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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

  • intracellular trafficking of DNA complexes in non-viral gene delivery

  • Author / Creator
    Hsu, Charlie Yu Ming
  • 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.

  • Subjects / Keywords
  • Graduation date
    2012-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R38Q0K
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biomedical Engineering
  • Specialization
    • Biomedical Sciences
  • Supervisor / co-supervisor and their department(s)
    • Hasan Uludag
  • Examining committee members and their departments
    • Unsworth, Larry (Chemical and Materials Engineering, Biomedical Engineering)
    • Hendzel, Michael (Experimental Oncology)
    • Marianna Foldvari (Pharmaceutical Sciences, School of Pharmacy & the Waterloo Institute for Nanotechnology)
    • Wilman, Alan (Biomedical Engineering)
    • Rancourt, Derrick (Oncology, Biochemistry & Molecular Biology, Medical Genetics)
    • Xiaoli Lilly Pang (Division of Medical Microbiology)