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Permanent link (DOI): https://doi.org/10.7939/R38Q0K

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Mechanistic studies on the uptake and intracellular trafficking of DNA complexes in primary cells using lipid-modified cationic polymers as non-viral gene carrier Open Access

Descriptions

Other title
intracellular trafficking of DNA complexes in non-viral gene delivery
Subject/Keyword
cationic polymers
transgene expression
non-viral gene delivery
targeted drug delivery
DNA topology
nuclear import
uptake pathways
intracellular trafficking
transfection
cell-based therapy
nucleic acid therapy
gene therapy
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Hsu, Charlie Yu Ming
Supervisor and department
Hasan Uludag
Examining committee member and department
Hendzel, Michael (Experimental Oncology)
Xiaoli Lilly Pang (Division of Medical Microbiology)
Unsworth, Larry (Chemical and Materials Engineering, Biomedical Engineering)
Rancourt, Derrick (Oncology, Biochemistry & Molecular Biology, Medical Genetics)
Marianna Foldvari (Pharmaceutical Sciences, School of Pharmacy & the Waterloo Institute for Nanotechnology)
Wilman, Alan (Biomedical Engineering)
Department
Department of Biomedical Engineering
Specialization
Biomedical Sciences
Date accepted
2012-07-23T08:30:42Z
Graduation date
2012-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
This thesis work is aimed at addressing some of the fundamental questions surrounding the intracellular fate of plasmid DNA in clinically-relevant primary cells, when delivered with lipid-modi!ed cationic polymers as gene carriers. We developed an optimized procedure for the transfection of primary cells, which included modi!cations designed to maximize the in vitro stability of the DNA-carrier complexes and enhance their transfection utility. These polymeric gene carriers bind to DNA through electrostatic interaction, which drives a self-assembly process that condenses DNA into sub-micron particles suitable for cellular uptake. This interaction is not DNA sequence or structurally speci!c, and thus able to package DNA molecules with different topologies and molecular weights with equal efficiency for uptake. However, circularized DNA performed better than its linearized equivalent in transfection, suggesting intracellular processing may be dependent on the physicochemical properties of the assembled complexes. Thus, we concentrated our effort on understanding the intracellular events leading to transfection. Using a linoleic acid substituted cationic polymer, we found that transfection efficiency is correlated with the amount of pDNA associated with the nucleus and that lipid-moieties is able to facilitate nuclear association of DNA to enhance transfection. Further, lipid modi!cation altered the uptake pathways of the complexes from ones that are predominantly driven by macropinocytosis to ones that are mediated by clathrin. Endosome escape continues to be an inefficient process with these polymeric gene carriers, suggesting methods to promote cytosolic release may be the most effective approach to enhance their transfection efficiencies.
Language
English
DOI
doi:10.7939/R38Q0K
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Hsu, C.Y., and Uludag, H. (2012) A simple and rapid non-viral approach to efficiently transfect primary tissue-derived cells using polyethylenimine. Nature Protocols. 7(5) 935-945.Hsu, CY and Uludag H. (2012). Nucleic-acid based gene therapeutics: delivery challenges and the modular design of non-viral gene carriers and expression cassettes to overcome intracellular barriers for sustained targeted expression. J. Drug Targ. Feb 6.Hsu, C. Y., and Uludag, H. (2008). Effect of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers. BMC Biotech. 8: 23.Hsu, C.Y., Hendzel, M., and Uludag, H. (2011) Improved Transfection Efficiency of an Aliphatic lipid Substituted 2 kDa Polyethylenimine is Attributed to Enhanced Nuclear Association and Uptake in Rat Bone Marrow Stromal Cell. J. Gene Med. Jan;13(1):46-59.Hsu C.Y., and Uludag H., (2013) Cellular Uptake Pathways of Lipid-Modified Cationic Polymers in Gene Delivery to Primary Cells. Biomaterials. In Press

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