UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Lipid polymorphism and intracellular delivery Hafez, Ismail Mahmoud 2000

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

Item Metadata

Download

Media
831-ubc_2000-565556.pdf [ 9.94MB ]
Metadata
JSON: 831-1.0089758.json
JSON-LD: 831-1.0089758-ld.json
RDF/XML (Pretty): 831-1.0089758-rdf.xml
RDF/JSON: 831-1.0089758-rdf.json
Turtle: 831-1.0089758-turtle.txt
N-Triples: 831-1.0089758-rdf-ntriples.txt
Original Record: 831-1.0089758-source.json
Full Text
831-1.0089758-fulltext.txt
Citation
831-1.0089758.ris

Full Text

LIPID POLYMORPHISM AND INTRACELLULAR DELIVERY by ISMAIL MAHMOUD HAFEZ B.Sc, McMaster University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry and Molecular Biology) We accepfttTtesthesis as^c^nT^iw§ /fo the recpred standard THE UNIVERSITY OF BRITISH COLUMBIA July 2000 © Ismail Mahmoud Hafez, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The role of lipid polymorphism and lipid-based systems used for intracellular delivery has been examined. First, the self-assembly properties of the anionic lipid cholesteryl hemisuccinate (CHEMS) were studied as a function of pH. CHEMS is an acidic cholesterol ester that self-assembles into bilayers in alkaline and neutral aqueous media and is commonly employed in mixtures with the nonbilayer lipid, dioleoylphosphatidylethanolamine (DOPE) to prepare pH-sensitive fusogenic liposomes. pH-sensitive liposomes can be used for the intracellular delivery of macromolecules through the endocytic pathway. It is shown that CHEMS itself adopts a nonbilayer phase at low pH. This is evident from the fusogenic properties of large unilamellar vesicles (LUVs) composed of CHEMS and direct visualization employing freeze-fracture electron microscopy. It is suggested that the pH-dependent phase preferences of CHEMS contributes to the pH-sensitive fusion of LUVs composed of mixtures of CHEMS and DOPE. Next, the pH-dependent fusion properties of LUVs composed of binary mixtures of anionic and cationic lipids was investigated. It was found that stable LUVs can be prepared from the ionizable anionic lipid CHEMS and the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pHf) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH f values that ranged from pH 4.0 to 6.7, respectively. This behaviour is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behaviour was observed for LUVs composed of the ionizable cationic lipid, 3cx-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and 3 1 P NMR evidence indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. Finally, the mechanism of nucleic acid transfection mediated by cationic lipids (lipofection) is elucidated. Cationic lipids are widely used as non-viral gene transfer agents, but the mechanism by which cationic liposomes promote the intracellular delivery of membrane impermeable macromolecules such as plasmid DNA or antisense oligonucleotides is not well understood. In this work it is demonstrated that cationic lipids can destabilize cell membranes by promoting the formation of nonbilayer lipid structures. Using 3 1 P NMR, it is shown that addition of cationic lipids to bilayer-adopting anionic phospholipids results in the formation of the nonbilayer inverted hexagonal (Hn) phase. Further, the presence of "helper" lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipid-mediated transfection, also facilitates the formation of the Hu phase. It is suggested that the ability of cationic lipids to promote nonbilayer structure in combination with anionic phospholipids leads to disruption of the endosomal membrane following uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating cytoplasmic release of the plasmid or oligonucleotide. iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS v LIST OF TABLES viii LIST OF FIGURES ix ABBREVIATIONS xi ACKNOWLEDGEMENTS xiii CHAPTER 1 - INTRODUCTION 1 A Lipid World 1 Lipids in Biomembranes 3 Liposomes as Model Membranes 5 LIPID POLYMORPHISM 9 Lipids Adopt a Variety of Structures 9 Detection of Nonbilayer Structure 11 Theory of Lipid Polymorphism 12 Nonbilayer Lipids in Nature 14 LIPID POLYMORPHISM AND MEMBRANE FUSION 16 Proposed Fusion Intermediates 16 Role of Lipids in Biological Membrane Fusion 18 pH-SENSITIVE LIPOSOMES 19 pH-Sensitive Formulations 20 Role of DOPE in pH-Sensitive Liposomes 22 Intracellular Delivery 23 STUDIES ON THE ROLE OF LIPID POLYMORPHISM IN INTRACELLULAR DELIVERY 25 Role of an Anionic Component of pH-Sensitive Liposomes 25 Tunable pH-Sensitive Liposomes 27 Cationic Lipid-Mediated Intracellular Delivery 28 v CHAPTER 2 - POLYMORPHIC PHASE BEHAVIOUR OF CHOLESTERYL HEMISUCCINATE 31 MATERIALS AND METHODS 31 Lipids and Chemicals 31 Large Unilamellar Vesicle (LUV) Preparation 33 pK Determination... 34 Lipid Mixing Assay 34 Effect of Lysophosphatidylcholine on the Stability of CHEMS Bilayers 35 Freeze-Fracture Electron Microscopy 37 RESULTS 37 pK of CHEMS 37 Fusion of CHEMS LUVs 38 Lysophosphatidylcholine Inhibits Acid-Induced Fusion of CHEMS LUVs 38 Freeze-Fracture Electron Microscopy Reveals the Phase Behaviour of CHEMS at Acidic pH 41 DISCUSSION 44 CHAPTER 3 - TUNABLE pH-SENSITIVE LIPOSOMES COMPOSED OF MIXTURES OF CATIONIC AND ANIONIC LIPID 46 MATERIALS AND METHODS 47 Lipids and Chemicals 47 Synthesis of DODAC 47 Preparation of large unilamellar vesicles (LUVs) 48 Lipid mixing fusion assays 49 Freeze-Fracture Electron Microscopy 50 3 1 P Nuclear Magnetic Resonance Spectroscopy 50 RESULTS 51 DODAC/CHEMS Lipid Mixtures can Form Bilayer Vesicles on Hydration 51 DODAC/CHEMS LUVs Undergo pH-Sensitive Fusion which can be Modulated by Adjusting the DODAC/CHEMS Ratio 53 pH-Sensitive Fusion that is Modulated by the Cationic-to-Anionic Lipid Ratio Can also be Observed for LUVs Containing an lonizable Cationic Lipid 55 Fusion of LUVs Composed of Cationic and Anionic Lipid is Accompanied by the Appearance of "Inverted" Nonbilayer Lipid Structures 59 vi DISCUSSION 65 CHAPTER 4 - MECHANISM OF ACTION OF CATIONIC LIPIDS 70 MATERIALS AND METHODS 71 Lipids 71 3 1 P nuclear magnetic resonance spectroscopy 71 RESULTS 72 Mixtures of Cationic Lipids and Anionic Phospholipids Adopt a Nonbilayer Phase 72 Effect of Helper Lipids 76 Influence of Cationic Lipid Structure on the Polymorphism of Mixtures of Cationic and Anionic Lipids 79 Influence of Anionic Lipid Structure on the Polymorphism of Mixtures of Cationic and Anionic Lipids 84 DISCUSSION 88 CHAPTER 5 - SUMMARY AND FUTURE DIRECTIONS 92 BIBLIOGRAPHY 95 vii L I S T O F T A B L E S T A B L E 1.1. pH-Sensitive Liposome Formulations 21 T A B L E 1.2 Intracellular Delivery Applications of pH-Sensitive Liposomes 24 T A B L E 4.1 Phase Transition Temperatures of Charge Neutral Mixtures of Cationic and Anionic Lipids 86 viii L I S T O F F I G U R E S F I G U R E 1.1 Structure of nananoic acid 2 F I G U R E 1.2 Structures of some naturally occurring lipids 6 F I G U R E 1.3 Schematic representation and representative freeze-fracture micrographs of liposome systems 8 F I G U R E 1 .4 Schematic representation of lipids organized into a bilayer, inverted hexagonal (HM), and micellar phases 1 0 F I G U R E 1.5 Molecular geometry lipids and predicted self-assembled morphological structures 1 3 F I G U R E 1.6 Proposed intermediates of membrane fusion 1 7 F I G U R E 2.1 Structure and apparent pKof cholesteryl hemisuccinate 3 2 F I G U R E 2.2 Acid-induced fusion of CHEMS LUVs 3 9 F I G U R E 2.3 LPC inhibits acid-induced fusion of CHEMS LUVs 4 0 F I G U R E 2 . 4 CHEMS adopts a lamellar phase on hydration at alkaline pH 4 2 F I G U R E 2.5 Acidified CHEMS bilayers adopt the hexagonal phase at acidic pH 4 3 F I G U R E 3.1 Structural characteristics of aqueous dispersions of DODAC/CHEMS 5 2 F I G U R E 3 .2 pH-dependent fusion properties of DODAC/CHEMS LUVs containing increasing amounts of DODAC 5 4 F I G U R E 3 .3 Correlation between membrane fusion and surface charge neutralization of DODAC/CHEMS LUVs 5 6 F I G U R E 3 . 4 pH-dependent fusion properties of DC-Chol/DOPA LUVs containing increasing amounts of DC-Choi 5 8 F I G U R E 3 .5 DODAC/CHEMS LUVs adopt nonbilayer phases at low pH values 6 1 F I G U R E 3.6 Equimolar mixtures of DODAC/CHEMS adopt nonbilayer structures following hydration at neutral pH values 6 3 IX FIGURE 3.7 Influence of pH on the polymorphic phase properties of aqueous dispersions of DC-ChoI/DOPA as detected by 3 1 P NMR 64 FIGURE 4.1 Equimolar mixtures of cationic lipids and anionic phospholipids exhibit nonbilayer phase preferences 75 FIGURE 4 . 2 Neutral co-lipids DOPC, DOPE and cholesterol modulate the nonbilayer phase behavior of mixtures of cationic and anionic lipids 78 FIGURE 4 . 3 Effect of cationic lipid acyl chain saturation on lipid phase behaviour in mixtures with DOPS 81 FIGURE 4 . 4 Effect of cationic lipid headgroup structure on lipid phase behaviour in mixtures with DOPS 83 FIGURE 4 .5 Temperature-dependent phase transition characteristics of an equimolar mixture of lysobisphosphatidic acid and DODAC 87 FIGURE 4.6 A model for the mechanism of action of cationic lipids 90 x Lipids Choi CHEMS CL DC-Choi DODAC DO PA DOPC DOPE DOPS DOTAP DOTMA DSDAC LPC LBPA NBD-PE OSDAC PE PI POPC POPG ABBREVIATIONS cholesterol cholesteryl hemisuccinate cardiolipin 3a-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride N,N-dioleoyl-N,N-dimethylammonium chloride 1,2-dioleoyl-sn-glycero-3-phosphate 1,2-dioleoyI-sn-glycero-3-phosphocholine 1,2-dioleoyl-SA7-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-phosphoserine 1,2-dioleoyloxy-3-(trimethylammonio) propane N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride N,N-stearyl-N,N-dimethylammonium chloride 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine lysobisphosphatidic acid 1,2-dioleoyl-sA7-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) N-oleoyl,N-stearyl-N,N-dimethylammonium chloride phosphatidylethanolamine phosphatidylinositol 1-palmitoyI-2-oleoyl-SA7-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-phosphoglycerol XI Rh-PE 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-lissamine rhodamine b sulfonyl SM sphingomyelin Triton X-100 t-octylphenoxypolyethoxyethanol Miscellaneous DTA Diphtheria toxin fragment A HM inverted hexagonal phase HEPES N-[2-hydroxyethyl]piprezine-N'-[2-ethanesulphonic acid] I FN interferon La-Hn lamellar to inverted hexagonal phase transition LUV large unilamellar vesicle MES 2-[N-morpholino]ethanesulfonic acid MLV multilamellar vesicle NMR nuclear magnetic resonance pH n pH at which the LUV exhibits a surface charge of zero pHf pH at which membrane fusion is half-maximal PolylC polyinosinic acid-polycytidylic acid SUV small unilamellar vesicle TNS 2-p-toluidinyl naphthalene 6-sulphonic acid t-SNARE target SNARE v-SNARE vesicle SNARE xii Acknowledgements I would like to thank my parents, Ehsan and Mahmoud for their generosity. You have supported me in so many ways throughout my studies. You guys are truly the outstanding parents. Special shout-out to Melanie Hafez, thanks for being my best pal, and putting up with me. To Pieter, I have to say that you're lab is a great place to work, and I am sure that there is not a comparable work environment anywhere. I feel lucky to have had the opportunity to work with you. Outrageous parties and I want to be invited to all of them! Kim. I buzzing off, man. What a fun place you make lab. Dave. I really think that you should resurrect your Sabbath albums. You guys make the lab a special place to work. Everyday is a day filled with adventure and lore. The humour-level routinely approaches Ti saturation. Norbert, thanks for your mentoring in the world of liposomes, ealcein, peptides, pH, and making buffers. I will never make a buffer again without using a volumetric flask. Elizabeth*, (see below). Lome, thanks for competing in the PRC Messiest Desk Contest with me. It made my mess just blend right in. Angel, you are like so nice. Delightful*, likeable*, sweet-tempered and kind-hearted*. Peter, thanks for everything, and I want you to invite me for fondue on your sailboat one day. Lenore, great outfits. I will never pay full price again. John, you've taught me a lot about strength and courage. First rule of FightClub... Ammen, see...plenty of evidence why UBC is a better place to work! Tabitha, I will always remember to eat a Zone-style meal of fish and rice for lunch. Thanks y'all. ism xiii Dedicated to my beloved sister Asmahan, Who continues to look out for me. x i v CHAPTER 1 INTRODUCTION A Lipid World. Living cells are surrounded by a bimolecular arrangement of lipids, first demonstrated by Gorter and Grendel, 1925. It has since been suggested that simple bilayer-bound compartments were prerequisite structures for the origin of early cellular life (Deamer & Oro, 1980; reviewed in Luisi et al., 1999). Organic acids capable of forming lipid vesicles on dispersion in water have been extracted from the Murchison carbonaceous chondrite (Deamer, 1985) indicating that a source of amphipathic components capable of forming vesicles was likely present on Earth 4,500 million years ago. One chemical constituent extracted from the Murchison meteorite which is capable of forming vesicles is nonanoic acid, a carboxylic acid with a nine carbon acyl chain (Figure 1.1). Under certain conditions of pH and temperature, single-chain fatty acids such as nonanoic acid self-assemble into closed bilayers capable of entrapment of aqueous solutes (Hargreaves & Deamer, 1978; Deamer & Pashley, 1989). It has therefore been hypothesized that primitive liposomes provided the precellular chambers for the compartmentalization of prebiotic materials necessary for the genesis of entities such as catalytic RNA. 1 FIGURE 1.1. Chemical structure of nonanoic acid. This lipid was extracted from the Murchison carbonaceous chondrite and is capable of self-assembly into vesicles upon dispersion in aqueous media (Deamer, 1985). 2 Lipids in Biomembranes. The lipid membrane def ines the boundary of a living cel l , a l lows the regulated interaction with its external environment, def ines specia l ized organel les, and acts as a scaffold for the anchoring of proteins and lipid signal ing molecules. The 'Fluid Mosa i c Mode l ' put forth by Singer and Nicholson, 1972 depicts the organizat ion of biological cell membranes as a two-dimensional fluid containing peripheral and integral membrane proteins. S ince their descript ion, lipid domains have a lso been recognized to exist in cell membranes. Spec ia l ized lipid domains or "lipid rafts" may mediate the clustering of supramolecular protein-lipid complexes capab le of execut ing speci f ic cellular functions (Simons & Ikonen, 1997; J a c o b s o n & Dietrich, 1999). The organizat ion of lipid molecules in most biological cell membranes is that of a bimolecular layer of amphipathic molecules, or a bi layer (Gorter & Grende l , 1925). The lipid bi layer of biological membranes also e n c o m p a s s e s an extra level of complexi ty in its relatively s imple arrangement. Biological membranes are asymmetr ic in composi t ion. For example, the inner lipid monolayer of the red blood cell is composed of phosphatidylserine (PS) and phosphat idylethanolamine (PE) , while the outer lipid monolayer harbours most of the phosphat idylchol ine (PC) and sphingomyel in (SM). A similar distribution of lipids is found in most eukaryot ic cell p lasma membranes . Membrane asymmetry in b iomembranes is maintained by lipid transport proteins or f l ippases which function to sequester aminol ipids such as P E and P S to the cytoplasmic membrane leaflet monolayer (reviewed in Da leke & 3 Lyles, 2000). One interesting application of the nearly absolute sequestration of PS to the cytoplasmic leaflet has been exploited to determine the onset of programmed cell death, or apoptosis. Cells undergoing apoptosis rapidly lose their plasma membrane asymmetry, and as a result begin to expose PS on the exoplasmic membrane leaflet. Externalized PS can be detected using annexin V probes which bind PS with high affinity in the presence of C a 2 + (Koopman et al., 1994). An extreme diversity of lipids exists throughout the domains of life. For instance, the lipid membranes of prokaryotic archaebacteria are unique to those found in eubacteria and eukaryotic cells. While in eubacteria and eukaryotic cell membranes, lipids are linked to headgroups predominantly via ester bonds, archaebacteria lipids are instead bonded via ether linkages. Furthermore, some of these lipids are characterized as being bipolar, tetraether lipids which have two headgroups and two very long C40 hydrocarbon chains (De Rosa, 1996). These lipids which defy the bimolecular architecture of a lipid "bilayer" form a membrane one molecule thick in which the bipolar tetraether lipids span the membrane. These modifications in lipid structure endow archaebacteria with the ability to endure habitats which generally exhibit extremes in both temperature and pH. Eubacteria and eukaryotic cell membranes are primarily composed of glycerolipids. A key distinction between the lipid composition of membranes in these domains of life is that membranes of eubacteria do not generally contain sterols, nor do eubacteria synthesize phosphatidylcholine (PC). For example, the membrane of the 4 gram-negative bacteria E. coli is characterized by having a very high proportion of phosphatidylethanolamine (PE) with phosphatidylglycerol (PG), cardiolipin (CL) and lipopolysaccharides (LPS) as minor components. The glycerophospholipids which are most abundant in eukaryotic cell membranes are zwitterionic lipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), and anionic phospholipids such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin (CL) and lysobisphosphatidic acid (LBPA). Cholesterol (Choi) is also a major constituent of animal cell membranes and comprises approximately 50% of the total plasma membrane lipids. The structures of these lipids is given in Figure 1.2. Liposomes as Model Membranes. Artificial lipid vesicles prepared from dispersions of phospholipids in aqueous media were first described by Bangham and Home, 1964. Since their pivotal discovery, research into membrane structure and function has been intense. Liposomes are widely accepted as models of biological cell membranes and have important biomedical applications as drug carriers and imaging agents. 5 Glycerophospholipids R,0 o-pc-x O" Acyl Chain R11R2 Palmitoyl C 1 5 H 3 1 C Stearoyl C 1 7 H 3 5 C Oleoyl C 8 » 1 7 C 7 H 1 4 C o II R10 I xo OH Lysophospholipid R,0 O - 0 _p-0 O Phospholipids Species X Phosphatidic Acid (PA) OH Phosphatidylcholine (PC) o^~ ^/N(CH 3)3 Phosphatidylethanolamine (PE) O^ ^^ N H 3 + O II Phosphatidylserine (PS) O" vs NH3+ Phosphatidylglycerol (PG) O" ^ Y ^ O H OH OH O II o OR, R,0 Cardiolipin (CL) R20 OH R,(K OH jf OH Lysobisphosphatidic acid (LBPA) Cholesterol (Choi) Sphingomyelin (SM) FIGURE 1.2. Chemical structures of some of the naturally occurring lipids. 6 Liposomes spontaneously form on hydration of amphipathic molecules such as phospholipids. Vesicles have also been prepared from a variety of amphipathic molecules including fatty acids (Hargreaves & Deamer, 1978), surfactants (Kaler et al., 1990) and even diblock copolymers (Discher etal., 1999). Artificial lipid vesicles are generally designated by size. Giant liposomes exhibit diameters in excess of several i^ m while large liposomes exhibit sub-micron diameters of 500-100 nm, and small liposomes exhibit diameters of 25-50 nm. Liposomes can also be designated by the lamellarity of the system as being unilamellar, oligolamellar or multilamellar. Freeze-fracture micrographs for some commonly described lipid vesicle systems is presented in Figure 1.3. Small and large unilamellar lipid vesicles (SUVs and LUVs) can be prepared by a variety of techniques including sonication (Huang, 1969), injection of lipids dissolved in ethanol or ether into aqueous buffer (Deamer, 1978; Batzri et al., 1973), detergent removal (Brunner et al., 1976), and reverse phase evaporation (Szoka & Papahadjopoulos, 1978). Perhaps the most convenient method to produce unilamellar vesicles is by the extrusion technique. The extrusion technique offers a solvent and detergent-free method for the preparation of LUVs and SUVs. Multilamellar vesicles (MLVs) are typically formed on mechanical hydration of dry lipid. In order to achieve solute equilibration across multiple lamellae, freeze-thaw steps are required (Mayer et al., 1985). In the extrusion procedure, frozen and thawed MLV systems are forced by high pressure through polycarbonate 7 FIGURE 1.3. Schematic representation and representative freeze-fracture micrographs of liposome systems. 8 membranes with defined pore-size allowing the rapid generation of vesicles with defined diameter (Olson et al., 1979; Mayer et al., 1986). Lipid vesicles prepared by this technique are generally useful as models of biological membranes or as vehicles for drug delivery. However, one disadvantage of these types of vesicles is that due to their sub-micron diameters they are inaccessible to detailed observation by optical microscopy. Giant unilamellar vesicles (GUVs), however, prepared by gentle hydration of lipid films are amenable to interrogation using optical fluorescence microscopy techniques (Akashi et al., 1996). GUVs can be prepared to be on the order of 25-100 u.m in diameter. GUVs have been studied using confocal microscopy to demonstrate the co-existence of separated lipid phase domains in mixed lipid systems (Korlach et al., 1999). GUVs can also be used to probe the mechanical properties of lipid bilayers (Needham etal., 1988). LIP ID P O L Y M O R P H I S M Lipids Adopt a Variety of Structures. Upon dispersion in water, amphipathic molecules can self-assemble into a variety of different structures. Many reviews have been written on the polymorphic phase behaviour of lipids (Cullis & de Kruijff, 1979; Gruner, 1985; Gruner et al., 1985; Lindblom & Rilfors, 1992; Epand, 1998). Lipids such as phosphatidylcholine (PC) adopt bilayer phases upon hydration (Figure 1.4), whereas fatty acids and lysolipids 9 FIGURE 1.4. Schematic representation of lipids organized into a bilayer, inverted hexagonal (Hn), and micellar phases. Corresponding 3 1 P NMR spectra of phospholipids organized in each phase is also presented. 10 adopt a micellar arrangement in water. Of particular interest are lipids such as unsaturated phosphatidylethanolamine (PE) which comprises a significant proportion of the lipids in biological membranes and adopts the nonbilayer inverted hexagonal (HM) phase in isolation. For example, dioleoylphosphatidylethanolamine (DOPE) adopts a bilayer phase below 10°C, while at elevated temperatures DOPE adopts the H M phase (Cullis & de Kruijff, 1978a). Formation of the H M phase is promoted by increasing acyl chain unsaturation and increasing temperature (Lewis etal., 1989). Lipids can also adopt some interesting non-vesicle, bilayer structures. Phosphatidylserine (PS), for example, adopts cochleate cylinders in the presence of calcium (Papahadjopoulos, 1975) and galactosylcerebroside (GalCer) lipids can self-assemble into helical ribbons and lipid nanotubes (Yager et al., 1995). Such novel lipidic structures such as lipid nanotubes may hold promise for rapid protein crystallization and structure determination using electron microscopy techniques (Wilson-Kubalek etal., 1998). Detection of Nonbilayer Structure. Nonbilayer structure can be determined by spectroscopic and microscopic techniques. A convenient method for probing the motional properties of phospholipid dispersions is 3 1 P nuclear magnetic resonance (NMR) spectroscopy. Characteristic phosphorus powder patterns are observed for phospholipids ^organized in bilayer or H M phase structure (Cullis & de Kruijff, 1979) (Figure 1.4). 11 Other nonbilayer phases such as micellar or cubic phases in which phospholipids experience isotropic motion cannot be definitively assigned by this method. Freeze-fracture electron microscopy can also be used to detect bilayer and nonbilayer phases. Characteristic fracture planes are observed for lipids in bilayer, HM and cubic lipid phases (Deamer et al., 1970; Rilfors et al., 1986; Hope et al., 1989). In addition, "lipidic particle" structures can be visualized by freeze-fracture electron microscopy (Verkleij, 1980) and are often noted in lipid bilayer systems undergoing fusion. Theory of Lipid Polymorphism. Molecular shape arguments have been used to rationalize the behaviour of lipids which adopt nonbilayer phases (Gruner et al., 1985). Lipids with a large headgroup area and a small hydrocarbon area have a cone-like geometry, exhibit positive membrane curvature and assemble into micelles (Figure 1.5). Lipids that are cylindrical in shape, having a ratio of the headgroup to hydrocarbon area nearly equal, pack into lipid bilayers. Alternatively, lipids with a small headgroup area with a larger hydrocarbon area exhibit negative membrane curvature and adopt "inverted" lipid phases such as the cubic or the inverted hexagonal (Hn) phase. In support of the shape hypothesis, mixtures of nonbilayer micellar lipids and nonbilayer Hn phase preferring lipids can adopt bilayer phases (Madden & Cullis, 1982). Such arguments can be used to explain the behaviour of lipids which assemble into micelles, bilayers, Hn and cubic phases, and in part to define the molecular assembly of lipid tubules and helices (Kulkarni etal., 1995). 12 A. Inverted hexagonal F I G U R E 1.5. Molecular geometry of lipids and the predicted self-assembly of morphologically distinct structures. 13 Nonbilayer Lipids in Nature. The presence of lipids which adopt nonbilayer phases such as the hexagonal Hn phase in isolation has fascinated researchers as to the functional roles of these components in biomembranes. The plasma membrane of E. coli is primarily composed of PE which in isolation adopts the nonbilayer HM phase. Similarly, the inner leaflet of the red blood cell membrane is composed in large part of PS and PE. This lipid mixture exhibits fusogenic and polymorphic phase behaviour in the presence of Ca 2 + or low pH when studied in isolation (Hope & Cullis, 1979; Hope et al., 1983). The mitochondrial membrane contains large amounts of cardiolipin, a lipid which can also adopt the inverted HM phase in the presence of C a 2 + (Rand & Sengupta, 1972; Cullis et al., 1978). Incorporation of the integral membrane protein glycophorin within cardiolipin bilayers inhibits the Hn phase-inducing effect of calcium Ca 2 +(Taraschi etal., 1983). Recently, related results were presented on the effect of the light harvesting complex (LHC-II) on the organization of the nonbilayer lipid monogalactosyldiacylglycerol (MGDG), a major lipid component of the thylakoid membrane (Simidjiev et al., 2000). Incorporation of the integral LHC-II protein complex promoted the structural reorganization of MGDG from the HM phase it prefers in isolation into an ordered bilayer phase. The stabilization of nonbilayer phases by integral membrane proteins may reflect a general property of some transmembrane polypeptides. 14 Nonbilayer lipids may play a role in the regulation of protein function, as has been suggested in the case of the integral membrane protein rhodopsin (Brown, 1994). Phosphatidylethanolamines with highly unsaturated acyl chains are required for the optimal activity of rhodopsin in reconstituted lipid membranes. Nonbilayer lipids present in biological membranes may modulate protein activity through alterations in bilayer physical properties such as bilayer membrane curvature. In studies on model organisms such as E. coli it was found that the lipid composition of cellular membranes is regulated to provide a balance between bilayer and nonbilayer lipids (Morein et al., 1996). In these experiments, E. coli grown at different temperatures underwent changes in the acyl chain composition of the three major lipids PE, PG and CL. At lower growth temperatures the phospholipid acyl chains became more unsaturated. A reverse of this trend was observed at higher growth temperatures with a decrease in unsaturated lipids and an increase in saturated lipid species. Acyl chain unsaturation promotes nonbilayer phases in lipids such as PE. 3 1 P NMR data indicated that regardless of growth temperature, the lipid composition of E. coli is maintained such that the lipid membrane is near a nonbilayer phase transition. This data shows that E coli have a homeostatic mechanism to maintain the physical state of their membrane. Such mechanisms were predicted to exist in previous reports on the role of nonbilayer lipids in biological membranes (Gruner, 1985). 15 LIPID POLYMORPHISM AND MEMBRANE FUSION Membrane fusion is a ubiquitous process in biological systems and involves the coalescence of two separate bilayers in order to complete processes such as exocytosis or viral infection. A local departure from the bilayer structure must take place in order to allow two lipid membranes to merge into one. Little is known about the structure of the lipid intermediates which are involved in membrane fusion in a biological setting. However, the study of fusion of model membrane systems has provided a guide to understanding some of factors which may underlie the dynamics of biological fusion events. Proposed Fusion Intermediates. Lipidic particle structures observed by freeze-fracture were first interpreted to be inverted micelles formed at the junctions between lipid bilayers undergoing membrane fusion (Cullis & Hope, 1978) (Figure 1.6). Alternatively, the lipidic particle observed by freeze-fracture techniques may be related to the formation of the "stalk" intermediate of membrane fusion as defined by Markin and colleagues (Markin et al., 1984) and later developed by Chernomordik and Siegel (Chernomordik & Zimmerberg, 1995; Siegel, 1999). In the stalk theory of membrane fusion, two apposed bilayers undergo a union of the contacting monolayers through the formation of a discrete, highly curved lipidic structure called the stalk (Figure 1.6). It has been proposed that the expansion of the stalk intermediate produces a transmonolayer contact (TMC) which ruptures due to increasing 16 INVERTED MICELLE INTERMEDIATE "STALK" INTERMEDIATE F I G U R E 1.6. Proposed intermediates of membrane fusion. Two apposed bilayers are schematically represented to undergo fusion through either an inverted micelle intermediate (IMI) or the stalk and transmembrane contact (TMC) intermediates. 17 mechanical tension to produce the fusion pore. Time-resolved cryoelectron microscopy has been used to directly visualize TMC-like structures formed in the early stages of lipid vesicle fusion (Siegel & Epand, 1997). The geometry of the stalk intermediate favours the incorporation of lipids which exhibit negative membrane curvature. Lipids such as unsaturated PE which have an inverted cone, or wedge structure have compatible shape to incorporate into the highly bent stalk intermediate. Conversely, micellar lipids which exhibit positive membrane curvature, have shapes which are complementary to that of PE and are incompatible with the orientation of lipids proposed in the stalk structure. Indeed, a correlation is observed between the shapes of lipids in the contacting monolayers and membrane fusion. Inverted hexagonal phase adopting lipids such as PE or protonated PS support the fusion of lipid vesicles (Ellens et al., 1985), while micellar lysolipids inhibit fusion of LUVs and Sendai virus-LUVs when applied to the outer lipid monolayers (Yeagle et al., 1994) lending indirect support to the stalk mechanism of membrane fusion. Role of Lipids in Biological Membrane Fusion. In biological systems, membrane fusion is tightly regulated by proteins which bring membranes into close apposition and initiate fusion. However, the mechanism that underlies membrane rearrangements in biological fusion events such as exocytosis remains the subject of debate. Studies of membrane fusion during exocytosis using patch-clamp techniques has allowed for the characterization of the exocytotic fusion 18 pore (Lindau & Aimers, 1995). These studies suggest that the initial fusion pore has properties similar to an ion channel and is comprised of proteins which incorporate lipids after opening. An alternative explanation proposed that if two membranes are brought into close proximity with protein tethers, membrane fusion will proceed spontaneously. Evidence for this model of membrane fusion is supported by studies in which v-SNARE and t-SNARE protein complexes, which were reconstituted into separate liposome populations, resulted in spontaneous membrane fusion (Weber etal., 1998). In an effort to directly identify the role of lipids in biological membrane fusion events, Chernomordik and colleagues have conducted a survey of the effect addition of nonbilayer lysolipids exerts on biological membrane fusion events. Addition of lysolipids to the contacting membrane monolayers inhibited sea urchin egg cortical exocytosis, mast cell degranulation, rat liver microscome-microsome fusion, and viral fusion (Chernomordik et al., 1993). A similar inhibitory effect of lysolipid is observed in the fusion of pure lipid systems (Yeagle et al., 1994). This indicates that membrane fusion in biological and model systems is highly dependent on the physical properties of the contacting lipid monolayers. pH-SENSITIVE LIPOSOMES Liposomes that exhibit triggered release properties have potentially important applications in drug delivery. It has been shown that liposomes can be constructed that are sensitive to a variety of physical and chemical stimuli, including 19 temperature, light, or pH (Gerasimov et al., 1996). Liposomes that can be triggered to release their contents or fuse in response to pH stimuli are of particular interest as they can potentially respond to acidic environments in vivo (Yatvin et al., 1980; Straubinger, 1993). Such environments include those encountered in tumor tissue (Tannock & Rotin, 1989) and primary endocytic vesicles (Tycko & Maxfield, 1982). pH-Sensitive Formulations. pH-sensitive liposomes are typically prepared from lipid mixtures containing dioleoylphosphatidylethanolamine (DOPE), a lipid that adopts the nonbilayer inverted hexagonal (Hu) phase in isolation (Cullis & de Kruijff, 1978a), and an ionizable acidic lipid such as cholesteryl hemisuccinate (Ellens et al., 1984). At pH values above the pKof the acidic lipid the negatively charged form of the acidic lipid can stabilize the DOPE in the bilayer organization, allowing the formation of bilayer vesicles. These vesicles then fuse as the pH is reduced towards the pK of the acidic lipid. Protonation of the anionic lipid diminishes the ability of this component to stabilize DOPE in the bilayer organization (Lai et al., 1985a). Lipids such as phosphatidylserine (Hope et al., 1983), palmitoylhomocysteine (Connor et al., 1984) and a-tocopherol hemisuccinate (Jizomoto et al., 1994) have also been used in concert with DOPE to prepare pH-sensitive liposomes. An overview of the different formulations of pH-sensitive liposomes is presented in Table 1.1. 20 TABLE 1.1. pH-Sensitive Liposome Formulations. Nonbilayer Lipid Anionic Stabilizing Lipid Reference DOPE Phosphatidylserine (Hope etal., 1983) DOPE Palmitoylhomocysteine (Connor etal., 1984) DOPE Cholesteryl hemisuccinate (Ellens etal., 1984) DOPE N-succinyldioleoylphosphatidylethanolamine (Nayerefa/., 1985) DOPE Oleic Acid (Duzgunes etal., 1985) DOPE Series of double-chain amphiphiles (Leventis etal., 1987) DOPE Dipalmitoylsuccinylglycerol (Collins etal., 1989) POPE a-tocopherol hemisuccinate (Jizomoto etal., 1994) DOPE Sulfatide (Wu etal., 1996) 21 Role of DOPE in pH-Sensitive Liposomes. Nearly all pH-sensitive liposome formulations contain a large amount of the nonbilayer lipid DOPE (Litzinger & Huang, 1992). Typically DOPE comprises between 50-80 mol % of the total vesicle lipid. Replacement of the nonbilayer lipid with a bilayer-forming lipid such as DOPC abolishes the pH-sensitivity and intracellular delivery capabilities of these systems (Straubinger et al., 1985). DOPE is a lipid which when dispersed in aqueous media preferentially adopts the hexagonal Hn phase above 10 °C (Cullis & de Kruijff, 1978a). Molecular shape arguments (Gruner et al., 1985) can be used to rationalize the phase behaviour of DOPE, which has a relatively small phosphoethanolamine headgroup and diunsaturated acyl chains which give the lipid an "inverted cone" structure. In terms of intrinsic membrane curvature arguments, DOPE exhibits a high degree of negative monolayer curvature. Lipids which exhibit opposite membrane curvature characteristics such as detergents like lysophosphatidylcholine or Triton X-100 have structures complementary to DOPE and in mixtures DOPE and detergents can form bilayer phases (Madden & Cullis, 1982). Similarly, bilayer-adopting lipids such as the zwitterionic lipid phosphatidylcholine can also stabilize DOPE into a bilayer phase (Cullis & de Kruijff, 1978b). Anionic (Ellens et al., 1984) and cationic lipids will also promote the stabilization of DOPE into a bilayer phase (Mok & Cullis, 1997). If ionizable lipids are incorporated into bilayer phases with DOPE, the stability of the bilayer is conditional on the pH which controls the structural preferences of the ionizable lipid. The first system described 22 as a fusogenic pH-sensitive liposome was composed of PS/DOPE (2:8 molar ratio) (Hope et al., 1983). These vesicles were stable at neutral pH, but underwent fusion at acidic pH values. PS itself adopts a bilayer phase on hydration at neutral pH values, however below pH 4, unsaturated PS species are known to adopt the inverted hexagonal phase (Hope & Cullis, 1980). Thus at acidic pH, PS/DOPE liposomes contain only lipids which prefer a nonbilayer phase, and as a result are unstable and fusogenic. Intracellular Delivery. The potential to use pH-sensitive liposomes to exploit intracellular delivery through the endocytic pathway was highlighted by Papahadjopoulos and co-workers who demonstrated that anionic liposomes are taken up by CV-1 cells through coated pits and encounter a low pH compartment (Straubinger et al., 1983). Shortly following this discovery, pH-sensitive liposomes prepared from the nonbilayer lipid DOPE and oleic acid were demonstrated to mediate the cytoplasmic delivery of the fluorescent dye calcein in CV-1 cells (Straubinger et al., 1985). pH-sensitive liposomes have since been used to deliver a variety of macromolecules including nucleic acids such as DNA and antisense oligonucleotides, protein toxins, and antibiotics. An overview of the various macromolecules introduced into cells using pH-sensitive liposomes is presented in Table 2.2. The mechanism of delivery via pH-sensitive liposomes through the endocytic pathway is not well defined (Litzinger& Huang, 1992; Straubinger, 1993). 23 TABLE 1.2. Intracellular Delivery Using pH-Sensitive Liposomes. Entrapped Molecule Assay Method pH-Sensitive Liposome Reference Calcein Fluorescence Microscopy Oleic acid/DOPE Straubinger et al., 1985 Calcein Fluorescence Microscopy PHC/DOPE Connor & Huang, 1985 Arabinoside-C Cell killing Oleic acid/DOPE Connor & Huang, 1986 Diphtheria Toxin A Cell killing Oleic acid/DOPE Collins & Huang, 1987 CAT-Plasmid DNA CAT activity Oleic acid/Chol/DOPE Wang & Huang, 1987 FITC-Dextran (4.2 kDa) Fluorescence Microscopy CHEMS/DOPE Chu etal., 1990 Ovalbumin MHC class-1 presentation DOSG/DOPE Nairef a/., 1992 Oligonucleotide Friend Retrovirus Inhibition Oleic acid/Chol/DOPE Ropert et al., 1992 PolylC RNA IFN production Oleic acid/Chol/DOPE Milhaud et al., 1992 Superoxide dismutase (SOD) Cell-associated SOD activity DOSG/DOPE Briscoe et al., 1995 Listeriolysin 0 / Ovalbumin and HPTS Fluorescence Microscopy/ MHC class-l presentation CHEMS/DOPE Lee etal., 1996 Gentamycin Bacterial killing N-succ-DOPE/DOPE Lutwyche et al., 1998 24 It is proposed that pH-sensitive liposomes undergo destabilization and leakage upon encountering an intracellular acidic stimulus. This may lead to the release of the liposomal contents within acidic endosomal compartments. Alternatively, if close proximity is achieved between the liposome and the lumenal membrane of the endosome at the time of acidification, destabilization of endosomal membrane may result from the preference of the pH-sensitive liposomal lipids for nonbilayer phases. STUDIES ON LIPID POLYMORPHISM AND INTRACELLULAR DELIVERY This thesis comprises three studies on the role of lipid polymorphism in the destabilization and fusion of both liposome delivery systems and biological cell membranes. Within each study, it is found that the ability of lipid bilayers to undergo polymorphic phase transitions is promoted by charge neutralization either by changes in pH, the presence of an oppositely charged lipid, or the combination. A particularly interesting finding of this work is that in the case of non-viral gene delivery promoted by cationic lipids, anionic cellular lipids common to biological membranes are necessary accomplices for the formation of membrane disruptive nonbilayer phases promoted by cationic lipids. Role of an Anionic Lipid Component of pH-Sensitive Liposomes. Cholesteryl hemisuccinate (CHEMS) is the anionic lipid most commonly used to prepare pH-sensitive liposomes (Straubinger, 1993). CHEMS can stabilize dioleoylphosphatidylethanolamine (DOPE), a lipid which preferentially adopts the 25 inverted hexagonal (Hu) phase above 10°C (Cullis & de Kruijff, 1978a), into the lamellar phase at pH 7.4 (Ellens et al., 1984; Lai et al., 1985a; Ellens et al., 1985). Lamellar CHEMS/DOPE systems can be prepared at neutral or slightly alkaline pH but these systems become unstable and fuse at acidic pH (Ellens et al., 1985). In these pH-sensitive liposomes the ionization state of CHEMS dictates the phase behaviour, and thus the fusogenic behaviour, of the lipid ensemble. This behaviour is usually rationalized as an ability of the anionic form of CHEMS to stabilize DOPE into a lamellar phase, whereas when CHEMS is in the neutral form this stabilizing property is reduced and DOPE can adopt the HM phase it prefers in isolation, thus promoting fusion. However, in mixtures of CHEMS with PE exhibiting higher La-Hn phase transition temperatures, it has been shown that CHEMS can modulate the La-Hn phase transition temperature depending on its state of ionization. In the anionic form CHEMS stabilizes against Hu formation as evidenced by an increase in the La-Hn phase transition temperature while the neutral form of CHEMS induces a marked reduction in the temperature of the La-Hn phase transition (Lai et al., 1985a; Cheetham et al., 1994). Cholesterol and other neutral sterols can also reduce the La-Hn transition temperatures in mixtures with PE (Cullis & de Kruijff, 1978b; Gallay & de Kruijff, 1982), but to a much lesser extent than protonated CHEMS (Lai et al., 1985a; Cheetham etal., 1994). In Chapter 2, the molecular mechanism of the potent HM phase-promoting activity of CHEMS at acidic pH was investigated. It was found that LUVs composed of CHEMS are fusogenic at acidic pH and CHEMS itself exhibited pH-dependent 26 polymorphic phase behaviour. Freeze-fracture electron microscopy supported the conclusion that CHEMS bilayers undergo a transition to the HM phase upon charge neutralization. The ability of CHEMS to adopt the HM phase in acidic conditions likely promotes the pH-sensitive fusion of liposomes composed of CHEMS and DOPE. Tunable pH-Sensitive Liposomes. The conventional design of pH-sensitive liposomes relies on the use of DOPE and an anionic stabilizing lipid. This approach gives limited control of the pH at which fusion will occur unless one resorts to using a number of anionic lipids with different pK values (Collins et al., 1989). Due to this limitation, an alternative approach in the design of pH-sensitive liposomes was investigated. In Chapter 3, results are presented which describe the properties of large unilamellar vesicles (LUVs) containing cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC). It is shown that these LUVs exhibited pH-sensitive fusion properties and that the pH at which fusion was observed can be modulated by the ratio of the component cationic and anionic lipids. pH-induced fusion of these LUVs correlated with a preference for nonbilayer inverted lipid structures. Related effects were observed for LUVs composed of dioleoylphosphatidic acid (DOPA) and the ionizable cationic lipid 3a-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi). It is concluded that the pH at which fusion of LUVs containing anionic lipids occurs can be readily modulated by inclusion of cationic lipid. It was also demonstrated that mixtures of cationic and anionic lipids exhibited interesting polymorphism that may 27 be related to the ability of cationic lipids to promote intracellular delivery of macromolecules. Cationic Lipid-Mediated Intracellular Delivery. The cell membrane presents a major barrier to the intracellular delivery of macromolecules such as plasmids, antisense oligonucleotides and therapeutic proteins. A number of approaches for enhancing the intracellular delivery of nucleic acids and proteins have been investigated, including the use of liposomes (Mayhew et al., 1977), membrane-translocating peptides (Fawell et al., 1994), cationic peptides (Wyman et al., 1997), cationic proteins (Pardridge & Boado, 1991), cationic polymers (Boussif et al., 1995) and cationic lipids (Feigner et al., 1987). Cationic lipids provide a particularly powerful tool for the introduction of polynucleic acids into cells. Cationic lipid-nucleic acid complexes, termed lipoplexes have been utilized to promote the intracellular delivery of plasmid DNA (Feigner et al., 1987), RNA (Malone et al., 1989), antisense oligonucleotides (Bennett et al., 1992), transcription factor decoy oligonucleotides (Park et al., 1999), ribozymes (Kariko et al., 1994) and transcription factors (Duzgunes & Feigner, 1993). Cationic lipids bind and condense nucleic acids into a variety of supramolecular structures (Radler et al., 1997; Koltover et al., 1998; Mok & Cullis, 1997; Xu et al., 1999; Sternberg et al., 1998) and impart a positive charge to the nucleic acid. Cationic lipoplexes associate with negatively charged cellular membranes through electrostatic interactions (Stamatatos et al., 1988). Entry of cationic lipoplexes into the cell is thought to occur primarily via endocytosis (Zabner 28 etal., 1995; Zhou & Huang, 1994; El Ouahabi etal., 1997; Wrobel & Collins, 1995; Friend et al., 1996), although delivery through pores in the plasma membrane has been proposed (van der Woude et al., 1995). In support of delivery through the endocytic pathway, endosomal membranes with a disrupted morphology are often observed in following intracellular delivery of lipoplexes (Zhou & Huang, 1994; El Ouahabi etal., 1997). The mechanism of nucleic acid delivery via cationic lipoplexes is thought to involve interaction with anionic components of the cell. Specifically, cationic liposomes fuse with liposomes containing anionic phospholipids (Stamatatos et al., 1988; Bailey & Cullis, 1997; Duzgunes et al., 1989). In addition, anionic lipids form ion pairs with cationic lipids (Bhattacharya & Mandal, 1998) which promotes the disassociation of cationic lipoplexes and release of nucleic acids (Xu & Szoka, Jr., 1996; Zelphati & Szoka, Jr., 1996; Bhattacharya & Mandal, 1998). Cationic lipids also induce the disruption of isolated lysosome membranes (Wattiaux et al., 1997). In vitro, cationic liposomes also enhance the transduction properties of retroviral vectors (Hodgson & Solaiman, 1996) and excess cationic liposomes administered separately can also enhance transfection efficiency of cationic lipoplexes (Farhood et al., 1995). This evidence suggests that cationic lipids play an active role in the disruption of cellular membrane barriers which involves an interaction with anionic phospholipids. 29 In Chapter 4, the structural characteristics of mixtures of synthetic cationic lipids and anionic phospholipids were investigated. Using 3 1 P NMR, a convenient non-perturbing technique for the study of phospholipid membrane structure (Cullis & de Kruijff, 1979; Yeagle, 1993), it was demonstrated that mixtures of anionic phospholipids in the presence of synthetic cationic lipids used for transfection adopted the nonbilayer inverted hexagonal (Hu) phase. It is suggested that cationic lipids promote the intracellular delivery of macromolecules through the disruption of cellular membranes by their association with anionic cellular lipids. 30 CHAPTER 2 POLYMORPHIC PHASE BEHAVIOUR OF CHOLESTERYL HEMISUCCINATE CHEMS consists of succinic acid esterified to the p-hydroxyl group of cholesterol (Figure 2.1 A). This chemical modification results in the ability of CHEMS to adopt a lamellar organization upon hydration in neutral or alkaline aqueous media (Lai et al., 1985b; Janoff et al., 1988), whereas cholesterol forms monohydrate crystals in an aqueous environment (Renshaw etal., 1983). In this chapter, the molecular mechanism of the strong inverted hexagonal (Hn) phase-inducing activity of CHEMS at acidic pH (Lai et al., 1985a; Cheetham et al., 1994) is investigated. It is shown that large unilamellar vesicles composed of CHEMS undergo membrane fusion upon acidification as indicated by lipid mixing, consistent with a preference of the neutral form for the Hn phase. This conclusion is supported by freeze-fracture electron microscopy studies. MATERIALS AND METHODS Lipids and Chemicals. Cholesteryl hemisuccinate (morpholine and Tris salt) was obtained from Sigma Chemical Company (St. Louis, MO) and was pure as assayed by thin layer chromatography. 2-p-toluidinyl naphthalene 6-sulphonic acid (TNS), N-[2-hydroxyethyl]piprezine-N'-[2-ethanesulphonic acid] (HEPES), 2-[N-morpholino]ethanesulfonic acid (MES), t-octylphenoxypolyethoxyethanol (Triton X-31 A FIGURE 2.1 Structure (A) and apparent pK determination (B) of cholesteryl hemisuccinate. The pK of CHEMS was determined from a pH titration of POPC/CHEMS (1:1 molar ratio) LUVs using the TNS assay. POPC/CHEMS LUVs (100 uJVI lipid) were prepared in media containing 1 uJvl TNS, 10 mM HEPES, 10 mM MES, 10 mM acetate and 140 mM NaCl. The pH was adjusted from 2.5 to 11. TNS fluorescence was monitored at excitation and emission wavelengths of 321 nm and 445 nm and expressed in relative fluorescence units. Data shown is representative of 3 experiments. 32 100) and glacial acetic acid were obtained from Sigma. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -palmitoyl-2-hydroxy-SA?-glycero-3-phosphocholine (LPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-Iissamine rhodamine b sulfonyl (Rh-PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). The concentration of phospholipid solutions was determined by phosphate assay (Bartlett, 1959). Large Unilamellar Vesicle (LUV) Preparation. CHEMS morpholine salt, POPC, NBD-PE and Rh-PE were dissolved separately in chloroform and stored at -20°C. CHEMS Tris salt was stored at -20°C in its powder form. CHEMS LUVs were either prepared by hydration of dried lipid films of CHEMS morpholine salt or by direct hydration of the CHEMS Tris salt. Both CHEMS salts gave identical results. For the pK determination, POPC was mixed with an equimolar amount of CHEMS morpholine salt in CHCI 3. For the lipid mixing assays, CHEMS morpholine salt was mixed with NBD-PE and Rh-PE (1 mol % each) in chloroform. The chloroform solutions were dried to a thin film under a stream of N 2 gas. The lipid films were further dried in the dark for at least 1 h under high vacuum (30-60 mtorr) to remove residual organic solvent. Dry lipid was hydrated using the appropriate buffer by vortex mixing to produce multilamellar vesicles (MLVs). LUVs were prepared by a freeze-thaw and extrusion technique (Mayer et al., 1986). The MLVs were subjected to 5 freeze-thaw cycles (liquid nitrogen/room temperature) 33 and extruded ten times through two stacked 0.1 jo.m pore-size polycarbonate filters using an extrusion device (Lipex Biomembranes, Vancouver, BC). Depending on the lipid formulation the mean diameter of the LUVs was 85-110 nm as determined by a quasi elastic light scattering device (Nicomp 270 submicron particle sizer) operating in the vesicle sizing mode. pK Determination. The apparent pK of CHEMS was determined employing TNS, a surface potential probe and pH stable CHEMS/POPC LUVs. It was not possible to perform the TNS assay on vesicles composed solely of CHEMS due to precipitation of the lipid at acidic pH. CHEMS/POPC (1/1 mol ratio) LUVs were prepared in 10 mM HEPES; 150 mM NaCl, pH 7.8. TNS was prepared as a 100 \M stock solution in distilled water. Lipid vesicles were diluted to 100 |j,M lipid in 2 ml of buffered solutions containing 1 TNS, 10 mM HEPES, 10 mM MES, 10 mM acetate, 140 mM NaCl, where the pH ranged from 2.5 to 11. Fluorescence intensity was monitored in a thermostated cuvette (25°C) in a Perkin Elmer LS-50 spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. The pH of each solution was measured after fluorescence measurements. Lipid Mixing Assay. Fusion of CHEMS vesicles was assayed by a lipid mixing assay employing fluorescence resonance energy transfer (Struck et al., 1981). Labeled CHEMS 34 LUVs were prepared to contain NBD-PE and Rh-PE (1 mol % each) in 10 mM HEPES, 150 NaCl, pH 7.8. Labeled and unlabeled CHEMS vesicles were mixed in a 1:5 mol ratio respectively, and introduced together to a final concentration of 150 u,M lipid by pipette injection into a stirred cuvette containing 2 ml of 10 mM HEPES, 10 mM MES, 10 mM acetate, 140 mM NaCl equilibrated to the desired pH values. Addition of the lipid did not alter the pH of the buffer. An increase of NBD-PE fluorescence indicates dilution of the membrane bound probes. NBD-PE fluorescence measurements were obtained at 25°C in a Perkin Elmer LS-50 using an excitation wavelength of 467 nm and emission wavelength of 540 nm using an emission filter at 530 nm. Lipid mixing was monitored for approximately 300 s, after which an aliquot of Triton X-100 (10% v/v) was added to a final concentration of 0.1% v/v. Lipid mixing as a percentage of infinite probe dilution was determined using the equation: Lipid mixing (%) = (Ft-Fo)/(Fmax-F0) x 100%, where F t is the fluorescence intensity of NBD-PE (at 540 nm) during the assay, F 0 is the initial value of NBD-PE fluorescence, and F m a x is the maximum fluorescence possible given by infinite probe dilution in the presence of 0.1% v/v Triton X-100. Effect of Lysophosphatidylcholine on the Stability of CHEMS Bilayers. Lysophosphatidylcholine (LPC) was dissolved in ethanol (8.0 mM) and stored at -20°C. Prior to each lipid mixing experiment, the ethanol stock of LPC was diluted with distilled water to a concentration of 0.5, 1.0 and 2.0 mM. Labeled and unlabeled CHEMS LUVs (1:5 mol ratio) were added to 2 ml of 5 mM HEPES, 150 35 mM NaCl, pH 7.5 to obtain a final lipid concentration of 150 |aM. Addition of LPC to preformed LUVs composed of CHEMS should lead to LPC incorporation only in the outer membrane leaflet owing to the slow transbilayer flip-flop of LPC (Bhamidipati & Hamilton, 1995). Fluorescence measurements were made at 25°C using excitation and emission wavelengths of 467 nm and 540 nm, respectively. After a stable signal was obtained, 20 JLXI of LPC was added from the 0.5, 1.0, or 2.0 mM LPC solutions to obtain a final LPC concentration of 5, 10 or 20 |j.M. The amount of LPC incorporation into CHEMS LUVs was estimated by fluorescence dequenching of NBD-PE in NBD-PE/Rh-PE labeled vesicles using the methodology of Chernomordik and colleagues (Chernomordik et al., 1997). The extent of NBD-PE fluorescence dequenching following the addition of LPC was used as a direct indication of LPC incorporation. Following the addition of LPC, the HEPES buffered saline was acidified with 1 M acetic acid to obtain the desired final pH. Light scattering measurements (90°) were made on a Perkin Elmer LS-50B spectrophotometer using excitation and emission monochromators set to 400 nm under continuous stirring at 25°C. This technique was used to assess dynamic changes in size of CHEMS LUVs in response to acidification. CHEMS vesicles were diluted to 150 lipid in 2 ml of 10 mM HEPES, 150 mM NaCl, pH 7.5. Addition of an aliquot of 1 M acetic acid was added to acidify the HEPES buffered saline. Measurements were made in the presence and absence of LPC. 36 Freeze-Fracture Electron Microscopy. The structure of the CHEMS dispersions was analyzed using freeze-fracture electron microscopy. MLV and LUV samples were prepared from 100 mM CHEMS Tris in 150 mM NaCl, pH 8.1. MLV and LUV samples were mixed with glycerol to a final concentration of 30% (v/v). LUV samples were also mixed at room temperature with acidic buffer containing 50 mM NaCl, 300 mM acetate, pH 2.7. Acidification of LUVs induced rapid aggregation and precipitation of the lipid. Glycerol was included to a final concentration of 30% v/v. The time interval between sample acidification and freezing was approximately 2 minutes. Samples were dispensed onto gold cups and rapidly frozen in Freon 22 cooled in liquid nitrogen. Platinum/carbon replicas were prepared using a Balzers Freeze-etching unit BAF 400D and observed by transmission electron microscopy as described elsewhere (Hope etal., 1989). RESULTS pK of CHEMS. The apparent pK of CHEMS in LUVs was measured using TNS to probe for changes in membrane surface charge in response to pH. TNS is a lipophilic anion that is essentially non-fluorescent in aqueous solution but exhibits an increase in fluorescent quantum yield upon membrane binding. TNS fluorescence has been used to assay the transmembrane distribution of acidic phospholipids (Eastman et al., 1991; Mui et al., 1995), and to determine pK values for ionizable lipid species in a membrane (Bailey & Cullis, 1994). The data in Figure 2.1 B shows the relative 37 change of TNS fluorescence in response to pH in the presence of pH-stable LUVs composed of equimolar amounts of POPC/CHEMS. Fitting the data to the Henderson-Hasselbach equation gives an apparent pK value of 5.8 for CHEMS. Fusion of CHEMS LUVs. CHEMS is commonly used in concert with DOPE to prepare pH-sensitive LUVs (Straubinger, 1993; Chu et al., 1990). The pH dependent fusogenic properties of LUVs composed of CHEMS alone were determined employing the lipid mixing assay (Struck et al., 1981). Lipid mixing of CHEMS LUVs occurred below pH 4.3 with extensive lipid mixing at pH 4.2 and below (Figure 2.2 A). Half-maximum lipid mixing of CHEMS LUVs occurs at pH 4.1 (Figure 2.2 B). Fusion of CHEMS/DOPE (3:7 mol ratio) LUVs occurs at slightly higher pH values (between pH 4 and 5) (Ellens et al., 1985), consistent with the Hn phase preference of DOPE. Lysophosphatidylcholine Inhibits Acid-Induced Fusion of CHEMS LUVs. Lysophosphatidylcholine (LPC) can inhibit membrane fusion by inhibiting the formation of fusion intermediates with a negative membrane curvature (Yeagle et al., 1994). Figure 2.3 A shows that acid promoted lipid mixing of CHEMS LUVs is increasingly blocked in the presence of up to 20 u,M LPC. Incorporated LPC is estimated to be 2 mol % of the outer membrane leaflet in CHEMS LUVs at a final concentration of 20 u,M LPC based on the dequenching of NBD-PE after LPC addition. 38 FIGURE 2.2 Acid-induced fusion of CHEMS LUVs. (A) Lipid mixing of CHEMS LUVs in response to acidic pH. (B) Extent of lipid mixing at 250 s as a function of pH. Labeled CHEMS LUVs containing Rh-PE and NBD-PE (1 mol % each) were mixed with unlabeled CHEMS LUVs (1:5 mol ratio) and introduced to a final concentration of 150 u,M lipid (at 50 s) into solutions containing 10 mM HEPES, 10 mM MES, 10 mM acetate and 140 mM NaCl equilibrated to desired pH values. NBD-PE fluorescence was monitored at excitation and emission wavelengths of 467 nm and 540 nm. Data shown is representative of 3 experiments. 39 FIGURE 2.3 LPC inhibits acid-induced fusion of CHEMS LUVs. (A) LPC dependent inhibition of acid induced lipid mixing of CHEMS LUVs. (B) Acid induced 90° light scattering changes in CHEMS LUVs in the presence and absence of LPC. CHEMS LUVs were diluted to 150 ixM lipid in 10 mM HEPES, 150 mM NaCl, pH 7.5. For lipid mixing experiments LPC was added prior to sample acidification. Lipid mixing was monitored by measuring NBD-PE fluorescence. 90° light scattering was observed using excitation and emission monochromators set to 400 nm. Data shown is representative of 3 experiments. 40 The ability of LPC to inhibit fusion of CHEMS LUVs was also analyzed by 90° light scattering using a fluorescence spectrometer with the emission and excitation set to 400 nm. In the absence of LPC, acidification (pH 4.15) caused an immediate increase in light scattering followed by a gradual decrease (Figure 2.3 B, -LPC). The solution initially became cloudy, followed by the precipitation of lipid aggregates in the buffer. Acidification in the presence of 20 |j.M LPC did not alter light scattering or the dispersion turbidity, indicating that the vesicle size remained unchanged (Figure 2.3 B, +LPC). The stabilization of CHEMS LUVs against fusion by trace amounts of LPC at low pH is consistent with the inhibition of the formation of nonbilayer structures required for the La-Hn phase transition to proceed. Detergents such as LPC can stabilize DOPE, an HM phase forming lipid, into a bilayer configuration (Madden & Cullis, 1982) by shape complementarity. The stabilization of acidified CHEMS bilayers by LPC is compatible with a preference of the neutral form of CHEMS for the inverted hexagonal (Hn) phase. Freeze-Fracture Electron Microscopy Reveals the Phase Behaviour of CHEMS at Acidic pH. i Freeze-fracture electron microscopy was used to identify the structure of the precipitates formed upon acidification of CHEMS LUVs. CHEMS forms MLVs upon hydration in alkaline solution as observed by the cross-fracture of multiple lamellae (Figure 2.4 A). This confirms earlier reports of the self-assembly of CHEMS into closed bilayers in aqueous media (Lai et al., 1985b; Janoff et al., 1988). Freeze-fracture micrographs of the LUVs produced by freeze-thaw and extrusion of 41 FIGURE 2.4 C H E M S adopts a lamellar phase on hydration at alkal ine p H . Transmiss ion electron micrographs of plat inum/carbon freeze-fracture repl icas of C H E M S M L V , pH 8.1 (A) and extruded C H E M S L U V , pH 8.1 (B). Sca le bars: 100 nm. Micrographs are representat ive images of repl icas obtained from 3 separate samp les . 4 2 FIGURE 2.5 Acid i f ied C H E M S bilayers adopt the hexagonal phase at ac id ic p H . Transmiss ion electron micrographs of plat inum/carbon freeze-fracture repl icas of C H E M S L U V incubated at (A) pH 4.3 and (B and C) pH 3.7. S c a l e bars represent 100 nm in (A), 87 nm in (B) and 36 nm in (C). Micrographs are representative images of repl icas obtained from a s ingle sample . 43 CHEMS MLVs are shown in Figure 2.4 B. Incubation of these LUVs in acidic media results in structures that display the characteristic striated Hu phase freeze-fracture pattern at pH 4.3 (Figure 2.5 A) and at pH 3.7 (Figure 5 B and C). The diameter of the hexagonal cylinders is ~ 6 nm as measured from Figure 2.5 C. DISCUSSION The results presented demonstrate an ability of CHEMS to adopt the Hu phase upon exposure to acidic pH. It is likely that the phase behavior of CHEMS at low pH is related to effects observed in mixtures with PE. For example, the preference of HM phase by CHEMS at or below pH 4.3 is consistent with the pronounced reduction of the La-Hn phase transition temperature induced by CHEMS at acidic pH in mixtures with PE (Lai era/., 1985a; Cheetham etal., 1994). Cholesterol levels of 50 mol % in DOPE membranes lower the La-Hn phase transition temperature by 10-20°C (Cullis & de Kruijff, 1978b), while only 25 mol % CHEMS at pH 4.5 reduces the La—Hu phase transition of egg PE by 30°C (Lai et al., 1985a). This is consistent with an ability of the neutral CHEMS to actively induce the HM phase. The polymorphism of CHEMS is closely related to that observed for dioleoylphosphatidylserine (DOPS) and a-tocopherol hemisuccinate (THS). DOPS and THS form bilayers under alkaline conditions, but undergo a phase transition to the Hn phase upon protonation of their acidic headgroups. THS forms a lamellar phase above pH 7, but below this pH, HM phase and inverted phase intermediates are favored (Boni et al., 1990). DOPS also forms a lamellar organization above pH 44 4.0 but below pH 3.5 DOPS adopts the H,, phase (Hope & Cullis, 1980; de Kroon et al., 1990). Qualitative molecular shape arguments have been used to rationalize such phase behaviour, and similar arguments can be used to rationalize the phase behaviour of CHEMS. In particular, protonation of the CHEMS headgroup may be expected to lead to a reduction of the headgroup area at the lipid-water interface due to reduced electrostatic repulsion between headgroups and reduced hydration. Since the area of the hydrophobic sterol domain presumably remains unaltered upon headgroup neutralization, the effective molecular shape may be modeled as changing from a bilayer forming cylinder to that of an inverted cone that preferentially adopts the HM phase. In summary, the results demonstrate that CHEMS adopts the HM organization at pH values below the pK of the succinate headgroup. This is consistent with an ability of the neutral form of CHEMS to actively induce the Hn organization in mixtures with DOPE, contributing to the pH-sensitive fusion of such systems. 45 CHAPTER 3 TUNABLE pH-SENSITIVE LIPOSOMES COMPOSED OF MIXTURES OF CATIONIC AND ANIONIC LIPIDS In this chapter, the design, construction, and characterization of the structural and fusogenic properties of LUVs containing cholesteryl hemisuccinate (CHEMS) and increasing amounts of the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) is described. It is shown that these LUVs exhibit pH-sensitive fusion properties and that the pH at which fusion is observed increases as the content of cationic lipid is increased. It is also demonstrated that pH-induced fusion correlates with a preference for nonbilayer inverted lipid structures. Related findings were observed for LUVs composed of dioleoylphosphatidic acid (DOPA) and the ionizable cationic lipid 3a-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi). It is concluded that the pH at which fusion of LUVs containing anionic lipids occurs can be readily modulated by inclusion of cationic lipid. Furthermore, mixtures of cationic and anionic lipids exhibit interesting polymorphism that may be related to the ability of cationic lipids to promote intracellular delivery of macromolecules. 46 Materials and Methods Lipids and Chemicals. DC-Choi, DOPA, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-lissamine rhodamine b sulfonyl (Rh-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesteryl hemisuccinate (morpholine salt), dimethylamine, oleoyl bromide, HEPES, 2-[N-morpholino]ethanesulfonic acid (MES), and t-octylphenoxypolyethoxyethanol (Triton X-100) were obtained from Sigma Chemical Company (St. Louis, MO). Synthesis of N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC). Oleoyl bromide (5 g) was stirred in a saturated solution of dimethylamine in methanol (200 ml) overnight. The solvent and most of the dimethylamine was removed on a rotovap. The residue was treated with 10 ml of 5 N NaOH solution and all the remaining dimethylamine removed under vacuum. Chloroform (40 ml) was added to the mixture, followed by oleoyl bromide (10 g). The mixture was refluxed overnight, diluted with water (50 ml) and extracted three times with chloroform. The organic phase was washed six times with 3% hydrochloric acid, decolorized with hydrochloric acid washed charcoal, and washed twice with saturated aqueous NaCl. The solvent was removed on a rotovap and the residue dried by azeotropic removal of water using ethanol. The residue was passed down a silica gel column (200 g) using a 2-20% MeOH/CH 2CI 2 gradient. Fractions 47 containing the hydroxyl analogue of DODAC were discarded. Fractions containing DODAC were combined and the solvent removed under vacuum. Fractions contaminated with the hydroxyl analogue of DODAC were stored for re-chromatography. The residue from the pure DODAC fractions was hydrated in water ( 5 0 mg/ml) at 6 0 ° C , passed through 0 . 1 u.m pore size polycarbonate filters using an Extruder (Lipex Biomembranes, Vancouver, BC) and lyophilized, yielding DODAC as a colourless powder ( 5 g). Purity was determined to be greater than 9 9 % b y 1 H NMR and TLC. Preparation of Large Unilamellar Vesicles (LUVs). LUVs were prepared from binary mixtures of DODAC and CHEMS or DC-Choi and DOPA. Lipids were dissolved in chloroform at the desired molar ratios and dried to a thin film under a stream of nitrogen gas. Typically 5 - 1 0 u.mol of total lipid was dried per 1 3 x 1 0 0 mm glass test tube. The resulting thin films were placed under high vacuum for at least 1 h to remove residual organic solvent. DODAC/CHEMS lipid films were hydrated by vortex mixing in 1 . 0 ml of aqueous buffer containing 1 0 mM HEPES, 1 5 0 mM NaCl, pH 8 . 1 . DC-Chol/DOPA lipid films were hydrated by vortex mixing in 1 . 0 ml of either 1 0 mM HEPES, 1 5 0 mM NaCl, pH 8 . 1 or 1 0 mM HEPES, 1 5 0 mM NaCl acidified to pH 3 . 9 with acetic acid. Following hydration the multilamellar vesicles (MLVs) were subjected to five freeze-thaw cycles (liquid nitrogen/room temperature). The MLV suspensions were then extruded 1 0 times through two stacked 0 . 2 jo.m pore size polycarbonate filters using an Extruder (Lipex Biomembranes, Vancouver, BC) to produce LUVs (Mayer et al., 1 9 8 6 ) . The mean 4 8 diameter of LUVs systems was determined using a Nicomp C270 submicron particle sizer operating in the particle mode. Lipid Mixing Fusion Assay. A membrane fusion assay based on fluorescence resonance energy transfer between two fluorescent lipid probes was used to assess the fusion of LUVs in response to pH (Struck et al., 1981). The fluorescent lipids NBD-PE and Rh-PE were included at 1 mol % each to produce labeled LUVs. Labeled and unlabeled vesicles were mixed at a 1:5 molar ratio and injected into 2 ml of buffer containing 10 mM HEPES, 10 mM MES, 10 mM acetate, 140 mM NaCl adjusted to pH values between 3.0 and 8.5. Ammonium chloride (10 mM) was included in the medium to eliminate transmembrane pH gradients for the DC-Chol/DOPA systems. The final lipid concentration was 150 [xM. NBD-PE fluorescence dequenching was monitored in a stirred cuvette in a Perkin-Elmer LS-50B at 25 °C using excitation and emission wavelengths of 467 nm and 540 nm, respectively. Lipid mixing was monitored for approximately 400 s. Complete dilution of the fluorescent probes was determined by the addition of Triton X-100 to a final concentration of 0.1 % (v/v). The extent of lipid mixing was calculated using the equation: Lipid mixing (%) = (F-F 0)/(Fm a x-Fo) x 100, where F is the NBD-PE fluorescence during the lipid mixing assay, F 0 is the initial fluorescence and F m a x is the NBD-PE fluorescence upon infinite probe dilution in the presence of 0.1 % (v/v) Triton X-100. To allow comparison of pH-sensitivity between different LUV compositions, the normalized lipid mixing (%) for each lipid 49 composition was expressed as a percentage of the maximum lipid mixing observed for a given LUV composition. Freeze-Fracture Electron Microscopy. The structures formed by hydrated DODAC/CHEMS mixtures were analyzed using freeze-fracture electron microscopy. DODAC/CHEMS lipid films were hydrated with 50 mM HEPES, 150 mM NaCl, pH 8.1, followed by five freeze-thaw cycles. DODAC/CHEMS LUVs that were subsequently acidified were formed from lipid films hydrated with 10 mM HEPES, 150 mM NaCl, pH 8.1, in order to reduce the buffering capacity. DODAC/CHEMS LUVs were treated with a volume of acidic buffer containing 50 mM acetate, 150 mM NaCl, pH 4.6, to obtain the final desired pH values. Glycerol was added to all samples to a final concentration of 30 % (v/v). Samples were dispensed onto gold cups and rapidly frozen in Freon 22 cooled in liquid nitrogen. Platinum/carbon replicas were prepared using a Balzers freeze-etching unit (BAF 400D) and observed by transmission electron microscopy as described elsewhere (Hope era/., 1989). 31P Nuclear Magnetic Resonance Spectroscopy. Lipid films composed of DC-Chol/DOPA (1.6 molar ratio) were hydrated in 20 mM HEPES, pH 7.6 or 20 mM HEPES, 20 mM acetate, pH 3.8, to achieve a final concentration of 15 mM phospholipid. The DC-Chol/DOPA dispersion hydrated at pH 3.8, was alkalized with approximately 60 u,l of 500 mM NaOH to achieve a final 50 pH of 6.1. P NMR spectra were obtained using a Bruker MSL-200 spectrometer operating at 81.3 MHz. Acquisition parameters included a 60° pulse, 10 kHz sweep width, a 1 s interpulse time and spectra were accumulated in the presence of broad band proton decoupling. The temperature was 300 K. RESULTS DODAC/CHEMS Lipid Mixtures can form Bilayer Vesicles on Hydration. It is well established that CHEMS adopts a bilayer structure on hydration at neutral pH (Lai et al., 1985b; Janoff et al., 1988) and can stabilize nonbilayer lipids such as DOPE in the bilayer organization (Ellens et al., 1984). Similarly, cationic lipids such as DODAC can form bilayers and stabilize DOPE in a bilayer structure (Mok & Cullis, 1997). Less is known concerning the structural properties of mixtures of bilayer-forming cationic and anionic lipids, which may be expected to exhibit unusual behaviour due to interactions between the positively and negatively charged headgroups. It was found that hydration of lipid films comprised of DODAC and CHEMS at cationic-to-anionic lipid molar ratios of less than one resulted in white milky dispersions visually similar to those observed for multilamellar vesicle (MLV) phospholipid dispersions. The structure of these DODAC/CHEMS lipid dispersions was examined by freeze-fracture electron microscopy, revealing the formation of MLVs (Figure 3.1 A). It was also found that MLVs consisting of DODAC/CHEMS at molar ratios less than one could be extruded through 0.2 u.m pore size filters to give rise to uniformly sized large unilamellar vesicle (LUV) 51 FIGURE 3.1 Structural characterist ics of aqueous d ispers ions of D O D A C / C H E M S (0.72 molar ratio). The lipid was hydrated in 50 m M H E P E S , 150 m M N a C l , pH 8.1, and freeze-fracture electron micrographs were prepared as descr ibed in Materials and Methods. (A) D O D A C / C H E M S M L V formed on hydration of the lipid film. (B) D O D A C / C H E M S L U V s formed after extrusion through two s tacked filters with 0.2 jxm pore s ize . S c a l e bar: 200 nm. Micrographs are representat ive images of repl icas obtained from 2 separate samp les . 52 structures (Figure 3.1 B). The mean diameter ± mean SD as determined .by-dynamic light scattering was 153 + 35 nm for DODAC/CHEMS molar ratios of 0 to 0.72 and 274 ± 94 nm for LUVs composed of DODAC/CHEMS at a molar ratio of 0.85. The mean diameter ± mean SD measurement was calculated from the average of the diameter ± SD obtained from the dynamic light scattering analysis of 3 separate samples for each DODAC/CHEMS formulation. DODAC/CHEMS LUVs Undergo pH-Sensitive Fusion which can be Modulated by Adjusting the DODAC/CHEMS Ratio. Previous work presented in Chapter 2 has shown that LUVs composed of CHEMS alone undergo pH-dependent fusion as the pH is lowered below pH 4.2. An objective of this investigation was to determine whether addition of cationic lipid resulted in an increase in the pH at which fusion occurred in these pH-sensitive LUVs. The next experiments therefore characterized the influence of increasing amounts of DODAC on the pH-dependent fusion properties of DODAC/CHEMS LUVs. Lipid mixing assays were performed as a function of pH for DODAC/CHEMS LUVs at molar ratios ranging from 0 to 0.85. As shown in Figure 3.2 A, increases in DODAC content resulted in an increase in the pH at which fusion occurred. For example, the pH for half-maximal fusion (pHf) for pure CHEMS LUVs is 4.0, whereas for LUVs composed of DODAC/CHEMS at a molar ratio 0.11 a pH f of 4.7 was observed. Further increases in the DODAC/CHEMS molar ratio to 0.85 resulted in LUVs with pHf values as high as 6.7. The fusion kinetics observed employing the 53 FIGURE 3.2 pH-dependent fusion properties of DODAC/CHEMS LUVs containing increasing amounts of DODAC. (A) DODAC/CHEMS LUVs were prepared at pH 8.1 were then introduced into buffer with the pH values indicated. The normalized lipid mixing (%) was determined as described in Materials and Methods. Data are presented for LUVs with DODAC/CHEMS molar ratios of 0 (O), 0.11 (•), 0.43 (•), 0.52 (A) , 0.61 (T) , 0.72 (•) and 0.85 (•). (B) Membrane fusion kinetics observed for LUVs composed of DODAC/CHEMS (0.11 molar ratio) and (C) LUVs composed of DODAC/CHEMS (0.85 molar ratio) following acidification. LUVs were added to buffer with the indicated pH at 50 s. The lipid mixing (%) was determined as described in Materials and Methods. Data presented is representative of at 3 separate experiments. 54 lipid mixing assay for LUVs composed of DODAC/CHEMS at molar ratios of 0.11 and 0.85 are shown in Figure 3.2 B and C, respectively. It is logical to suggest that fusion between DODAC/CHEMS LUVs will not proceed unless the surface charge is neutralized, allowing close contact between opposing bilayers. For the surface charge to be zero the proportion of CHEMS that is negatively charged must equal the DODAC content of the membrane. Under these conditions it is straightforward to show that the pH at which the surface charge is neutral (pHn) can be written as: pH n= PKCHEMS + logio [XDODAC/(XCHEMS~XDODAC)] (Eq. 1) where PKCHEMS is the apparent pK of CHEMS and X DODAC and XCHEMS are the molar fractions of CHEMS and DODAC, respectively. In Chapter 2 it was shown that the apparent pK of CHEMS in a lipid bilayer is 5.8, allowing a comparison between pHf and pH n. The assumption that the apparent pK of CHEMS remains constant is only an approximation. As shown in Figure 3.3, pH f correlates well with pH n, supporting the conclusion that fusion proceeds between DODAC/CHEMS LUVs when the surface charge is zero. pH-Sensitive Fusion that is Modulated by the Cationic-to-Anionic Lipid Ratio Can Also be Observed for LUVs Containing an lonizable Cationic Lipid. pH-sensitive fusion of DODAC/CHEMS LUVs is modulated by the neutralization of CHEMS, the anionic lipid component, which is in excess compared to the cationic 55 FIGURE 3.3 Correlation between membrane fusion and surface charge neutralization of DODAC/CHEMS LUVs. Half-maximum fusion (pHf) values were determined from Figure 3.2 for each DODAC/CHEMS LUVs formulation as the pH at which lipid mixing was 50% of the maximum observed lipid mixing and plotted as a function of the DODAC/CHEMS molar ratio (•). The pH at which the surface charge was predicted to be zero (dashed line) was determined employing Eq. 1 using P K C H E M S = 5.8. 56 lipid. These systems exhibit progressively lower levels of negative surface charge as the pH is lowered, fusing when the surface charge is zero. It is of interest to examine the properties of systems in which the cationic lipid species is the ionizable component and is present in excess over the anionic lipid species. By analogy with the behaviour of DODAC/CHEMS LUVs, such systems should form stable bilayers at low pH where the cationic lipid species is fully charged, and should fuse as the pH is raised towards the pKof the cationic lipid. DC-Choi, which was chosen as the ionizable cationic lipid has a pK of -8.0 (Zuidam & Barenholz, 1997). The anionic lipid chosen was DOPA which exhibits pK values of -3.0 and -8.0 (Tocanne & Teissie, 1990). It was found that DC-Chol/DOPA LUVs at molar ratios ranging from 1.6 to 4 could be prepared at pH 3.9, and as predicted, fusion between these LUVs was observed as the pH was raised (Figure 3.4 A). The fusion kinetics observed employing the lipid mixing assay for LUVs composed of DC-Chol/DOPA at molar ratios of 1.6 and 4.0 are shown in Figure 3.4 B and C, respectively. Variations in the DC-Chol/DOPA molar ratio over the range 1.6 to 4.0 resulted in changes in the pHf from 4.9 to 7.7. It was not possible to correlate pH f with pH n, the pH giving rise to zero surface charge, due to the indeterminate nature of the pK values of DC-Choi and DOPA in these mixed systems. 57 FIGURE 3.4 pH-dependent fusion properties of DC-Chol/DOPA LUVs containing increasing amounts of DC-Choi. (A) DC-Chol/DOPA LUVs were prepared at pH 3.9 and were then introduced into buffer with the pH values indicated. The normalized lipid mixing (%) was determined as described in Materials and Methods. Data are presented for LUVs with DC-Chol/DOPA LUVs molar ratios of 1.6 (•), 2.0 (A) , 3.0 (•) and 4.0 (T) . (B) Membrane fusion kinetics observed for LUVs composed of DC-Chol/DOPA (1.6 molar ratio) and (C) LUVs composed of DC-Chol/DOPA (4.0 molar ratio) following the pH increase. LUVs were added to buffer with the indicated pH at 50 s. The lipid mixing (%) was determined as described in Materials and Methods. Data is representative of 3 separate experiments. 58 Fusion of LUVs Composed of Cationic and Anionic Lipid is Accompanied by the Appearance of "Inverted" Nonbilayer Lipid Structures. The structural features of LUVs composed of cationic and anionic lipids when the pH is adjusted to levels that promote fusion are of interest. While the absence of surface charge may be necessary to allow fusion, it is unlikely that this condition alone is sufficient to induce fusion. It was observed during the lipid mixing assays that fusion of DODAC/CHEMS LUVs was accompanied by an increase in the turbidity of the LUV dispersion, followed by precipitation of the lipid. The structures formed by DODAC/CHEMS (0.85 molar ratio) LUVs following incubation at pH 6.9 (the approximate pH f for this system) were examined employing freeze-fracture electron microscopy. As shown in Figure 3.5 A-C, large structures containing characteristic "lipidic particle" (Verkleij et al., 1980) or "interlamellar attachment site" (Siegel, 1999) structures are observed. Such structures are commonly observed in fusion between vesicles induced by the tendency of component lipids to adopt nonbilayer structures (Verkleij et al., 1980). Nonbilayer lipidic particle structures may form due to the lateral phase separation of distinct lipid domains. The propensity of DODAC/CHEMS (0.85 molar ratio) LUVs to adopt nonbilayer structure was further demonstrated by examining the structures formed following incubation at pH 5.4, a value below the pHf for this system. As shown in Figure 3.5 D, the freeze-fracture 59 micrographs obtained reveal the characteristic striated pattern of lipid organized in the hexagonal Hn phase. The ability of mixtures of cationic lipid and anionic lipid to adopt nonbilayer structures under conditions of zero surface charge is intriguing, and suggests that equimolar mixtures of charged cationic and anionic lipids may prefer nonbilayer structure on hydration, whereas either species in isolation adopts a lamellar organization. This possibility was investigated for aqueous dispersions of equimolar amounts of DODAC and CHEMS prepared at pH 8 . 1 . Freeze-fracture electron microscopy studies revealed systems containing lipidic particle structures (Figure 60 FIGURE 3.5 DODAC/CHEMS LUVs adopt nonbilayer phases at low pH values. Freeze-fracture electron micrographs of DODAC/CHEMS LUVs (0.85 molar ratio) prepared at pH 8.1 and then incubated at lower pH values. (A-C) Incubation at pH 6.9 induces fusion and lipidic particle structures. (D) Incubation at pH 5.4 results in fusion and formation of the inverted hexagonal phase. Scale bars: 200 nm. Micrographs obtained at each pH are representative images of replicas obtained from 2 separate samples. 61 3.6 A). Extensive regions of linear arrays of lipidic particles were also observed (Figure 3.6 B and C); such structure has been associated with lipids in the cubic phase (Rilfors etal., 1986; Ellens etal., 1989). The polymorphic phase preferences of DC-Chol/DOPA lipid mixtures could be investigated directly employing 3 1 P NMR due to the presence of the phosphate group of the phosphatidic acid. As shown in Figure 3.7 A, DC-Chol/DOPA (1.6 molar ratio) dispersions prepared at pH 3.8 reveal the characteristic asymmetric lineshape with a low field shoulder and a high field peak associated with bilayer structure (Cullis & de Kruijff, 1979). Adjustment of the pH to 6.1 (Figure 3.7 B) results in the appearance of a 3 1 P NMR signal with reversed asymmetry that is a factor of two narrower and is characteristic of phospholipid in the hexagonal Hu phase (Cullis & de Kruijff, 1979). Integration of the spectra shown in Figure 3.7 A and B indicated a constant 3 1P NMR signal intensity after correction for the number of scans accumulated for each spectrum. At pH 7.6, the DC-Chol/DOPA mixture adopts a bilayer organization as indicated by 3 1 P NMR (Figure 3.7 C). These results illustrate the ability of the charged form of either DC-Choi or DOPA to stabilize the ensemble into a bilayer organization whereas when the amounts of charged DC-Chol and DOPA are equal the hexagonal Hu phase is adopted. Changes in the width of the chemical shift anisotropy of the spectra indicating a bilayer organization at acidic and alkaline pH values (Figure 3.7 A and C), may be ascribed to changes in the ionization of phosphatidic acid (Pott etal., 1995). 62 FIGURE 3.6 Equimolar mixtures of DODAC/CHEMS adopt nonbilayer structures following hydration at neutral pH values. Freeze-fracture electron micrographs of equimolar mixtures of DODAC/CHEMS hydrated in 50 mM HEPES, 150 mM NaCl, pH 8.1 reveal (A, B) lipidic particle structures and (C) regular arrays of lipidic particles. Freeze-fracture replicas were prepared and observed as described in Methods and Materials. Scale bars: 200 nm. Micrographs obtained at each pH are representative images of replicas obtained from 2 separate samples. 63 PPM FIGURE 3.7 Influence of pH on the polymorphic phase properties of aqueous dispersions of DC-Chol/DOPA as detected by 3 1 P NMR. (A) 3 1 P NMR spectrum obtained from an aqueous dispersion of DC-Chol/DOPA (1.6 molar ratio) hydrated at pH 3.8. (B) 3 1 P NMR spectrum of the sample employed in (A) alkalized to pH 6.1 with NaOH. (C) 3 1 P NMR spectrum of an aqueous dispersion of DC-Chol/DOPA (1.6 molar ratio) hydrated at pH 7.6. A 50 Hz line broadening was applied to each spectrum. For other acquisition parameters see Materials and Methods. Each spectrum presented is representative of 3 separate experiments. 64 DISCUSSION The results provide information on a new class of liposomal systems comprised of mixtures of cationic and anionic lipids. There are three major points of interest. First, stable liposomes can be generated from mixtures of cationic and anionic lipids when either the cationic or anionic lipid species is in excess. Second, fusion between these liposomes is stimulated by a preference for nonbilayer "inverted" lipid phase structures when the surface charge is zero. Finally, if an ionizable cationic or anionic lipid is employed, pH-dependent fusion between them is observed and varying the proportions of cationic and anionic lipids can modulate the pH at which this fusion occurs. These features are discussed in turn. The observation that stable liposomal systems can be generated from mixtures of cationic and anionic lipids is perhaps surprising given the possibility of forming phase-separated crystalline domains of neutral cationic-anionic lipid pairs in these mixed lipid systems. The evidence is consistent with formation of liquid crystalline lipid bilayers from charged mixtures of cationic and anionic lipids, with no evidence for crystalline domains of cationic-anionic lipid pairs. Perhaps the most compelling evidence in this regard is provided by the 3 1 P NMR lineshapes observed for DOPA in mixtures with DC-Choi (Figure 3.7) as well as the constant 3 1 P NMR signal intensity arising from the DOPA. The bilayer and hexagonal Hn 3 1 P NMR lineshapes are signatures of liquid crystalline phospholipids that are free to rotate rapidly around their long axes, with additional motional averaging in the hexagonal HM phase due to lateral diffusion around the aqueous cores of the Hn phase cylinders 65 (Cullis & de Kruijff, 1979). The constant 3 1 P NMR signal intensities that are observed independent of phase structure suggest that all of the DOPA, even if it is part of a DC-Chol:DOPA lipid pair, is contributing to these liquid crystalline lineshapes in both the bilayer or hexagonal phases. Previous work has shown that phospholipids in crystalline domains give rise to much broader 3 1 P NMR lineshapes with increased Ti values (Tilcock etal., 1984). The observation that LUVs containing cationic and anionic lipids fuse when the net surface charge is zero, and that this fusion is due to a preference for nonbilayer structure, is of particular interest for three reasons. First, fusion of LUVs when component lipids can adopt inverted lipid structures is consistent with the extensive literature indicating that membrane fusion proceeds via nonbilayer structures such as inverted micelles or stalks (see Chernomordik & Zimmerberg, 1995; Siegel, 1999 for reviews). These structures are favored by the presence of lipids able to adopt, inverted lipid phases. Second, the ability of mixtures of cationic and anionic lipids to adopt cubic and hexagonal Hn phases under conditions of zero surface charge is also fully consistent with the phase behaviour of mixtures of cationic and anionic surfactants (Kaler et al., 1989; Zemb et al., 1999) and intrinsic curvature or lipid shape arguments used to rationalize the phase preferences of lipids (Gruner et al., 1985). In particular, it has been shown that equimolar mixtures of single chain cationic and anionic detergents can spontaneously form closed bilayer vesicles in aqueous solution (Kaler et al., 1989). In these systems, the cationic and anionic surfactants are suggested to form ion pairs that result in a diacyl zwitterion with a 66 cylindrical molecular shape that is compatible with lamellar structure. Using the language of Gruner et al. (1985), due to decreases in the headgroup area the intrinsic curvature of the lipid monolayers decreases substantially when the ion pairs are formed, leading to transitions from micellar to lamellar structures. Analogous behaviour would be expected for ion pairs formed from mixtures of cationic and anionic lipids that adopt bilayer structure in isolation. In this case the reduction in intrinsic curvature on formation of zwitterions composed of cationic lipid and anionic lipid ion pairs would be expected to lead to transitions from bilayer structure to inverted lipid phases such as the hexagonal Hn phase or the cubic phase, as observed experimentally. Using shape arguments, this corresponds to a transition from charged lipids with a cylindrical shape to neutral ion pairs with a "cone" shape that is compatible with inverted lipid phase structures. Related behaviour is observed, for example, when the negative headgroup charge of cardiolipin, a bilayer forming tetra-acyl phospholipid, is neutralized by the addition of C a 2 + , triggering a bilayer-to-Hn phase transition (Rand & Sengupta, 1972; Cullis etal., 1978). The third reason why fusion between LUVs containing cationic and anionic lipids which reflects a preference for nonbilayer structure is of interest is that it represents the first demonstration that mixtures of two species of lipids that both adopt the bilayer configuration in isolation can form nonbilayer phases in combination. It is likely that this ability is basic to the mechanism whereby cationic lipids increase the intracellular delivery and transfection potency of nucleic acid polymers following incubation of cells with cationic lipid-nucleic acid polymer complexes (Feigner et al., 67 1987). Previous work has shown that negatively charged lipids found in cell membranes can displace cationic lipid from nucleic acids in these complexes (Xu & Szoka, 1996; Zelphati & Szoka, 1996). The work presented here gives rise to the possibility that the displaced cationic lipid combines with the negatively charged lipid in cell membranes to actively promote formation of nonbilayer structures such as the hexagonal Hu phase. This would be expected to assist in the membrane disruption process that allows anionic polymers to penetrate the plasma or endosomal membrane to gain access to the cell cytoplasm. The observations that LUVs composed of mixtures of DODAC/CHEMS and DC-Chol/DOPA undergo pH-dependent fusion at pH values that are regulated by the cationic-to-anionic lipid ratios has obvious potential for design of pH-sensitive liposomes for tumor delivery or intracellular delivery applications. Tumor pH values measured using microelectrode techniques reveal that these tissues are acidic compared to normal tissue with a mean pH of 7.0 (range 5.8 to 7.6) (Tannock & Rotin, 1989) indicating the potential usefulness of liposomes that become unstable and release their contents, such as anticancer drugs, at these pH values. Alternatively, a major impediment to the utility of macromolecular drugs such as antisense oligonucleotides for the down-regulation of pathogenic genes (Loose-Mitchell, 1988) or plasmid expression vectors for gene therapy applications (Ledley, 1995) is the inability of these charged macromolecules to penetrate target cell membranes. Previous work has demonstrated the encapsulation of plasmid DNA within anionic CHEMS/DOPE pH-sensitive liposomes (Legendre & Szoka, 1992). 68 This suggests that the encapsulation of nucleic acids within tunable pH-sensitive DODAC/CHEMS vesicle systems containing an excess of anionic lipid is possible. Delivery using liposomes that are tuned to become unstable in mildly acidic endocytic compartments could be of considerable utility. The particular advantage of the systems described here is that the pH value at which the liposome becomes unstable can be adjusted in a straightforward and systematic manner. In summary, the studies show that stable liposomes can be constructed from mixtures of cationic and anionic lipids and that these liposomes fuse at pH values that can be readily adjusted by varying the ratio of the cationic and anionic lipid components. Fusion is accompanied by formation of nonbilayer structures formed under conditions of zero surface charge. These liposomes have potential utility in their own right as intracellular delivery vehicles. Further, the ability of mixtures of cationic and anionic lipids to adopt nonbilayer structures gives potentially important insight into the mechanism of action of cationic lipids used for intracellular delivery of nucleic acids. 6 9 CHAPTER 4 MECHANISM OF ACTION OF CATIONIC LIPIDS Cationic lipids are widely used as non-viral gene transfer agents. However, the mechanism by which cationic liposomes promote the intracellular delivery of membrane impermeable macromolecules such as plasmid DNA or antisense oligonucleotides remains obscure. In this chapter, the mechanism by which cationic lipids destabilize cell membranes was investigated. Using 3 1 P NMR, it was demonstrated that mixtures of synthetic cationic lipids routinely used for gene transfer and naturally occurring anionic phospholipids adopt the nonbilayer inverted hexagonal (HM) phase. Lipids such as dioleoylphosphatidylethanolamine and cholesterol which have been empirically found to enhance cationic lipid-mediated transfection further encourage the formation of the HM phase. The nonbilayer structural properties of mixtures of cationic and anionic lipids is dependent on lipid headgroup structure and the degree of acyl chain saturation. It is suggested that the structural preferences of mixtures of synthetic cationic lipids and cellular anionic phospholipids facilitates membrane destabilization leading to fusion between cationic lipid complexes and anionic cellular membranes or gross breach of intracellular membrane barriers. 70 MATERIALS AND METHODS Lipids. The cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was synthesized as previously described (Mok & Cullis, 1997). Dioleoyldimethylammonium chloride (DODAC), N-oleoyl,N-stearyl-N,N-dimethylammonium chloride (OSDAC) and distearyldimethylammonium chloride (DSDAC) were gifts from Steven Ansell at Inex Pharmaceuticals (Vancouver, BC, Canada). 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) and 3a-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi) and the phospholipids, dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid (DOPA), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG); phosphatidylinositol-liver derived (PI); tetraoleoylcardiolipin (CL), sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1'-oleoyl-2'-hydroxy)-glycerol (lysobisphosphatidic acid; S,R isomer), dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesterol was obtained from Sigma Chemical Company (St. Louis, MO). 31P Nuclear Magnetic Resonance Spectroscopy. Lipids were dissolved in chloroform and stored at -20 °C. Phospholipid concentration was determined by the method of Bartlett, 1959. Lipid films were prepared by co-dissolving lipids to desired molar ratios and drying under a stream of N 2 . Typically 20 ixmoles of total phospholipid was dried in a 16 x 100 mm test tube. 71 Lipid films were further dried under high vacuum to remove residual solvent. Dried lipid films were hydrated with 20 mM HEPES, pH 7.4 with five freeze-thaw cycles (liquid N2/room temperature). 3 1 P NMR spectra were obtained using a Bruker MSL-200 spectrometer operating at 81.3 MHz. Acquisition parameters included a 60° pulse, 10 kHz sweep width, a 1 s interpulse time and spectra were accumulated in the presence of broad band proton decoupling. Sample temperature was regulated using a Bruker VT-100 temperature controller. 0 ppm was set using an external phosphoric acid standard. RESULTS Mixtures of Cationic Lipids and Anionic Phospholipids Adopt a Nonbilayer Phase. In Chapter 3, it was shown that mixtures of the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) with the anionic lipid cholesteryl hemisuccinate (CHEMS) can adopt nonbilayer structures such as arrays of lipidic particles and the hexagonal HM phase under conditions of neutral surface charge. Initial studies were conducted to determine whether this behaviour is a general property of mixtures of cationic and anionic lipids. In the first set of experiments dioleoylphosphatidylserine (DOPS) was used as the anionic lipid and was mixed with equimolar amounts of a variety of cationic lipids, including DODAC, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Feigner et al., 1987) , 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) (Stamatatos et al., 1988) and 3a-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride 72 (DC-Choi) (Gao & Huang, 1991). The phase behaviour of these dispersions could be investigated using 3 1 P NMR techniques. As shown in Figure 4.1 A, a dispersion of the anionic phospholipid dioleoylphosphatidylserine (DOPS) prepared at pH 7.4 reveals the characteristic 3 1 P NMR asymmetric lineshape with a low field shoulder and a high field peak associated with bilayer structure (Cullis & de Kruijff, 1979). However, the samples composed of equimolar mixtures of DOPS and DODAC, DOTMA and DOTAP (Figure 4.1 B-D) all give rise to 3 1 P NMR spectra that are a factor of two narrower and exhibit reversed asymmetry compared to the bilayer lineshape and are characteristic of phospholipids organized in the inverted hexagonal (Hn) phase (Cullis & de Kruijff, 1979). It may be noted that the 3 1 P NMR spectrum obtained for the equimolar mixture of DC-Chol/DOPS is consistent with the co-existence of both HM and bilayer phases (Figure 4.1 E). Mixtures of DOPS with the permanently charged DODAC, DOTMA and DOTAP all exhibit well defined 3 1 P NMR spectra indicative of Hn phase. These cationic lipids have quaternary amine headgroups and dioleoyl acyl chains, whereas DC-Choi has a tertiary amine headgroup with a pK value of 8.0 (Zuidam & Barenholz, 1997) and a sterol hydrophobic group. These criteria may contribute to the 3 1 P NMR spectra obtained for the equimolar mixture of DC-Chol/DOPS at pH 7.4 (Figure 4.1 E). The second set of experiments were performed to establish the generality of the observation that mixtures of cationic and anionic lipids adopt HM phase structure and 73 examined the structural preferences of mixtures of DODAC with a variety of different species of anionic phospholipids. These included dioleoylphosphatidic acid (DOPA), lysobisphosphatidic acid (LBPA; a major component of late endosomal membranes) (Kobayashi et al., 1998), tetraoleoylcardiolipin (CL), liver-derived phosphatidylinositol (PI) and 1-palmitoyl-2-oleoyl phosphatidylglyercol (POPG). As shown in Figures 4.1 F, 4.1 G, 4.1 H, 4.1 I and 4.1 J for mixtures of DODAC with DOPA, LBPA, CL, PI and POPG respectively, all gave 3 1 P NMR spectra consistent with lipids organized into the HM phase. For these experiments, anionic phospholipids were mixed with an equimolar amount of DODAC, with the exception of cardiolipin which is a bivalent, tetra-acyl lipid which was mixed with two molar equivalents of DODAC. The potency of DODAC and, by extension, other cationic lipids for inducing Hn phase structure is inherent in the fact that PS, PA, CL, PI and PG all adopt the bilayer organization in isolation at neutral pH and all can stabilize nonbilayer lipids such as dioleoylphosphatidylethanolamine (DOPE) into the bilayer organization (Hope et al., 1983). It should be noted that PI has never before been observed to adopt the HM phase under any condition. Equimolar mixtures of anionic and cationic lipids such as DODAC/DOPS dispersions prepared in buffer containing 150 mM NaCl also gave indistinguishable 3 1 P NMR spectra consistent with lipid organized in the Hu phase. 74 A F 1 1—I 1—I—1—I 1—I—I—L J I I I I 1 I I I L 0 0 PPM PPM F I G U R E 4.1 Equimolar mixtures of cationic lipids and anionic phospholipids exhibit nonbilayer phase preferences. 3 1 P NMR spectrum obtained from an aqueous dispersion of (A) DOPS, (B) DODAC/DOPS, (C) DOTMA/DOPS, (D) DOTAP/DOPS, (E) DC-Chol/DOPS, (F) DODAC/DOPA, (G) DODAC/LBPA, (H) DODAC/CL (2:1 molar ratio), (I) DODAC/PI, (J) DODAC/POPG. A 50 Hz line broadening was applied to each spectrum. For other acquisition parameters see Methods and Materials. Each spectrum presented is representative of at least 3 separate experiments. 75 Effect of Helper Lipids. Plasmid DNA-cationic lipid complexes used for transfection are usually derived from mixtures of plasmid with liposomes composed of cationic lipids and "helper" lipids such as DOPE (Feigner etal., 1987; Zhou & Huang, 1994; Feigner et al., 1994; Farhood etal., 1995) or cholesterol (Sternberg etal., 1998; Li etal., 1999; Liu et al., 1997; Templeton et al., 1997). Cholesterol containing lipoplexes exhibit serum stability (Li et al., 1999) and transfection competence in vivo (Liu et al., 1997; Templeton et al., 1997; Sternberg et al., 1998). These helper lipids give rise to improved transfection potencies of the resulting complex. If the mechanism of action of cationic lipids relies on an ability to induce HM phase or related nonbilayer structure to destabilize target membranes such as the endosomal membrane it would be expected that these helper lipids would also promote HM organization. Conversely, it would be expected that lipids such as dioleoylphosphatidylcholine (DOPC) which give rise to reduced levels of transfection when present in complexes (Feigner etal., 1994; Mok & Cullis, 1997; Xu etal., 1999) would hinder the ability of cationic lipids to induce nonbilayer structure. That this is indeed the case is illustrated in Figure 4.2. As shown in Figure 4.2 A, aqueous dispersions of DOPS/DOPC (1:1 molar ratio) adopt a bilayer structure (Figure 4.2 A). The addition of equimolar amounts of DODAC (with respect to DOPS) does not result in induction of HM phase structure as shown in Figure 4.2 B for a lipid dispersion containing DODAC/DOPS/DOPC (1:1:1 molar ratio), which also exhibits bilayer structure (Figure 4.2 B). Thus the presence of DOPC prevents the DODAC-DOPS ion pairs from adopting the HM phase they adopt in isolation (Figure 4.1 B). 76 This behaviour contrasts with the properties of lipids used as helper lipids. As shown in Figure 4.2 C, aqueous dispersions of DOPS/DOPE (1:1 molar ratio) adopt the bilayer phase, consistent with the ability of DOPS to stabilize DOPE in the lamellar organization as noted previously (Hope and Cullis, 1979). The addition of equimolar amounts of DODAC to DOPS results in a complete transition to HM phase organization as shown in Figure 4.2 D for a DODAC/DOPS/DOPE (1:1:1 molar ratio) dispersion. Furthermore, the addition of DOPE to the DOPC-containing lipid dispersion of Figure 4.2 B results in a complete transition to Hn organization as shown in Figure 4.2 E for a DODAC/DOPS/DOPC/DOPE (1:1:1:1 molar ratio) lipid dispersion. This is consistent with the preference of DOPE for the HM phase in isolation (Cullis and de Kruijff, 1978a) and again points to the ability of the DOPE helper lipid to facilitate Hn organization. Cholesterol has similar abilities to promote Hn phase organization. This is illustrated by the influence of cholesterol on the structure of the DODAC/DOPS/DOPC (1:1:1 molar ratio) system which, as previously indicated, adopts the bilayer organization (Figure 4.2 B). As shown in Figure 4.2 F for a DODAC/DOPS/DOPC/Cholesterol (1:1:1:1 molar ratio) lipid dispersion, the inclusion of cholesterol induces Hn organization. This is consistent with the previously noted ability of cholesterol to promote the HM phase in a variety of mixed phospholipid systems (Cullis & de Kruijff, 1978b; Gallay & de Kruijff, 1982). These results illustrate the ability of different neutral co-lipids to modulate the bilayer-to-Hn phase transition promoted by the synthetic cationic lipid DODAC in the 77 FIGURE 4.2 Neutral co-lipids DOPC, DOPE and cholesterol modulate the nonbilayer phase behavior of mixtures of cationic and anionic lipids. Lipid components were present at equal molar amounts in each mixture. 3 1 P NMR spectrum obtained from aqueous dispersions of (A) DOPS/DOPC, (B) DODAC/DOPS/DOPC, (C) DOPS/DOPE, (D) DODAC/DOPS/DOPE, (E) DODAC/DOPS/DOPC/DOPE, (F) DODAC/DOPS/DOPC/Chol. A 50 Hz line broadening was applied to each spectrum. For other acquisition parameters see Materials and Methods. Each spectrum presented is representative of at least 3 separate experiments. 78 presence of the anionic phospholipid DOPS. DOPC, a co-lipid which inhibits transfection was found to stabilize bilayer lipid phases, while DOPE and cholesterol which have been empirically determined to enhance transfection of lipoplexes, encourage the formation of nonbilayer phases. Our results support a correlation between the ability of co-lipids to enhance transfection and their ability to induce the formation of nonbilayer structure. Influence of Cationic Lipid Structure on the Polymorphism of Mixtures of Cationic and Anionic Lipids. It is well known that the chemical structure of cationic lipids can strongly influence the transfection potency of plasmid DNA-cationic lipid complexes formed from them. Although a detailed correlation between the transfection potency of complexes formed from different cationic lipids and the polymorphic phase preferences of these lipids in mixtures with anionic lipids is outside the scope of this work, it is important to point out that the ability of cationic lipids to induce the Hn phase in combination with anionic phospholipids does offer the possibility of obtaining a measurable parameter to correlate with transfection potency. Briefly, the relative tendency of lipid monolayers to adopt Hu organization is reflected by parameters such as the intrinsic radius of curvature which is a measure of the radius of the Hu phase cylinders. A related parameter is the bilayer-to-Hn transition temperature (TBri) (Tate & Gruner, 1989). Lipids that have smaller intrinsic radii of curvature exhibit lower T B H values, and it is of interest to relate the TBH values of mixtures of cationic and 79 anionic lipids with the transfection properties of the cationic lipid species. In this regard, the transfection potency of cationic lipids has been found to decrease with the increasing acyl chain length and saturation of the cationic lipid (Feigner et al., 1994; Hope etal., 1998). Using a series of diacyl-dimethylammonium chlorides, di-C18:1(A9) (DODAC), C18:1(A9)/C18:0 (OSDAC) and di-C18:0 (DSDAC), the temperature dependent phase behaviour of these lipids in equimolar mixtures with DOPS was examined by 3 1 P NMR. It was found than an equimolar mixture of DODAC/DOPS exhibits a T B H between 1-6 °C (Figure 4.3 A). The introduction of a single saturated acyl chain on the cationic lipid resulted in an increase in T B H to between 25-31 °C OSDAC/DOPS (1:1 molar ratio) (Figure 4.3 B), whereas for DSDAC/DOPS (1:1 molar ratio) a T B H between 43-48 °C was observed (Figure 4.3 C). These data are therefore consistent with the hypothesis that the enhanced transfection efficiency observed for unsaturated cationic lipids (Feigner et al., 1994; Hope et al., 1998) is due to their ability to readily promote nonbilayer structure in the presence of anionic cellular phospholipids at physiological temperatures. 80 A B C 40 20 0 -20 -40 40 20 0 -20 -40 40 20 0 -20 -40 P P M PPM P P M FIGURE 4.3 Effect of cationic lipid acyl chain saturation on the bilayer to hexagonal Hu phase transition temperature in mixtures with DOPS. Lipid components were present at equal molar amounts in each mixture. 3 1 P NMR spectrum obtained from aqueous dispersions of (A) DODAC/DOPS, (B) OSDAC/DOPS, and (C) DSDAC/DOPS. Temperature is as indicated on each figure. A 50 Hz line broadening was applied to each spectrum. For other acquisition parameters see Materials and Methods. Each spectrum presented is representative of at least 2 separate experiments. 81 The majority of commercially available cationic lipid reagents are chemically configured with dioleoyl acyl chains, and differ only in headgroup structure. Modification of cationic lipid headgroup structure has been shown to profoundly effect cationic lipoplex transfection efficiency (Feigner et al., 1994; Wheeler et al., 1996; Remy et al., 1994). The effect of cationic lipid headgroup structure on the phase behaviour of mixtures of cationic and anionic phospholipids was therefore studied. The temperature dependence of mixtures of different of cationic lipids in combination with DOPS was investigated. As stated above, the mixture of DODAC/DOPS (1:1 molar ratio) was found to have a bilayer to hexagonal phase transition temperature (TBri) between 1-6 °C (Figure 4.3/4.4 A). Mixtures of DOPS and DOTMA and DOTAP were both found to have similar bilayer to Hn phase transition temperatures of between 7-13 °C (Figure 4.4 B-C). This result indicates that the structure of the cationic lipid headgroup effects formation of the Hn phase produced in mixtures of cationic and anionic lipids. 82 A B C I I « I.. -I .1 ,. - J . J I it it » t fci .ill i nit I t i i * 40 20 0 -20 -40 40 20 0 -20 -40 40 20 0 -20 -40 PPM PPM PPM F I G U R E 4.4 Effect of cationic lipid headgroup structure on the bilayer to hexagonal Hn phase transition temperature in mixtures with DOPS. Lipid components were present at equal molar amounts in each mixture. 3 1 P NMR spectrum obtained from aqueous dispersions of (A) DODAC/DOPS, (B) DOTMA/DOPS, and (C) DOTAP/DOPS. 3 1 P NMR spectra were recorded at 1, 6 and 13 °C. Temperature is as indicated on each figure. A 50 H z line broadening was applied to each spectrum. For other acquisition parameters see Materials and Methods. Each spectrum presented is representative of at least 2 separate experiments. 83 Influence of Anionic Lipid Structure on the Polymorphism of Mixtures of Cationic and Anionic Lipids In the same way that the acyl chain composition and headgroup structure of cationic lipids can influence the propensity for Hn phase of their mixtures with an anionic lipid, it would be expected that the structure of the anionic lipid could influence the polymorphism of these mixtures. This has important implications as it could explain the differing susceptibility of cells to transfection as due to differences in the acyl chain or headgroup composition of component anionic lipids. It has been demonstrated that cationic lipid acyl chain saturation and headgroup structure modulates the bilayer to Hn phase transition in mixtures with the anionic phospholipid DOPS. It would also be expected that anionic phospholipid headgroup structure also influences the formation of nonbilayer phases in the presence of cationic lipids. Using 3 1 P NMR, the T B H of equimolar mixtures of various anionic phospholipids in combination with DODAC (Table 1) was determined. DODAC in combination with the anionic phospholipids DOPA and CL which have a small phosphate headgroup exhibited the lowest T B H , which could not be determined below - 3 °C. PI which has a bulkier phospho-carbohydrate headgroup was found to have a T B H in the range of 12-17 °C in an equimolar mixture with DODAC. Equimolar mixtures of DODAC with POPG and LBPA (which is the major anionic phospholipid component of late endosomal membranes) (Kobayashi et al., 1998) did not exhibit a well-defined T B H value, as narrow "isotropic" 3 1 P N M R spectra were observed below ~ 20 °C, with HM phase spectra at higher temperatures 84 (DODAC/LBPA shown in Figure 4.5). Such isotropic spectra have been associated with nonbilayer structures such as cubic phases, which basically consist of three-dimensional intercalated networks of lipid tubules or inverted micelles (Rilfors et al., 1986), which like the HM phase, have been associated with lipid intermediates involved during membrane fusion (Siegel, 1999). 85 TABLE 4.1. Temperature-dependent phase transition temperatures of charge neutral mixtures of cationic and anionic lipid. Each transition temperature determination is representative of 2 separate experiments. Cationic Lipid Anionic Lipid Transition Type Determined by 3 1 P NMR Transition Temperature DSDAC DOPS Bilayer-Hn 43-38 °C OSDAC DOPS Bilayer-Hn 25-31 °C DODAC DOPS Bilayer-Hn 1-6 °C DODAC DOPA N.D. Hn < -3 °C DODAC CL N.D. Hn < -3 °C DODAC PI Bilayer-Hn 12-17 °C DOTMA DOPS Bilayer-Hn 6-13 °C DOTAP DOPS Bilayer-Hn 6-13 °C DODAC LBPA Cubic-Hn 22-26 °C DODAC POPG Cubic-Hn 17-23 °C 86 1 I I I I I 1 I 1 10 0 -10 PPM F I G U R E 4 .5 . Temperature-dependent phase transition characteristics of an equimolar mixture of the anionic phospholipid lysobisphosphatidic acid (LBPA) and the cationic lipid DODAC. 3 1 P NMR spectra obtained of an aqueous dispersion of LBPA/DODAC (1:1 molar ratio) at various temperatures as indicated. A 50 Hz line broadening was applied to each spectrum. For other acquisition parameters see Materials and Methods. Spectrum presented are representative of 2 separate experiments. 87 DISCUSSION The propensity of mixtures of bilayer forming cationic and anionic lipids to adopt the Hn phase (Figure 4.1 B-J) likely arises due to a decrease in headgroup area by ion pairing (Bhattacharya & Mandal, 1998) resulting in the decrease of the intrinsic curvature of the lipid monolayers. Using shape arguments (Gruner et al., 1985), this corresponds to a transition from charged lipids with a cylindrical shape to neutral ion paired tetra-acyl zwitterion with a "cone" shape that is compatible with the formation of inverted lipid phases. Related behavior was discussed in Chapter 3, with mixtures of anionic or cationic sterols paired with an oppositely charged diacyl lipids. Formation of a similar sterol-diacyl lipid zwitterion with a molecular shape compatible with assembly of inverted lipid phases is also expected. Furthermore, in this study, it is suggested that a putative octa-acyl zwitterion is formed from the charge neutral mixture of DODAC/CL (2:1 molar ratio) which may be expected to adopt an HM phase with extreme curvature characteristics. The observation that mixtures of synthetic cationic lipids and anionic phospholipids adopt the nonbilayer Hn phase suggests that cellular anionic phospholipids are complicit to the membrane destabilizing activity induced by cationic lipids. For the cationic and anionic lipid mixtures studied here, the bilayer to Hn phase transition temperatures (TBH) may be used as a measure of the spontaneous monolayer curvature, or the tendency of the lipid membrane to "bend" (Tate & Gruner, 1989). As the temperature increases above the T B H , the spontaneous radius of curvature of the lipids organized into the Hn phase decreases (Tate & Gruner, 1989). Using 88 these arguments and the results obtained in this study (Table 1), the spontaneous monolayer curvature for mixtures of a series of diacyl-dimethyammonium chlorides cationic lipids and DOPS is DODAC/DOPS > OSDAC/DOPS > DSDAC/DOPS. In other words, the presence of an unsaturated cationic lipid DODAC incorporated into a cellular membrane containing anionic lipid is expected have a greater membrane perturbing effect than the saturated analogue DSDAC. This result is corroborated by transfection studies which show higher levels of transfection using unsaturated cationic lipids versus saturated analogues (Feigner etal., 1994; Hope etal., 1998). It has been shown that cationic lipids induce disruption of purified lysosomes (Wattiaux er al., 1997) and endosomal compartments (Zhou & Huang, 1994; El Ouahabi er al., 1997). Time-resolved studies of the mixing of lipid components of cationic lipoplexes with intracellular membranes suggests that this step occurs within late endosomal compartments (Wrobel & Collins, 1995). The anionic lipid content of endosomal membranes increases dramatically with the appearance of lysobisphosphatidic acid (LBPA) as a major anionic component of and late endosome (Kobayashi et al., 1998) and lysosome membranes (Brotherus & Renkonen, 1977). Here it is directly shown that LBPA in the presence of an equimolar content of cationic lipid adopts inverted cubic and Hn phases (Figure 4.5). It is tempting to speculate that due to its abundance in late endosomes that the anionic lipid LBPA plays an important role in membrane disruption induced by cationic lipids. 89 5 FIGURE 4.6. Mechanism for disruption of cellular membranes mediated by cationic lipoplexes. Following binding (Step 1 ) and endocytosis (Step 2 ) into a target cell, cationic lipoplexes are transferred to late endosomal compartments (Step 3 ) . Cationic lipids induce destabilization of the endosomal membrane leading to possible fusion (Step 4) of the lipoplex with the endosomal membrane, or complete remodeling of the endosomal membrane into a nonbilayer phase (Step 5 ) . 90 A model for the mechanism of action of cationic lipids which is an extension of that previously suggested by Szoka and co-workers (Xu & Szoka, 1996; Zelphati & Szoka, 1996) is proposed (Figure 4.6). Following endocytosis (step 1), the lipoplex is transferred from early to late endosomes (step 2), where the content of anionic phospholipid is relatively high (Kobayashi et al., 1998). Within the late endosomal compartment (step 3), the anionic phospholipids form ion pairs with the cationic lipids and promote dissociation of the lipoplex (Xu & Szoka, 1996; Zelphati & Szoka, 1996; Bhattacharya & Mandal, 1998). Concomitantly, the charge neutral mixtures of cationic anionic lipid which adopt nonbilayer structure promote the destabilization of the endosomal membrane producing fusion (step 4) or gross rupture of the endosomal membrane through the formation of nonbilayer structure (step 5). The presence of additional nonbilayer lipids within the lipoplex, or the endosomal membrane would enhance the destabilization induced by cationic lipids. In this study it was demonstrated that synthetic cationic lipids used in lipoplex formulations promote the formation of nonbilayer phases in combination with a variety of anionic phospholipids. This is likely the mechanism by which cationic lipids disrupt cellular membranes and promote the intracellular delivery of foreign macromolecules. 3 1 P NMR analysis of structural properties of anionic phospholipids in combination with cationic lipids may be used as a convenient tool to rationally test existing and design novel cationic lipid reagents. 91 CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS In this work the role of lipid polymorphism in the activity, design and mechanism of three distinct lipid-based intracellular delivery systems is described. In Chapters 2 and 3, the propensity of systems containing ionizable anionic lipids either alone or in combination with cationic lipid to adopt nonbilayer phase structure under conditions of neutral surface charge was used to explain the behaviour of a well characterized pH-sensitive liposome system and then exploited in the design of a novel tunable pH-sensitive liposome system. In Chapter 4, the discovery of nonbilayer phases observed in mixtures of cationic and anionic lipids was investigated in detail, and related to the mechanism of how cationic lipids disrupt cell membranes. In this latter case it was found that the ability of cationic lipids to disrupt cell membranes was dependent upon cellular anionic lipids as a co-requirement for the formation of membrane disruptive nonbilayer phases. These results will be summarized in turn and future directions outlined. In Chapter 2, a common anionic component of pH-sensitive liposomes was studied. Cholesteryl hemisuccinate (CHEMS) was shown to assemble into large unilamellar vesicles (LUVs) which exhibit pH-dependent fusion characteristics. The ability of CHEMS bilayers to undergo' membrane fusion was found to be related to the nonbilayer phase preferences of this lipid at acidic pH. CHEMS is not a unique lipid in its ability to adopt nonbilayer phases upon charge neutralization of the 92 headgroup, however, CHEMS is the first sterol lipid described to adopt the inverted hexagonal (HM) phase in isolation. The ability of CHEMS to adopt a nonbilayer phase under conditions of headgroup neutralization likely contributes to the fusion observed in pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and CHEMS. In Chapter 3, the design of novel pH-sensitive liposomes is described in which CHEMS was mixed with increasing amounts of a cationic lipid. It was observed that bilayer vesicles could be prepared from mixtures of anionic and cationic lipid which contained excess negative or positive surface charge, and that these systems underwent fusion as the surface charge was decreased. It was also discovered that fusion of lipid systems composed of cationic and anionic lipid reflected a propensity of these charge neutral mixtures to adopt nonbilayer phases. Similarities exist between stabilized DOPE pH-sensitive liposomes and tunable pH-sensitive liposomes. First the anionic lipid in these systems plays a similar bilayer-stabilizing role. In DOPE-based systems, the anionic stabilizing lipid comprises the total amount of the anionic lipid, while in mixed cationic/anionic liposomes, the stabilizing lipid reflects only the anionic lipid that is present in excess over the cationic lipid. In tunable pH-sensitive liposomes, the role of DOPE, which is the nonbilayer lipid in traditional pH-sensitive liposome systems, is replaced by the cationic:anionic lipid pairs, which prefer nonbilayer phases in isolation. There is considerable versatility in this platform technology. Tunable pH-sensitive liposomes 93 can be prepared from cationic and anionic lipids to prepare a variety of fusogenic systems, negative or positive in charge, sensitive to acidic or basic conditions. In Chapter 4, the ability of cationic and anionic lipids to adopt nonbilayer phases was investigated and related to the mechanism of action of cationic lipids. It was determined that many criteria which impact the transfection efficiency mediated by cationic lipids such as cationic lipid headgroup structure, acyl chain saturation, and the presence of additional lipids also impact the ability of cationic lipids to adopt nonbilayer phases in the presence of anionic phospholipids. A strong correlation is observed between the structural characteristics of cationic lipids which mediate potent transfection activities and the ability of cationic lipids to readily promote the formation of nonbilayer phases in mixtures with anionic phospholipids. The structural characteristics of anionic phospholipids were also demonstrated to modulate the nonbilayer phase preferences in the presence of cationic lipids. This suggests that the differences in transfection mediated by cationic lipids may be in large part determined by the lipid composition of the membranes in a cell of interest. Comparisons may then be made based on the cellular phospholipid distribution and the efficacy of cationic-lipid mediated transfection systems. The elucidation of how cationic lipids remodel the molecular architecture of anionic lipid bilayers should also be pursued towards the design of novel chemical entities which have potent and precise membrane perturbing activities. 94 BIBLIOGRAPHY Akashi, K., Miyata, H., Itoh, H. & Kinosita, K.J. (1996) Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys J . , 71, 3242-3250. Bailey, A.L. & Cullis, P.R. (1994) Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids. Biochemistry, 33, 12573-12580. Bailey, A.L. & Cullis, P.R. (1997) Membrane fusion with cationic liposomes: effects of target membrane lipid composition. Biochemistry, 36, 1628-1634. Bangham, A. D. & Home, R. W. (1964) Negative staining of phospholipids and their structural modification by surface agents as observed in the electron microscope. J Mol Biol., 8, 660-668. Bartlett, M.G. (1959) Phosphorus assay in column chromatography. J Biol Chem., 234, 466-468. Batzri, S. & Korn, E.D. (1973) Single bilayer liposomes prepared without sonication. Biochim Biophys Acta, 298, 1015-1019. Bennett, C.F., Chiang, M.Y., Chan, H., Shoemaker, J.E. & Mirabelli, C.K. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol., 41, 1023-1033. Bhamidipati, S.P. & Hamilton, J.A. (1995) Interactions of lyso 1-palmitoylphosphatidylcholine with phospholipids: a 13C and 31P NMR study. Biochemistry, 34, 5666-5677. Bhattacharya, S. & Mandal, S.S. (1998) Evidence of interlipidic ion pairing in anion-induced DNA release from cationic amphiphile-DNA complexes. Mechanistic implications in transfection. Biochemistry, 37, 7764-7777. Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B. & Behr, J.P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA, 92, 7297-7301. Brotherus, J. & Renkonen, O. (1977) Subcellular distributions of lipids in cultured BHK cells: evidence for the enrichment of lysobisphosphatidic acid and neutral lipids in lysosomes. J Lipid Res., 18, 191-202. Brown, M.F. (1994) Modulation of rhodopsin function by properties of the membrane bilayer. Chem Phys Lipids, 73,159-180. 95 Brunner, J . , Skrabal, P. & Hauser, H. (1976) Single bilayer vesicles prepared without sonication. Physico-chemical properties. Biochim Biophys Acta, 455, 322-331. Cheetham, J.J., Nir, S., Johnson, E., Flanagan, T.D. & Epand, R.M. (1993) The effects of membrane physical properties on the fusion of Sendai virus with human erythrocyte ghosts and liposomes. Analysis of kinetics and extent of fusion. J Biol Chem., 269, 5467-5472. Chernomordik, L.V., Leikina, E., Frolov, V., Bronk, P. & Zimmerberg, J . (1997) An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J Cell Bio., 136, 81-93. Chernomordik, L.V., Vogel, S.S., Sokoloff, A., Onaran, H.O., Leikina, E.A. & Zimmerberg, J. (1993) Lysolipids reversibly inhibit Ca(2+)-, GTP- and pH-dependent fusion of biological membranes. FEBS Letters, 318, 71-76. Chernomordik, L.V. & Zimmerberg, J. (1995) Bending membranes to the task: structural intermediates in bilayer fusion. CurrOpin Struct Biol., 5, 541-547. Collins, D., Litzinger, D.C. & Huang, L. (1990) Structural and functional comparisons of pH-sensitive liposomes composed of phosphatidylethanolamine and three different diacylsuccinylglycerols. Biochim Biophys Acta, 1025, 234-242. Connor, J . , Yatvin, M.B. & Huang, L. (1984) pH-sensitive liposomes: acid-induced liposome fusion. Proc Natl Acad Sci U S A, 81, 1715-1718. Cullis, P.R. & de Kruijff, B. (1978a) The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study. Biochim Biophys Acta, 513, 31-42. Cullis, P.R. & de Kruijff, B. (1978b) Polymorphic phase behaviour of lipid mixtures as detected by 31P NMR. Evidence that cholesterol may destabilize bilayer structure in membrane systems containing phosphatidylethanolamine. Biochim Biophys Acta, 507, 207-218. Cullis, P.R. & de Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim Biophys Acta, 559, 399-420. Cullis, P.R. & Hope, M.J. (1978) Effects of fusogenic agent on membrane structure of erythrocyte ghosts and the mechanism of membrane fusion. Nature, 271, 672-674. Cullis, P.R., Verkleij, A.J. & Ververgaert, P.H. (1978) Polymorphic phase behaviour of cardiolipin as detected by 31P NMR and freeze-fracture techniques. Effects of calcium, dibucaine and chlorpromazine. Biochim Biophys Acta, 513, 11-20. 96 Daleke, D.L. & Lyles, J.V. (2000) Identification and purification of aminophospholipid flippases. Biochim Biophys Acta, 1486, 108-127. de Kroon, A.I., Timmermans, J.W., Killian, J.A. & de Kruijff, B. (1990) The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR. Chem Phys Lipids, 54, 33-42. De Rosa, M. (1996) Archaeal lipids: structural features and supramolecular organization. Thin Solid Films 284-285, 13-17. Deamer, D.W. (1978) Preparation and properties of ether-injection liposomes. Ann N YAcad Sci., 308, 250-258. Deamer, D. W. (1985) Boundary structures are formed by organic components of the Murchison carbonaceous chondrite. Nature, 317, 792-794. Deamer, D.W., Leonard, R., Tardieu, A. & Branton, D. (1970) Lamellar and hexagonal lipid phases visualized by freeze-etching. Biochim Biophys Acta, 219, 47-60. Deamer, D.W. & Oro, J. (1980) Role of lipids in prebiotic structures. Biosystems, 12, 167-175. Deamer, D.W. & Pashley, R.M. (1989) Amphiphilic components of the Murchison carbonaceous chondrite: surface properties and membrane formation. Orig Life Evol Biosph., 19, 21-38. Discher, B.M., Won, Y.Y., Ege, D.S., Lee, J . C , Bates, F.S., Discher, D.E. & Hammer, D.A. (1999) Polymersomes: tough vesicles made from diblock copolymers. Science, 284, 1143-1146. Duzgunes, N. & Feigner, P.L. (1993) Intracellular delivery of nucleic acids and transcription factors by cationic liposomes. Methods Enzymol., 221, 303-306. Duzgunes, N., Goldstein, J.A., Friend, D.S. & Feigner, P.L. (1989) Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles. Biochemistry, 28, 9179-9184. Duzgunes, N., Straubinger, R.M., Baldwin, P.A., Friend, D.S. & Papahadjopoulos, D. (1985) Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes. Biochemistry, 24, 3091-3098. Eastman, S.J., Hope, M.J. & Cullis, P.R. (1991) Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients. Biochemistry, 30, 1740-1745. 97 El Ouahabi, A., Thiry, M., Pector, V., Fuks, R., Ruysschaert, J.M. & Vandenbranden, M. (1997) The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett, 414, 187-192. Ellens, H., Bentz, J. & Szoka, F.C. (1984) pH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact. Biochemistry, 23, 1532-1538. Ellens, H., Bentz, J. & Szoka, F.C. (1985) H+- and Ca2+-induced fusion and destabilization of liposomes, Biochemistry, 24, 3099-3106. Ellens, H., Siegel, D.P., Alford, D., Yeagle, P.L, Boni, L, Lis, LJ, Quinn, P.J. & Bentz, J . (1989) Membrane fusion and inverted phases. Biochemistry, 28, 3692-3703. Epand, R.M. (1998) Lipid polymorphism and protein-lipid interactions. Biochim Biophys Acta, 1376, 353-368. Farhood, H., Serbina, N. & Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim Biophys Acta, 1235, 289-295. Fawell, S., Seery, J . , Daikh, Y., Moore, C , Chen, L.L., Pepinsky, B. & Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A, 91, 664-668. Feigner, J.H., Kumar, R., Sridhar, C.N., Wheeler, C.J., Tsai, Y.J. , Border, R., Ramsey, P., Martin, M. & Feigner, P.L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem., 269, 2550-2561. Feigner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. & Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA, 84, 7413-7417. Friend, D.S., Papahadjopoulos, D. & Debs, R.J. (1996) Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. Biochim Biophys Acta, 1278, 41-50. Gallay, J . & de Kruijff, B. (1982) Correlation between molecular shape and hexagonal HII phase promoting ability of sterols. FEBS Lett, 143, 133-136. Gao, X. & Huang, L. (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun., 179, 280-285. 98 Gerasimov, O.V., Rui, Y. & Thompson, D.H. (1996) Triggered Release from Liposomes Mediated by Physically and Chemically Induced Phase Transitions. Vesicles (ed. by Morton Rosoff), p. 679. Marcel Dekker, Inc., New York. Gorter, E. & Grendel, F. (1925) On Biomolecular Layers of Lipoids on the Chromocytes of the Blood. J Exp Med, 41, 439-443. Gruner, S.M. (1985) Intrinsic curvature hypothesis for biomembrane lipid composition: a role for nonbilayer lipids. Proc Natl Acad Sci USA, 82, 3665-3669. Gruner, S.M., Cullis, P.R., Hope, M.J. & Tilcock, O P . (1985) Lipid polymorphism: the molecular basis of nonbilayer phases. Ann Rev Biophysics & Biophysical Chem, 14, 211-238. Hargreaves, W.R. & Deamer, D.W. (1978) Liposomes from ionic, single-chain amphiphiles. Biochemistry, 17, 3759-3768. Hazemoto, N., Harada, M., Suzuki, S., Kaiho, F., Haga, M. & Kato, Y. (1993) Effect of phosphatidylcholine and cholesterol on pH-sensitive liposomes. Chem Pharm Bull., 41, 1003-1006. Hodgson, O P . & Solaiman, F. (1996) Virosomes: cationic liposomes enhance retroviral transduction. Nat Biotechnol., 14, 339-342. Hope, M.J. & Cullis, P.R. (1979) The bilayer stability of inner monolayer lipids from the human erythrocyte. FEBS Lett, 107, 323-326. Hope, M.J., Mui, B., Ansell, S. & Ahkong, Q.F. (1998) Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs. Mol Mem Bio., 15, 1-14. Hope, M.J., Walker, D.C. & Cullis, P.R. (1983) Ca2+ and pH induced fusion of small unilamellar vesicles consisting of phosphatidylethanolamine and negatively charged phospholipids: a freeze fracture study. Biochem Biophys Res Comm., 110, 15-22. Hope, M.J., Wong, K.F. & Cullis, P.R. (1989) Freeze-fracture of lipids and model membrane systems. J Electron Micro Tech., 13, 277-287. Huang, C. (1969) Studies on phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry, 8, 344-352. Jacobson, K. & Dietrich, C. (1999) Looking at lipid rafts? Trends Cell Biol., 9, 87-91. Janoff, A.S., Kurtz, C.L., Jablonski, R.L., Minchey, S.R., Boni, L.T., Gruner, S.M., Cullis, P.R., Mayer, L.D. & Hope, M.J. (1988) Characterization of cholesterol 99 hemisuccinate and alpha-tocopherol hemisuccinate vesicles. Biochim Biophys Acta., 941, 165-175. Jizomoto, H., Kanaoka, E. & Hirano, K. (1994) pH-Sensitive liposomes composed of tocopherol hemisuccinate and of phosphatidylethanolamine including tocopherol hemisuccinate. Biochim Biophys Acta., 1213, 343-348. Kaler, E.W., Murthy, A.K., Rodriguez, B.E. & Zasadzinski, J.A. (1989) Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants. Science, 245,1371-1374. Kariko, K., Megyeri, K., Xiao, Q. & Barnathan, E.S. (1994) Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA. FEBS Lett, 352, 41-44. Kobayashi, T., Stang, E., Fang, K.S., de Moerloose, P., Parton, R.G. & Gruenberg, J. (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature, 392, 193-197. Koltover, I., Salditt, T., Radler, J.O. & Safinya, C R . (1998) An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science, 281, 78-81. Koopman, G., Reutelingsperger, C P . , Kuijten, G.A., Keehnen, R.M., Pals, S T . & van Oers, M.H. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood, 84, 1415-1420. Korlach, J. , Schwille, P., Webb, W.W. & Feigenson, G.W. (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci USA, 96, 8461-8466. Kulkarni, V.S., Anderson, W.H. & Brown, R.E. (1995) Bilayer nanotubes and helical ribbons formed by hydrated galactosylceramides: acyl chain and headgroup effects. Biophys J, 69, 1976-1986. Lai, M.Z., Duzgunes, N. & Szoka, F.C. (1985b) Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes. Biochemistry, 24, 1646-1653. Lai, M.Z., Vail, W.J. & Szoka, F.C. (1985a) Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate. Biochemistry, 24, 1654-1661. Ledley, F.D. (1995) Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther., 6, 1129-1144. 100 Legendre, J.-Y. & Szoka, J.F. (1992) Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: Comparison with cationic liposomes. Pharm Res., 9, 1235-1242. Lewis, R.N., Mannock, D.A., McElhaney, R.N., Turner, D.C. & Gruner, S.M. (1989) Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions. Biochemistry, 28, 541-548. Li, S., Tseng, W.C., Stolz, D.B., Wu, S.P., Watkins, S.C. & Huang, L. (1999) Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther., 6, 585-594. Lindau, M. & Aimers, W. (1995) Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. CurrOpin Cell Biol., 7, 509-517. Lindblom, G. & Rilfors, L. (1992) Nonlamellar phases formed by membrane lipids. Adv Colloid Interface Sci., 41, 101-125. Litzinger, D.C. & Huang, L. (1992) Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications. Biochim Biophys Acta, 1113, 201-227. Liu, Y., Mounkes, L.C., Liggitt, H.D., Brown, C.S., Solodin, I., Heath, T.D. & Debs, R.J. (1997) Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat Biotechnol., 15, 167-173. Loose-Mitchell, D.S. (1988) Antisense nucleic acids as a potential class of pharmaceutical agents. Trends Pharmacol Sci., 9, 45-47. Luisi P.L., Walde, P., and Oberholzer, T. (1999) Lipid vesicles as posible intermediates in the origin of life. Curr Opin Coll Inter Sci., 4, 33-39. Lutwyche, P. Cordeiro, C , Wiseman, D.J., St-Louis, M., Uh, M., Hope, M.J., Webb, M.S. & Finlay, B.B. (1999) Intracellular delivery and antibacterial activity of gentamicin encapsulated in pH-sensitive liposomes. Antimicrol Agents Chemotherapy, 42, 2511-2520. Madden, T.D. & Cullis, P.R. (1982) Stabilization of bilayer structure for unsaturated phosphatidylethanolamines by detergents. Biochim Biophys Acta, 684, 149-153. Malone, R.W., Feigner, P.L. & Verma, I.M. (1989) Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci U S A, 86 , 6077-6081. Markin, V.S., Kozlov, M.M. & Borovjagin, V.L. (1984) On the theory of membrane fusion. The stalk mechanism. Gen Physiol Biophys, 3, 361-377. 101 Mayer, L.D., Hope, M.J. & Cullis, P.R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta, 858, 161-168. Mayer, L.D., Hope, M.J., Cullis, P.R. & Janoff, A.S. (1985) Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles. Biochim Biophys Acta, 817, 193-196. Mayhew, E., Papahadjopoulos, D., O'Malley, J.A., Carter, W.A. & Vail, W.J. (1977) Cellular uptake and protection against virus infection by polyinosinic-polycytidylic acid entrapped within phospholipid vesicles. Mol Pharmacol., 13, 488-495. Morein, S., Andersson, A., Rilfors, L. & Lindblom, G. (1996) Wild-type Escherichia coli cells regulate the membrane lipid composition in a "window" between gel and non-lamellar structures. J Biol Chem., 271, 6801-6809. Mui, B.L., Dobereiner, H.G., Madden, T.D. & Cullis, P.R. (1995) Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles. Biophys J., 69, 930-941. Nayar, R. & Schroit, A.J. (1985) Generation of pH-sensitive liposomes: use of large unilamellar vesicles containing N-succinyldioleoylphosphatidylethanolamine. Biochemistry, 24, 5967-5971. Needham, D., Mcintosh, T.J. & Evans, E. (1988) Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. Biochemistry, 27, 4668-4673. Olson, F., Hunt, C.A., Szoka, F.C, Vail, W.J. & Papahadjopoulos, D. (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta, 557, 9-23. Papahadjopoulos, D., Vail, W.J., Jacobson, K. & Poste, G. (1975) Cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles. Biochim Biophys Acta, 394, 483-491. Pardridge, W.M. & Boado, R.J. (1991) Enhanced cellular uptake of biotinylated antisense oligonucleotide or peptide mediated by avidin, a cationic protein. FEBS Lett, 288, 30-32. Park, Y.G., Nesterova, M., Agrawal, S. & Cho-Chung, Y.S. (1999) Dual blockade of cyclic AMP response element- (CRE) and AP-1-directed transcription by CRE-transcription factor decoy oligonucleotide, gene-specific inhibition of tumor growth. J Biol Chem., 274, 1573-1580. Pott, T., Maillet, J.C. & Dufourc, E.J. (1995) Effects of pH and cholesterol on DM PA membranes: a solid state 2H- and 31P-NMR study. Biophys J., 69, 1897-1908. 102 Radler, J.O., Koltover, I., Salditt, T. & Safinya, C R . (1997) Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science, 275, 810-814. Rand, R.P. & Sengupta, S. (1972) Cardiolipin forms hexagonal structures with divalent cations. Biochim Biophys Acta, 255, 484-492. Remy, J.S., Sirlin, C , Vierling, P. & Behr, J.P. (1994) Gene transfer with a series of lipophilic DNA-binding molecules. Bioconjug Chem., 5, 647-654. Renshaw, P.F., Janoff, A.S. & Miller, K.W. (1983) On the nature of dilute aqueous cholesterol suspensions. J Lipid Res., 24, 47-51. Rilfors, L, Eriksson, P.O., Arvidson, G. & Lindblom, G. (1986) Relationship between three-dimensional arrays of "lipidic particles" and bicontinuous cubic lipid phases. Biochemistry, 25, 7702-7711. Scherer, P.G. & Seelig, J. (1989) Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles. Biochemistry, 28, 7720-7728. Siegel, D.P. (1999) The modified stalk mechanism of lamellar/inverted phase transitions and its implications for membrane fusion. Biophys J., 76, 291-313. Siegel, D.P. & Epand, R.M. (1997) The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: Implications for membrane fusion mechanisms. Biophys J., 73, 3089-3111. Silvius, J.R. & Zuckermann, M.J. (1993) Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion. Biochemistry, 32, 3153-3161. Simidjiev, I., Stoylova, S., Amenitsch, H., Javorfi, T., Mustardy, L, Laggner, P., Holzenburg, A. & Garab, G. (2000) Self-assembly of large, ordered lamellae from nonbilayer lipids and integral membrane proteins in vitro. Proc Natl Acad Sci USA, 97, 1473-1476. Simons, K. & Ikonen, E. (1997) Functional rafts in cell membranes. Nature, 387, 569-572. Singer, S. J. and Nicholson, G. L. (1972) The fluid mosaic model of cell membranes. Science, 175, 720-731. Stamatatos, L, Leventis, R., Zuckermann, M.J. & Silvius, J.R. (1988) Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry, 27, 3917-3925. 103 Sternberg, B., Hong, K., Zheng, W. & Papahadjopoulos, D. (1998) Ultrastructural characterization of cationic liposome-DNA complexes showing enhanced stability in serum and high transfection activity in vivo. Biochim Biophys Acta, 1375, 23-35. Straubinger, R.M. (1993) pH-Sensitive liposomes for delivery of macromolecules into cytoplasm of cultured cells. Methods Enzymol., 221, 361-376. Straubinger, R.M., Duzgunes, N., Papahadjopoulos, D. (1985) pH-sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules. FEBS Lett., 179, 148-154. Straubinger, R.M., Hong, K., Friend, D.S., Papahadjopoulos, D. (1983) Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles. Cell, 32, 1069-1079. Struck, D.K., Hoekstra, D. & Pagano, R.E. (1981) Use of resonance energy transfer to monitor membrane fusion. Biochemistry, 20, 4093-4099. Szoka, F.J. & Papahadjopoulos, D. (1978) Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA, 75, 4194-4198. Tannock, I.F. & Rotin, D. (1989) Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res., 49, 4373-4384. Taraschi, T.F., de Kruijff, B. & Verkleij, A.J. (1983) The effect of an integral membrane protein on lipid polymorphism in the cardiolipin-Ca2+ system. Eur J Biochem., 129, 621-625. Tate, M.W. & Gruner, S.M. (1989) Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes. Biochemistry, 28, 4245-4253. Templeton, N.S., Lasic, D.D., Frederik, P.M., Strey, H.H., Roberts, D.D. & Pavlakis, G.N. (1997) Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol., 15, 647-652. Tilcock, C P . , Bally, M.B., Farren, S.B. & Cullis, P.R. (1982) Influence of cholesterol on the structural preferences of dioleoylphosphatidylethanolamine-dioleoylphosphatidylcholine systems: a phosphorus-31 and deuterium nuclear magnetic resonance study. Biochemistry, 21, 4596-4601. 104 Tilcock, C P . , Bally, M.B., Farren, S.B., Cullis, P.R. & Gruner, S.M. (1984) Cation-dependent segregation phenomena and phase behavior in model membrane systems containing phosphatidylserine: influence of cholesterol and acyl chain composition. Biochemistry, 23, 2696-2703. Tocanne, J.-F. and Teissie, J. (1990) Ionization of phospholipids and phospholipid-supported interfacial lateral diffusion of protons in model membrane systems. Biochim Biophys Acta, 1031, 111 -142. Tycko, B. & Maxfield, F.R. (1982) Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell, 28, 643-651. van der Woude, I., Visser, H.W., ter Beest, M.B., Wagenaar, A., Ruiters, M.H., Engberts, J.B. & Hoekstra, D. (1995) Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system. Biochim Biophys Acta, 1240, 34-40. Verkleij, A.J. , van Echteld, C.J., Gerritsen, W.J., Cullis, P.R., de & Kruijff, B. (1980) The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions. Biochim Biophys Acta, 600, 620-624. Wattiaux, R., Jadot, M., Warnier-Pirotte, M.T. & Wattiaux-De, C S . (1997) Cationic lipids destabilize lysosomal membrane in vitro. FEBS Lett, 417, 199-202. Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H. & Rothman, J.E. (1998) SNAREpins: minimal machinery for membrane fusion. Cell, 92, 759-772. Wheeler, C.J., Sukhu, L, Yang, G., Tsai, Y., Bustamente, C , Feigner, P., Norman, J. & Manthorpe, M. (1996) Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes. Biochim Biophys Acta, 1280, 1-11. Wilson-Kubalek, E.M., Brown, R.E., Celia, H. & Milligan, R.A. (1998) Lipid nanotubes as substrates for helical crystallization of macromolecules. Proc Natl Acad Sci USA, 95, 8040-8045. Wrobel, I. & Collins, D. (1995) Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim Biophys Acta, 1235, 296-304. Wu, X., Lee, K.H. & Li, Q.T. (1996) Stability and pH sensitivity of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles. Biochim Biophys Acta, 1284, 13-19. 105 Wyman, T.B., Nicol, F., Zelphati, O., Scaria, P.V., Plank, C. & Szoka, F.C.J. (1997) Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry, 36, 3008-3017. Xu, Y., Hui, S.W., Frederik, P. & Szoka, F.C.J. (1999) Physicochemical characterization and purification of cationic lipoplexes. Biophys J., 77, 341-353. Xu, Y. & Szoka, F .C, Jr. (1996) Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry, 35, 5616-5623. Yager, P., Chappell, J . & Archibald, D.D. (1991) When lipid bilayers won't form liposomes: tubules, helices and cochleate cylinders. Biomembrane Structure and Function-The State of the Art. (ed. by B.P. Gaber and K.R.K. Easwaran), p.1. Adenine Press, Schenectady, New York. Yatvin, M.B., Kreutz, W., Horwitz, B.A. & Shinitzky, M. (1980) pH-sensitive liposomes: possible clinical implications. Science, 210, 1253-1255. Yeagle, P.L. (1993) Phosphorus-31 nuclear magnetic resonance in membrane fusion studies. Methods Enzymol., 220, 68-79. Yeagle, P.L., Smith, FT., Young, J.E. & Flanagan, T.D. (1994) Inhibition of membrane fusion by lysophosphatidylcholine. Biochemistry, 33, 1820-1827. Zelphati, O. & Szoka, F.C, Jr. (1996) Mechanism of oligonucleotide release from cationic liposomes. Proc Natl Acad Sci USA, 93, 11493-11498. Zemb, T , Dubois, M., Deme, B. & Gulik-Krzywicki, T. (1999) Self-assembly of flat nanodiscs in salt-free catanionic surfactant solutions. Science, 283, 816-819. Zhou, X. & Huang, L. (1994) DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim Biophys Acta, 1189, 195-203. Zuidam, N.J. & Barenholz, Y. (1997) Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim Biophys Acta, 1329, 211-222. 106 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089758/manifest

Comment

Related Items