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Structural and functional studies of lipid nanoparticles

University of British Columbia

Macromolecules such as nucleic acids and inorganic nanoparticles require a delivery vector to capitalize on their true therapeutic and diagnostic potential. In order to ensure seamless clinical application of such systems, robust manufacturing practices and potent delivery are essential. Herein, the design of lipid nanoparticle (LNP) vectors for the intracellular delivery of plasmid DNA (pDNA) is discussed. Building on the clinical formulation of short-interfering RNA (siRNA), LNP composition and charge-ratios were varied in order to optimize transfection of dividing cells. A major hurdle to improving the efficacy of such LNP systems was understanding the mechanism whereby ionizable cationic lipids entrap negatively charged macromolecules and the resulting structure generated. A novel mechanism of siRNA entrapment and particle morphology is proposed in this thesis. From these studies, formulations were designed to entrap and deliver inorganic metal nanoparticles, which are otherwise unable to act as therapeutics. A significant amount of research has focused on the intracellular delivery of siRNA which enables gene-silencing of problematic genes, while little attention has been given to LNP systems that enable gene expression. The first part of this dissertation explores the design of lipid nanoparticle formulations of pDNA. Such systems bear incredible potential in therapeutic or vaccine applications, and gene-editing approaches through CRISPR/Cas9. We showed that formulations designed for delivery of siRNA perform quite poorly for the delivery of pDNA. Improvements in the design of the formulation led to a major investigation into the mechanism of entrapment and the delivery of nucleic acid. Previous studies into the core of lipid nanoparticles suggested the presence of inverted micellar structures coating siRNA with a stabilizing surface monolayer of lipids. Here we deconstruct the proposed structure and systematically examine the evidence for the formation of inverted micellar structures. From these studies, a novel oil-in-water emulsion structure was proposed. Finally, from this work, the capacity of oil-in-water emulsions to act as payload carriers of therapeutics was explored. LNP composed of phosphatidylcholine and triglycerides were explored as carriers for hydrophobic inorganic nanoparticles such as iron oxide, gold, and semiconductor quantum dots. Taken together, this work highlights the broad applicability of oil-in-water LNP formulations.

Text

2023-11-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/65841

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/