  Ill  4.2.5 Rate of Exchange of P E G - P E from The Carrier: Influence of Vesicle Composition O n the basis of the time-course of elimination of P E G - P E and that o f the carrier from the plasma (Figure 4.3 A , B and Figure 4.4A, B), it is possible to estimate the extent o f 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 P F V s and conventional liposomes, respectively. The rate of transfer o f P E G - P E from P F V s 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% o f 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% o f 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% o f 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 o f exchange  of PEG-conjugated lipids from conventional  liposomes were similar to those seen for P F V s . Within 2 hours, 45% o f 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 P F V s 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 P E G - D P P E [ H ] ) . 3  On  the basis of the results presented so far, two important conclusions can be made. First, that the process o f 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  component ( D O D A C )  was designed to determine whether the  cationic  of programmable fusogenic vesicles could also be lost from the  bilayer. Since P F V s 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 P F V s , D O D A C was traced using a [ C ] - D O D A C label 1 4  and [ H ] - C H E was used as the marker for P F V s . 3  A s 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 P F V s .  A t 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 P F V s , 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).  A t 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 (Figure 4.3A).  PFVs  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  PEG-DMPE  for  PEG-DMPE-PFV.  113  to 0.075 E -§. 0.060  B  0.00  10  0  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 [ C ] - D O D A C and [ H ] - C H E were determined at 0.5, 1, 2, 4, 8 and 24 hours by 1 4  3  dual label scintillation counting. Shown in panel (A) are plasma levels of D O D A C present in PEG-DMPE-PFVs  (•)  PEG-DPPE-PFVs  (•)  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 P F V s ([ C]/[ H]). Each point represents mean ± S.E, n=4. 14  3  114  Furthermore, the kinetics o f elimination of P F V s ([ H]-CHE) from plasma again were very 3  similar to those shown in Figure 4.3B, indicating that regardless o f the radiolabeled marker ( [ H ] - C H E or [ C ] - C H E ) used to trace the liposomal lipid, the results obtained were the same 3  14  (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 o f D O D A C from circulating P F V was influenced by the P E G - P E component of PFV.  Exchange of D O D A C occurred rapidly from P E G - D M P E - P F V and was slower from  PEG-DSPE-PFV.  A t 4 hours, 70% and 20% of initial D O D A C had exchanged out o f 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 P F V ) between those of P F V s 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 P F V s as a function o f the acyl chain length of the P E G - l i p i d component o f P F V s was lower than the corresponding ratio of P E G - P E to P F V s .  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 o f PEG-Ceramide and D O D A C over a 010% 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: T o 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 P F V s is based on exchange o f 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 o f 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 B D F - 1 mice injected with P F V s 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).  O n 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 o f plasma and is followed by a single peak containing both low density lipoproteins ( L D L ) and intermediate density lipoproteins (IDL). High density lipoproteins ( H D L ) 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 [ H ] 3  (Figure 4.7A).  was observed to co-elute with D S P C : C H O L  liposomes  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 P F V s containing P E G - D M P E .  A t 1 hour, P E G - D M P E [ H ] 3  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). time of 8 hours (Figure 4.7D), P E G - D M P E [ H ] 3  Similarly, at the later  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.  P F V s 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 I  further in section 4.3.  The elution profile of P E G - D P P E and P F V s 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). A t 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. O n the basis of the data presented in Figure 4.7E and F , the ratio of P E G - D P P E [ H ] to P F V [ C ] 3  was estimated, the ratios being 0.16 and 0.1 at 1 and 8 hours, respectively.  1 4  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 P F V s 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 [ C ] - C H E , 14  ( • ) and P E G - D M P E ( P E G - D M P E [ H ] , 3  profiles ratio)  of P E G - D M P E - P F V s , radiolabeled  symbols).  with  [ C]-CHE, 14  open symbols).  Panels C and D show fractionation  DOPE:CHOL:DODAC:PEG-DMPE, (•)  and  PEG-DMPE  Panels E and F show P E G - D P P E - P F V s , 14  D M P E [ H ] ) , (open symbols).  3  (open  and P E G - D M P E ( P E G -  In each fraction, the levels of liposomal lipid radiolabeled  with [ C ] - C H E and the corresponding tritiated P E G - P E 1 4  (PEG-DMPE[ H]),  DOPE:CHOL:DODAC:PEG-DPPE  (30:45:15:10, molar ratio) radiolabeled with [ C ] - C H E , (•) 3  (30:45:15:10, molar  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 P F V s , 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 P F V s 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 .  O n 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. L i p i d mixtures which in isolation form the non-lamellar hexagonal H phase, can be n  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 phase-forming lipids, such as phosphatidylethanolamine, play a major role in n  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 P E G - l i p i d , 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 P F V s 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 P F V s 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  1900  -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 o f 10 relative to the length o f the polymer, (varies between 6 to 8 nm for P E G  1 9 0 0  ) the distribution o f 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 o f P E G - l i p i d would be  expected to occur from the outer leaflet of the P F V s triggering the destabilization o f the vesicle.  In this regard, P E G - P E s possessing shorter acyl chains exchange out o f 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 M o r i et al. (1998)  respectively.  The importance of the nature of the anchor o f 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 o f which are relatively longer chain anchors ( H S P E , 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 o f PEG-lipids.  Silvius and Zuckermann (1993) have reported an approximately 6.3-fold  decrease in the rate o f intervesicle transfer with the extension o f 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 o f PEG-lipids have been described.  First, there is an increase in translational and rotational entropy associated with  the exchange o f 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 o f the free polymer. Silvius and Zuckermann (1993) have pointed out the contribution o f an additional factor: interactions of the anchored polymer with other membrane components. Further, the exchange o f 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 P E G - P E s from conventional (stable) vesicles.  While it  has been reported by Connor et al. (1986) that the inclusion o f cholesterol in pH-sensitive fusogenic systems causes these vesicles to become acid-insensitive, it is shown here that P F V s are stable systemically despite the inclusion o f cholesterol. The exchange of D O D A C from P F V s is also influenced by the hydrophobicity o f the PEG-anchor and may occur instantaneously as in the case o f P E G - D M P E - P F V s . The role of P E G - P E s 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 P F V s is the initiation o f exchange o f P E G - l i p i d . 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 P F V s , 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 o f contact with soluble proteins. Second, on the basis o f the F P L C results (Figure 4.7B and C) showing the presence o f P E G D M P E in fractions corresponding to lipoproteins and other plasma proteins, the possibility of overestimating the levels o f 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 P F V s 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 P F V s 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 P E G - l i p i d 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 o f PEG-lipids together with H D L implies that following exchange o f P E G - l i p i d from the vesicle surface,  125  PEG-lipids bind to lipoproteins and/or plasma proteins. This is especially true in the case of P E G - D M P E which co-elutes with L D L , H D L 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 P E G D M P E . Therefore, the plasma elimination data for P F V containing P E G - D M P E 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 PEGPE), 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[ H] being similar to PEG-DPPE-containing 3  vesicles (Figure 4.5B). These results support the occurrence of exchange of PEG-PE from P F V 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 P E G 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 P E G regulates the rate of exchange of PEG-lipids from the surface of a vesicle, whether P F V or a conventional, sterically stabilized vesicle. The partitioning of the cationic component of PFVs from the P F V surface is also influenced by the chemical composition of the PEG-lipid anchor. The desorption of D O D A C 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 T H E A N T I C A N C E R PROPERTIES OF M I T O X A N T R O N E E N C A P S U L A T E D 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 P F V accumulation in the liver. Therapy of liverlocalized 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 P F V s 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 P F V s 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.  A s 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 ( L i m 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 druginduced reductions in liposome accumulation in liver will affect treatment outcomes ( L i m , 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.  in vitro does not appear to be affected by temperature, lipid  Retention  composition, presence or absence of a p H gradient, or the presence of serum proteins ( L i m 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,  PEG-DSPE)  is  more  effective  in delaying  tumor progression  than  mitoxantrone encapsulated in P F V s 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 P F V s .  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 5.2.1.1 Mitoxantrone and Lipid Levels in the Plasma  Mitoxantrone and lipid levels were measured in the plasma of S C I D / R A G - 2  mice  bearing established L S I 8 0 solid tumors over a 48 hour time period following a single i.v. injection o f free mitoxantrone (22.5 umole/kg), mitoxantrone encapsulated in P E G - D M P E P F V s 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 o f mitoxantrone obtained after injection o f 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 mitoxantroneloaded 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 P F V s following i.v. administration in tumor-bearing mice is illustrated in Figure 5.IB.  The PEG-DSPE-containing P F V s 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).  5.2.1.2 Mitoxantrone and Lipid Accumulation in LSI 80 Solid Tumors The extent of drug and liposome accumulation in solid L S I 8 0 tumors following i.v. administration  was  studied  in S C I D / R A G - 2  mice  bearing established  LSI80  tumor  xenografts and the results are shown in Figure 5.2 (panels A and B , respectively). maximum concentration ( C  max  The  ) 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 P F V s , 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 ( P F V ) 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 P F V s . 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 ) and the mean area under the drug L  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 P F V s or D S P C : C H O L liposomes. Second, the mean A U C drug is administered in conventional liposomes.  D  in the tumors is greatest when the  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.  A s 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 S C I D / R A G - 2 mice were injected bilaterally with 1 x 10 L S I 8 0 cells subcutaneously. 6  the tumors were ~0.3 encapsulated  c m , mice were injected with free drug (•) 3  within P E G - D M P E - P F V s  (22.5pmol/kg) via the lateral tail vein.  (•)  or P E G - D S P E - P F V s  When  or mitoxantrone  ( A ) at  10  mg/kg  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 [ C ] 14  radiolabelled drug as a tracer, Panel B : The liposomal lipid marker ( [ H ] - C H E ) was used to 3  measure the rate of elimination of P F V s from plasma. Mean ± S.E, n=6.  135  o  r  0  i  10  i  i  i  i  20  30  40  50  Time (hours) Figure 5.2 Tumor accumulation of mitoxantrone and liposomal lipid in the human LS180 solid tumor xenograft S C I D / R A G - 2 mice were injected bilaterally with 1 x 10 L S I 8 0 cells subcutaneously. 6  When  the tumors reached approximately 0.3 c m , mice were injected with free drug ( • ) 3  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  (•)  or  at 10  mg/kg (22.5pmol/kg) via the lateral tail vein. Mice were terminated using C 0 asphyxiation 2  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 S C I D / R A G - 2 mice bearing established L S I 8 0 tumors. AUC  TUMOR  AUC  1  D  TUMOR  2  (pmole lipid /g tumor) hour  (pmole drug /g tumor) hour  16.7  2.2  PEG-DMPE-PFV  3.8  0.63  PEG-DSPE-PFV  16.5  1.25  DSPC:CHOL  1  L  A U C was determined by trapezoidal integration of the mean of the lipid concentrationL  time. 2  AUC  D  was determined by trapezoidal integration of the mean of the drug concentration-  time.  5.2.2 Therapeutic Activity of Mitoxantrone-Loaded PFVs 5.2.2.1 Efficacy of Mitoxantrone-loaded PFVs against LSI 80 human colon carcinoma model It was important to assess how the efficacy of mitoxantrone encapsulated within P F V s 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 P F V s or D S P C : C H O L vesicles were examined against a human colon carcinoma, L S I 8 0 , grown as a xenograft in immune deficient S C I D / R A G - 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 S C I D / R A G - 2 mice were injected bilaterally with 1 x 10 L S I 8 0 cells subcutaneously. 6  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 P F V s ( 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, L S I 8 0 cells were  inoculated subcutaneously  in the  flank  of  S C I D / R A G - 2 animals and tumor growth measured following i.v. administration o f three injections o f 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 P F V s was evaluated against non-established tumors at this dose. Tumor volumes were calculated using the formula of Tomayko and Reynolds (1989) following measurements o f 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 o f mice.  This  normalization accounts for animals that had to be killed as a consequence o f tumor ulceration (see Chapter 2) and is also the reason for the lack o f error bars in Figure 5.3. this analysis are shown in Figure 5.3.  The results o f  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. B y day 17, tumor volumes increased at a rate similar to tumors in untreated animals, suggesting that treatment o f 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 P E G - D M P E - P F V formulation would be less active than the PEG-DSPE-PFV formulation, the value of the P F V technology is highlighted when results between animals treated with mitoxantrone loaded in the DSPC:CHOL and the PEG-DSPE-PFVs are compared.  5.2.2.2 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 PEGDSPE-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 o f drug encapsulated in  D S P C : C H O L liposomes was less than that observed for free drug (median survival time o f 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 o f 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 o f mitoxantrone encapsulated in conventional liposomes ( D S P C : C H O L ) was also evaluated.  A s 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 o f the reinoculated 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 B D F - 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  o CO  o o  CD  <  bB cd  "S  ON OO  ^1-  Q  Q  CD  T3 CD  •s CD H  O  (-1 CD  T3 CD td <D  CO  ro  ro ro IT)  ON  o  U  oo vo  oo in  Q  Q  u  o co CD  a to Q  >  CO  |3  > to  a  CO Q  CD  s £H CD  X3  PH  'O  &  CD  ii « jo •  a  |S  c  CD  c o  O  a  CO  CD  bO bD  o  valu  CO <D  CQ 4>  a>  o  I/O  x  cn cd  J3  u  +H CO  %  • i-H  VO CO +-»  o  CD CO cd CD  *c  s  o  o  bO  1—1  (N 1—I  o I  <-l  O  a o  lo  CD CD  i «->  >  fl  I—1  fe  > fe  o  W  W  e ILS  c  CO  cd -»-»  o o  PH . „  Q  PH I PH  Q 1  O  w  PH  PH  PH  Q 6  PH  hH^  CD  PH to  en CM  CO CO  CD CO  «  cd^ -4-»  73  o  T3  bo  C  cd  b  licable.  00  he ani mal  00  hH  up:  ON  ere  ro ro  S  :e experi  CD  ved n  CO  ,w  a  ro ro  o  es H  o  CD  M-H  w  3  CD  M  in Li  CD  u  _c  c3  o  >r,  CD CD o cd  PO  -aa  CD  were  ©  was i  CD on  a  ninei  TS  o >-  00  CM CH  VO  p c O  cfcl cd cd  CD  Pool  a  5.3 Discussion The results presented illustrate the therapeutic benefits that can arise when using P F V s 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 ( L i m 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 P F V destabilization. Thus, in contrast to conventional liposomes, transformations  in P F V 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 P E G 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 P F V s containing this short acyl chain lipid, this results in carrier accumulation in M P S organs such as the liver and spleen. A s 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.  A s with the solid tumor, provided that the P F V s 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 o f P F V s , 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 o f the carrier at an even slower rate than P E G - D S P E and, possibly, by incorporation o f targeting elements. Second, it is not possible at present to identify whether mitoxantrone delivered to the tumor site by P F V s is delivered specifically into neoplastic cells. A s illustrated in Chapter 1 (Figure 1.10), destabilization o f P F V s that have accumulated within tumors could trigger vesicle fusion with adjacent cells, thereby directly introducing encapsulated mitoxantrone into the cell cytoplasm. Alternatively, destabilized P F V s 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 P F V s and the therapeutic properties o f the encapsulated drug are improved when compared to conventional liposome formulations. The loss of PEG-modified lipids, the presence o f 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 o f transformation of P F V s from circulation-stable vesicles into destabilized ones, capable of releasing their contents.  Recent studies have demonstrated fusion between P F V s and  eukaryotic cells in culture and shown that this can allow introduction o f 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: D I S C U S S I O N 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 o f 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 P F V s with respect to morphology, ability to encapsulate mitoxantrone and further to demonstrate controlled destabilization. properties o f P F V s were examined  Second, the  in vivo as a function o f the P E G - l i p i d component. The  final study examined the efficacy of mitoxantrone-loaded P F V s against animal models o f cancer. In characterizing programmable fusogenic vesicles  in vitro, I show that P F V s can be  formulated and that P E G conjugates are essential for the structural integrity o f P F V s .  This  was anticipated because there is evidence to show that PEG-lipids stabilize non-bilayerforming lipids into a bilayer conformation (Cullis et al., 1991; Holland et al., 1996a). Given the composition o f P F V s , the observation that mitoxantrone could be stably encapsulated into P F V s using a transmembrane p H gradient was somewhat more surprising. Central to testing the hypothesis was determining that the mechanism by which P F V s acted was indeed as proposed in section 1.8.1.2. Towards this end it was established in vitro that PEG-lipids play a critical role in determining the characteristics of release o f aqueous contents o f P F V (Chapter 3).  This was a significant finding from the standpoint of enhancing the  bioavailability o f 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 o f P F V .  The  phenomenon o f exchange of PEG-lipids is shown to be common to vesicles of different lipid composition ( P F V and conventional liposomes; Chapter 4).  However, P F V s have an  inherent advantage over conventional liposomes in being transiently stable and composed of primarily fusogenic  lipids.  The demonstration that controlled destabilization o f 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 o f contents (Chapter 5).  This is a meaningful advance in the context o f  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 o f this work is that P F V technology relies on component lipids and membranespecific 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 o f intravenously injected  carriers in  intracellular delivery.  I here  characterized a multi-functional,  transformable liposome on the basis of the exchangeability o f 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 o f P E G - l i p i d component (Chapter 4 and 5). Furthermore, the contrast between the therapeutic activity of P F V s 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  DOPE:DODAC:PEG-Ceramide-containing  Mori  et  liposomes  al.  (1998)  with  have  shown  erythrocyte  fusion  ghosts.  of  Taken  together with the information gained from the present studies of various steps in the mechanism of action of P F V s , it may be possible to demonstrate fusion of P F V s 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 plasmids.  such as proteins, oligonucleotides  and  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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