Many steps are involved in transfection and must be overcome to provide a successful gene delivery agent: DNA binding/compaction, cellular recognition and uptake, endosomal escape, trafficking through the cell and eventual release of cargo. All of these potential hurdles are being investigated by the Reineke group using our polymeric vectors, which are composed of carbohydrate and oligoamine monomers; however, the crucial first step to efficient transfection, DNA binding, is an event rarely dissected quantitatively.
The purpose of this dissertation research was to carefully elucidate aspects of the mechanism of interaction between our polymers and plasmid DNA (pDNA) through biophysical techniques, such as microcalorimetry, dynamic light scattering, zeta potential, circular dichroism, and FTIR spectroscopy. The combination of these results revealed the significance of the carbohydrate hydroxyl stereochemistry and amide spacing to the DNA affinity, as a result of hydrogen bonding to the backbone and base pairs. As the amine number increased from 1 to 4, the DNA binding mechanism of trehalose-containing polymers became less dependent upon electrostatics and more on hydrogen bonding. This change is a result of decreasing charge fraction with increasing amine density. To determine the necessity of charge-charge interaction between our polycations and nucleic acids, a new series of polymers, replacing the oligoamine with an analogous oligo(ethylene glycol), was synthesized and its characterization compared to the poly(glycoamidoamine)s previously studied. It was shown that removing the electrostatic component prevented DNA binding. Therefore, we conclude that long-range Coulombic attraction initiates the interaction between our polymeric vectors and nucleic acids, but then hydrogen bonding becomes a dominant contributor.
Stabilization of our delivery agents to physiological salt and serum conditions is also critical to their clinical use, so another aspect of this thesis project was to conjugate poly(ethylene glycol) to the vectors to provide charge shielding and steric hindrance to thwart aggregation. This stabilization was successfully accomplished with the poly(glycoamidoamine)s using two different formulations and the cell transfection and uptake was monitored with and without cell-targeting moieties. All of these research efforts contribute both to the fundamental knowledge of interaction mechanisms and practical applicability of using these agents for therapeutic delivery of nucleic acids.
|Advisor:||Reineke, Theresa M.|
|Commitee:||Ault, Bruce S., Beck, Thomas L., Halsall, H. Brian, Lynch, Matthew L., Reineke, Theresa M.|
|School:||University of Cincinnati|
|Department:||Arts and Sciences : Chemistry|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Biophysics|
|Keywords:||Binding mechanisms, DNA, Electrostatics, Gene delivery, Hydrogen bonding, Microcalorimetry, Polymers, Secondary structure|
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