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Title:Quo vadis: quantitative evaluation of intracellular kinetics of siRNA delivered by poly-and-lipoplexes
Author(s):Lazebnik, Mihael
Director of Research:Pack, Daniel W;
Doctoral Committee Chair(s):Kong, Hyunjoon
Doctoral Committee Member(s):Yang, Hong; Underhill, Gregory H
Department / Program:Chemical & Biomolecular Engineering
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):endocytosis
siRNA delivery
Non-viral gene delivery
polyplexes
lipoplexes
polyethylenimine (PEI)
N-[1-(2,3-iii dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP)
cholesterol
Abstract:Small interfering RNA (siRNA), when delivered to cells, is able to elicit a potent post transcriptional gene silencing with enormous therapeutic potential. It has been demonstrated that siRNA is able to tackle a wide range of conditions that include viral infections, cancer, immunological disorders, and even alcoholism. Still, siRNA is too large to passively internalize into the cells by means of diffusion, and its electric charge is of the same polarity as the net-anionic plasma membranes, making it exceedingly difficult to even approach the cells. As such, various materials have been developed to allow the delivery of the siRNA molecules to their primary sites of activity in cytosol. This, however, only solved the problem in part, since such vectors interact with oligonucleotides to form particles that, as commonly believed, require endocytosis to enter the cells. Endocytosis is a process by which cells communicate with their surroundings and internalize nutrients. The fundamental principle in this process is that the internalized material is secluded from the cytosol in vesicles formed by invaginations of sections of plasma membranes. These vesicles transport and sort material in a highly organized manner and most commonly either recycle the internalized material out of the cells or deliver it to be degraded in lysosomes. Once again, in order to enter cytosol, siRNA molecules must cross a biological membrane. Thousands of chemical formulations have been adapted or de novo synthesized to overcome the various barriers to siRNA delivery. These include targeting to specific cells, evasion from immune system, cell internalization, and escape from the endocytic vesicles into cytosol. Still, since endocytosis occurs inside of the cells and consists of steps that last time intervals of different orders of magnitude, it is has become a great challenge to design a vector material capable of mediating the delivery of siRNA into the cytosol. The research presented in this dissertation will illuminate the reasons behind the success of some of the most common delivery materials: polyethylenimine (PEI) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) liposomes with various amounts of cholesterol as “helper lipids.” In order to gain new insights into the endocytosis of siRNA-based synthetic particles, I exploited the ability of horseradish peroxidase (HRP) to elicit crosslinking and subsequent isolation of different groups of vesicles within the endocytic system. This method allows for a quantitative description of the intracellular kinetics of internalized complexes, and was demonstrated to be superior to other commonly used methods. The data acquired by this method were used to construct a mathematical model describing endocytosis of the investigated vectors that revealed a series of interesting results; the success of PEI is based on the ability of the PEI-based particles to attach to plasma membranes. PEI-based particles internalize into early endosomes and gain access to cytosol primarily from early endosomes 14 minutes after the internalization. On the contrary, DOTAP/cholesterol liposomes primarily gain access to cytosol through direct fusion with plasma membranes, and increasing the content of cholesterol in the vectors was shown to enhance the fusion of the lipid-based particles with the biological membranes. However, once the liposomes enter the cells, their ability to escape the internal vesicles is not a substantial contributor to their success. The methods and the kinetics parameters presented in this dissertation will aid in rational development of siRNA vectors and provide means by which the intracellular trafficking of the internalized particles can be investigated.
Issue Date:2015-12-03
Type:Thesis
URI:http://hdl.handle.net/2142/88965
Rights Information:Copyright 2015 Mihael Lazebnik
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12


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