Smith, Silvia
(2023)
Characterisation and Intercellular Mechanistic Analysis of Supramolecular, Cationic, mRNA-Loaded Polyplexes for Efficient Transfection in HEK293T and A549 Cells.
MRes thesis, University of Nottingham.
Abstract
Part A:
The approval of two mRNA vaccines for COVID-19 by the U.S. FDA generated significant attention from the scientific community for RNA technology, with its safety, efficacy, and rapid production capabilities. Beyond infectious disease, RNA therapeutics hold great promise in cancer treatment and various other diseases. However, the physiochemical properties of free mRNA present significant challenges in achieving efficient intracellular delivery in vivo. Therefore, the development of effective delivery systems for exogenous RNA is crucial. Among various delivery methods, lipid and polymer-based nanoparticles (LNPs and PNPs) have emerged as prominent candidates. This review discusses different classes of PNPs, providing a comprehensive analysis of their molecular design criteria and plausible internalisation pathways within cells. Despite their favourable pharmacokinetics, precise tunability of chemical properties, and potential for cellular targeting, PNPs are behind LNPs in clinical advancement. This review explores the key challenges encountered in PNP development, with a particular emphasis on the importance of understanding PNP endosome escape mechanisms, which currently represent a bottleneck for improving transfection efficiency. By identifying how PNP composition, shape, and size influence endosome escape, PNP formulations can be optimised to deliver RNA therapeutics effectively and safely. Future perspectives and research directions necessary for the clinical translation of PNPs are also discussed.
Part B:
The demand for effective mRNA delivery methods has surged due to rapid developments in RNA therapeutics. Cationic polymers, forming protective mRNA complexes, have emerged as promising carriers. This study explores the transfection efficiency of supramolecular TETA-2L2 and TEPA-2L2 polyplexes within HEK293T and A549 cells, determined by luciferase assays and imaging GFP expression. The stability, size, surface charge and low cytotoxicity of these polyplexes were advantageous characteristics observed at the optimal nucleotide to polymer (N/P) ratios, around 8 to 16. Understanding their uptake mechanisms and endosomal escape is crucial for tailoring therapeutic applications. This investigation indicates the involvement of multiple endocytic pathways for TETA-2L2 and TEPA-2L2 polyplex uptake. Observations of cells exposed to chloroquine revealed how the polyplexes rely on acidic environments for endosomal membrane destabilisation and efficient escape. Notably, variations in transfection efficiency were observed among different cell types, highlighting the significance of cell-specific considerations in polyplex design. Future research should focus on imaging uptake inhibition, evaluating co-localisation with endosomes and comparative assessments with other endosomal disruptive agents. These findings hold significant potential for advancing mRNA delivery methods required for therapeutic applications.
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