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Lipid polymorphism and intracellular delivery Hafez, Ismail Mahmoud


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 pHdependent 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 pHf 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, 3α-[N-(N',N'- dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Choi) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and ³¹P 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 ³¹P NMR, it is shown that addition of cationic lipids to bilayer-adopting anionic phospholipids results in the formation of the nonbilayer inverted hexagonal (H[sub ii] phase. Further, the presence of "helper" lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipidmediated transfection, also facilitates the formation of the H[sub ii] 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.

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