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Mechanisms of Cytotoxicity and Intracellular Trafficking for Gene Delivery Polymers
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Herein, different polymer libraries were examined to determine the effect polymer structure has on intracellular events. The effect of different polyamine lengths in copolymers on cellular uptake, the effect of modifying end groups of trehalose-containing polymers on transfection efficiency, and the effect of different linker lengths between galactose and a hepatocyte-targeted polymer on transfection efficiency were studied. Furthermore, it was demonstrated that polymers with terbium chelated in their repeat units could potentially be used for Förster resonance energy transfer (FRET) studies to monitor pDNA release from the polymer. Much of the work in this dissertation focuses on elucidating the intracellular mechanisms of linear poly(ethylenimine) (PEI) and how it compares to poly(L-tartaramidopentaethylenetetramine) (T4) and poly(galactaramidopentaethylenetetramine) (G4), two poly(glycoamidoamine)s synthesized by our group. The long-term goal of this project is to develop structure-function relationships between polymers and pDNA delivery efficacy that will result in the rational design of safe, efficient vehicles for therapeutic nucleic acid delivery. Many polymers used as DNA delivery vehicles display high cytotoxicity. Often, the polymers with the highest transfection efficiency are the most toxic, as demonstrated herein by PEI and T4 with varying polymer lengths. Therefore, it was of interest to study how polymer structure influences mechanisms of cytotoxicity. To this end, studies on several mechanisms of cytotoxicity, including nuclear envelope permeabilization, were conducted. Longer polymers induced more cytotoxic responses than shorter ones, and it appears that hydroxyl groups in the repeat unit of polymers play a role in polyplex formation. This research has also led us to a potential link between transfection efficiency and cytotoxicity; the polymers with the highest transfection efficiency were also the most toxic, and were also able to induce the most nuclear envelope permeability. It is possible that these polymers' ability to permeabilize the nuclear envelope is what causes their high transfection efficiency and high toxicity. In addition, flow cytometry and confocal microscopy studies revealed that polymer structure plays a role in nuclear trafficking; poly(glycoamidoamine)s G4 and T4 more dependent on intracellular machinery than PEI. This research demonstrates the impact that changes in polymer structure have on intracellular mechanisms.
- Doctoral Dissertations