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Title:Endocytosis mechanism of poly- and lipoplexes in siRNA delivery in HeLa cells
Author(s):Lazebnik, Mihael
Advisor(s):Pack, Daniel W.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):small interfering ribonucleic acid (siRNA)
gene delivery
ribonucleic acid interference (RNAi)
Abstract:RNA interference (RNAi) is a method that can be used to treat various medical conditions. This method can silence expression of virtually any gene, and it is initiated by small interfering RNA (siRNA) molecule. siRNA is a 19 nucleotides-long double stranded RNA-molecule. In order to begin gene silencing, siRNA needs to be delivered to the cytosol. The first of the barriers that stand on the path of the siRNA delivery is endocytosis. Endocytosis is a process by which nutrients, signals and pathogens enter the cells. This process consists of multiple pathways that lead the objects that were ingested by the cells to different intracellular destinations. The chemical environment encountered by the endocytosed objects also depends on the specific pathways. Because of the large size, negative charge and other chemical properties, siRNA cannot readily enter the cells. One of the most promising techniques to deliver siRNA is to employ its anionic property to electrostatically complex it with cationic lipids or polymers and create lipid- or polymer based particles (lipoplexes or polyplexes). The materials that are used as the delivery vectors can mediate the uptake and the release of siRNA in the cytosol. In order to synthesize newer generation of the delivery materials, it is paramount to know through which endocytosis pathways the current materials deliver their cargo and which limitations they encounter. In the study presented here, two representatives of the siRNA delivery vectors were chosen, and the investigation into their endocytosis in HeLa cells was performed. Chemical and biological (RNAi) methods were used to selectively inhibit endocytosis pathways and investigate the influence of inhibition on total uptake of and RNAi mediated by polyplexes and lipoplexes. Fluorescence microscopy was also used to determine the predominant pathways in the siRNA delivery by two different materials. The experiments revealed that polyplexes enter the cells through multiple endocytosis pathways simultaneously, and the expression of the delivered siRNA is the most prominent when polyplexes enter through caveolin-, flotillin- and ARF6-mediated endocytosis pathways. ARF6 might play multiple roles in the delivery and release of the delivered siRNA in cells. Lipoplexes, on the other hand, enter the cells and release siRNA through a method of direct fusion with the plasma membrane rather than endocytosis. The data and conclusions presented in this study are consistent with the current knowledge and expand on the understanding of the complicated endocytosis mechanisms of siRNA-based particles.
Issue Date:2012-09-18
Rights Information:Copyright 2012 Mihael Lazebnik
Date Available in IDEALS:2012-09-18
Date Deposited:2012-08

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