UBC Theses and Dissertations
Biophysical characterization of lipid nanoparticles containing nucleic acid polymers as produced by microfluidic mixing Leung, Alex Kar-Kei
Lipid nanoparticles (LNP) are currently the most advanced delivery systems for enabling siRNA to be used for therapeutic applications. However, the structure of these siRNA-LNP systems has not been well defined previously. The objective of this thesis is to determine the structure of nucleic acid-LNP systems produced by a novel microfluidic mixing technique and to use this structural understanding to develop systems with improved gene silencing efficacy. The first part of the thesis focuses on determining the structure of siRNA-LNP systems produced by microfluidic mixing and the effects of varying lipid components on the structure and encapsulation properties. SiRNA-LNP were formulated using an ionizable cationic lipid, distearoylphosphatidylcholine, cholesterol and a polyethylene glycol-lipid. Cryo-TEM of siRNA-LNP produced by microfluidic mixing exhibit a solid, electron-dense core with siRNA encapsulation efficiency close to 100%. Molecular dynamics modeling indicates that the core of the particle consists of periodic aqueous compartments containing siRNA. The ability of the lipid mixture to adopt non-bilayer phases seems to be crucial for the encapsulation of siRNA. The microfluidic mixing technology was also extended to the encapsulation of plasmid DNA and mRNA. These results provide an understanding of the structure and the mechanism of formation for siRNA-LNP produced by microfluidic mixing. Since it is clear that formation of siRNA-LNP by microfluidic mixing does not require any bilayer-forming lipids, it is possible to generate particles with high cationic lipid content and bilayer-destabilizing lipids without compromising particle stability. The last part of the thesis aims to improve the gene silencing efficacy of siRNA-LNP by enhancing the endosomolytic properties of the LNP. This was attempted by two way: first by increasing the cationic lipid content of the LNP and second, by the incorporation of bilayer-destabilizing "helper" lipids in the formulation. A novel "helper" lipid dioleoyl-four amino butyric acid (DOFAB) was synthesized for this purpose. In contrary to the hypothesis, both increasing cationic lipid content and the incorporation of "helper" lipids in siRNA-LNP formulations led to decreased in vitro gene silencing activity. This leads to new questions as to role of cationic lipids and "helper" lipids play in the intracellular release of siRNA.
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Attribution-NonCommercial-NoDerivs 2.5 Canada