Intracellular Considerations in the Development of Non-Viral Nucleic Acid Delivery Systems for Systemic Administration

Citation

Mishra, Swaroop (2006) Intracellular Considerations in the Development of Non-Viral Nucleic Acid Delivery Systems for Systemic Administration. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/7MP8-JJ24. https://resolver.caltech.edu/CaltechETD:etd-09222005-123557

Abstract

Non-viral nucleic acid delivery systems must condense nucleic acids into small particles, confer protection from degrading factors in serum and in cells, achieve uptake to targeted cells, direct nucleic acids to appropriate intracellular destinations, release this cargo to permit its action, and exhibit minimal toxicity. The nature of synthetic vectors allows for facile addition of new features, but these modifications can affect performance in unanticipated ways. The effective combination of functional components necessitates a systems approach, where the materials design simultaneously considers the functional environment of and the various barriers to delivery. This thesis facilitates and promotes a systems approach by undertaking development of an improved mechanistic understanding of non-viral gene transfer in vitro, emphasizing elucidation of delivery vehicles’ interactions with and behavior within cells. Special attention is given to the gene delivery behavior of cyclodextrin-containing polycations.

Simple modifications to delivery systems can have unanticipated consequences. In Chapter 2, it is shown that greater distance between toxicity-reducing cyclodextrin moieties and amidine charge centers increases both the transfection efficiency and toxicity of a polycationic vector. Chapter 3 shows data demonstrating that modification with poly(ethylene glycol) for extracellular salt-stabilization alters non-viral gene delivery particles’ intracellular trafficking and resulting gene expression. Taken together, the results reveal that non-viral gene delivery vehicles behave as assembled, multifunctional systems.

pH-buffering components exhibit complex behavior in non-viral gene delivery. In Chapter 4, the intracellular activity of such components is quantified using confocal microscopy. Analysis of chloroquine and its chemical analogues demonstrates in Chapter 5 that chloroquine improves non-viral gene transfer through pH-buffering as well as through enhanced nucleic acid unpackaging and its own interactions with nucleic acids. Chapter 6 gives results that characterize delivery behavior of analogous vectors with and without pH-buffering capacity and show that factors beyond buffering activity contribute to improved transfection efficiency. Collectively, these results emphasize consideration of new system components’ effects on all functions of a non-viral gene delivery system.

A systems approach requires comprehensive consideration of the gene delivery process. Chapter 7 reviews current understanding of intracellular barriers to non-viral gene delivery, and Chapter 8 offers recommendations for future work.

Thesis Files