UBC Theses and Dissertations
Development of stabilized plasmid lipid particles as intracellular gene delivery vehicles Palmer, Lorne R.
This thesis focuses on the development of an efficient, non-viral system capable of delivering genetic information (i.e. plasmid DNA) to cells in vitro and eventually in vivo. The approach taken is to develop small liposomal systems containing plasmid that can deliver intact DNA to disease sites in vivo, and then to examine methods to improve the transfection potential of these systems. In Chapter 2, a detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These "stabilized plasmid-lipid particles" (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid molecule per particle and fully protect the encapsulated plasmid from digestion by serum nucleases. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyl-dimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of "fusogenic" lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. In Chapter 3, the ligand employed is a cationic poly (ethylene glycol) (PEG) lipid (CPL) consisting of a lipid anchor and a PEG3400 spacer chain with 1, 2, 4, or 8 positive charges at the end of the PEG (CPL1, CPL2 , CPL4 , and CPL8). These CPL are introduced into preformed LUVs by a post-insertion method. The uptake of LUV-CPL by BHK cells is assessed both quantitatively and qualitatively. The LUV-CPL4 system (containing 4 positive charges) exhibits dramatically improved uptake compared to LUV in the absence of CPL. Chapter 4 examines the influence of CPL4 on the transfection potency of SPLP. It is shown that up to 4 mol% CPL4 can be inserted into preformed SPLP, resulting in up to 50-fold enhancements in uptake into baby hamster kidney (BHK) cells. In the presence of Ca2+ this results in up to 10[superscript 6]-fold enhancements in transgene expression as compared to SPLP in the absence of CPL4 or Ca2+. These transfection levels are comparable to those observed for plasmid DNA-cationic lipid complexes (lipoplexes) but without the toxic effects noted for lipoplex systems. It is concluded that in the presence of Ca2+ and appropriate ligands to stimulate uptake, SPLP are highly transfection potent, supporting their potential as in vivo gene therapy vectors.
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