| The importance of gene therapy research, with its potential of offering cures for cancer, tumor, cystic fibrosis and other such hereditary diseases, hardly needs explication. Gene therapy tackles genetic diseases through the identification of the errant genes) and the corresponding target cell(s). What remains then is the delivery of the former to the latter, and this question of “how” has drawn attention from many different scientific disciplines in recent years. Several methods have been explored, the most promising and clinically feasible of which center on the idea of using a carrier to deliver DNA to cell nuclei. Since their induction into the field of gene therapy in the mid 1980's, cationic lipids, with their ability to carry large pieces of DNA, versatility with respect to different types of cells, and ease of application have shown great potential as a powerful DNA carrier. This dissertation addresses the physical interactions involved in the process of DNA delivery using cationic lipid carriers and presents our current understanding of the mechanisms and pathways of DNA uptake by mammalian cells, which aids the optimization of the DNA carrier.; X-ray diffraction and biological protein assays, combined with conventional optical microscopy and confocal laser scanning microscopy techniques, have enabled us to establish correlations between structure, function, and behavior of cationic lipid/DNA complexes before, during, and after delivery to cells. Upon mixing, cationic liposomes and DNA spontaneously self-assemble into two condensed structures, lamellar Lcα and inverted hexagonal HcII, driven by the entropic gain through the release of the counter-ions. HcII complexes exhibit fusion and DNA release in the cytoplasm, resulting in high transfection efficiency (TE). Lcα complexes enter the cell through endocytosis, after which complexes with low membrane charge density (σ) remain trapped by the endosome, causing low TE, and those with high σ escapes the endosome, inducing high TE. TE increases exponentially with σ, indicating an activated process where the energy barrier is lowered as σ increases. Further probing of subcellular interactions in live cells promises completion of the map of transfection pathway. |
