UBC Theses and Dissertations
Biophysical characterization of catonic liposome/plasmid DNA complexes for gene therapy Wasan, Ellen K.
The goal of gene therapy is to achieve expression of an exogenous gene that results in a specific functional change. Cationic liposome-based carriers of plasmid DNA ("lipoplexes") are the most widely used method of nonviral DNA delivery. Lipoplexes form through electrostatic interactions between DNA and liposomes. This thesis investigates how lipid composition and ionic environment affect the biophysical behavior of lipoplexes and their transfection ability. A major concern regarding lipoplex development is their instability in a physiological environment or salt solutions. Lipoplexes containing a cationic lipid and the transfection-helper lipid dioleoylphosphatidylethanolamine (DOPE) exhibit heterogeneous morphology and variable activity. A charge ratio near neutrality or the presence of salts promotes super-aggregation (particle mean diameter > 1 µm). Cryo-transmission electron microscopy showed that liposome morphology differs when plasmid, oligodeoxynucleotides or phosphate anions are added. In conjunction with lipid mixing assays and particle size analysis, these observations demonstrate that charge ratio and charge density are critical for lipoplex structure. The extent of lipid mixing and aggregation, during or after lipoplex formation, is influenced by lipid composition as well as the presence of salt or serum. A multi-step lipid-mixing assay to model in vitro transfection demonstrated that lipoplexes with relatively high in vitro transfection undergo lipid-mixing reactions after salt or serum interactions. Significantly, liposomal internal aqueous contents were retained in the lipoplexes. This may allow the codelivery of drugs within the lipoplexes during transfection of cells. Smaller, salt-stable lipoplexes are desirable for systemic in vivo use. It was also of great interest to pursue development of lipoplexes capable of trapping drugs for codelivery (e.g. transfection enhancers). Such lipoplexes must be transfection-competent, possess a large trapped volume and have the ability to maintain an ion gradient for drug loading. This was achieved by replacing DOPE with cholesterol and including poly(ethylene glycol) to stabilize the lipoplexes against aggregation. A model amine drug, vincristine, was loaded into lipoplexes via a pH gradient. This research introduces the novel concept that salt-stable lipoplexes can be generated which can be loaded with secondary compounds for codelivery, adding a new functionality to cationic liposomes as carriers of DNA for gene therapy.
Item Citations and Data