UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Influence of calcium on the transfection properties of lipid-based gene delivery systems Lam, Angela Man Iu

Abstract

This thesis is focused on examining the influence of calcium on the transfection potencies and the intracellular processing of two lipid-based gene delivery systems: plasmid DNA-cationic lipid complexes, and stabilized plasmid-lipid particles (SPLP). Results in Chapter 2 demonstrate that calcium can increase the in vitro transfection potency of plasmid DNA-cationic liposome complexes up to 20-fold. The effect is Ca²⁺-specific: other cations such as Mg² and Na⁺ did not give rise to enhanced transfection and the presence of EGTA inhibited the stimulatory effect. It was shown that Ca²⁺ increased cellular uptake of the DNA-lipid complexes, suggesting that increased transfection potency arose from increased intracellular delivery of both cationic lipid and plasmid DNA in the presence of Ca²⁺. In particular, the levels of intact intracellular plasmid DNA were significantly enhanced when Ca²⁺ was present. The generality of the Ca²⁺ effect for enhancing complex-mediated transfection is demonstrated for a number of different cell lines and different cationic lipid formulations. The influence of Ca²⁺ on SPLP, a system in which plasmid DNA is encapsulated within unilamellar lipid vesicles, is investigated in Chapter 3. It was shown that the transfection potency of SPLP in baby hamster kidney cells could be enhanced several hundred-fold by the presence of Ca²⁺. Interestingly, Ca²⁺- enhanced transfection did not result from enhanced uptake of SPLP into the baby hamster kidney cells. Evidence from fluorescent microscopy studies employing rhodamine-labeled SPLP, Southern blots analysis of delivered DNA, and ³¹P NMR studies of appropriate model membrane systems suggested that Ca²⁺ enhances the transfection potency of the encapsulated system by assisting in the destabilization of the endosomal membrane. This work was extended to .encapsulated systems that contained higher amounts of the cationic lipid and that contained a cationic pegylated lipid on its surface. Increases in potency of 3 to 5 orders of magnitude at the optimal Ca²⁺ concentrations were observed, indicating that Ca²⁺ and cationic lipid act synergistically to increase the transfection potency of lipid-based gene transfer systems. In summary, results of this work show that Ca²⁺ acts as an efficient cofactor for enhancing the transfection properties of lipid-based gene delivery systems.

Item Media

Item Citations and Data

Rights

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics