Date of Award

8-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Webb, Ken

Committee Member

Kindy , Mark

Committee Member

Alexis , Frank

Abstract

Scaffold-mediated nonviral gene delivery avoids several drawbacks of systemic injection such as clearance by the reticulo-endothelial system and serum aggregation. Existing synthetic and natural polymers used in gene delivery scaffolds are primarily derived from other tissue engineering applications with design parameters focused on the physicochemical properties of the scaffold and its biocompatibility. Common synthetic materials used in polymeric scaffolds such as PEG are practically bio-inert with minimal cell and protein interaction. Therefore, an opportunity exists for the rational design novel gene delivery scaffolds with components capable of increasing the expression of incorporated transgene by including scaffold components that interact with cellular processes and pathways. Amphiphilic block copolymers including triblock poloxamers and 4-arm poloxamines have recently emerged as potent biological response modifiers capable of interacting with cellular pathways in order to address several intracellular barriers to nonviral gene delivery. In particular, poloxamines are attractive candidates for scaffold incorporation due to the tetrafunctional structure of the copolymer. While the activity and mechanism of poloxamers in nonviral gene delivery with polymer gene carriers is relatively well understood, the use of poloxamines in polymer- or lipid-mediated gene delivery has not been studied. In this dissertation, the activity and mechanism of one poloxamine formulation, Tetronic T904, in polyplex transfection was thoroughly investigated. We found that like poloxamers, T904 had the ability to significantly enhance the expression of transgene. However, T904 was not limited by the promoter design of the vector and its application extended to lipid-based transfection as well. It also differed mechanistically from poloxamers, having no effect on vector internalization in the cell or nuclear import. Instead, T904 enhanced transgene expression by significantly increasing the production of transgene mRNA transcripts. Having validated the efficacy of Tetronics in nonviral gene delivery, a hybrid hydrogel was developed incorporating the cell adhesive properties of fibrin with T904 to provide scaffold-mediated nonviral gene delivery. The resulting hybrid hydrogel maintained the enzymatic degradability of fibrin and allowed the controlled release of incorporated DNA and polymer/DNA complexes. T904/fibrin hydrogels containing polymer/DNA complexes were able to transfect clusters of cells seeded in 3D with transgene expression detected for up to one month. Hybrid hydrogels implanted in a cutaneous wound model maintained the bioactivity of incorporated polymer/DNA complexes and facilitated the migration of cells and expression of transgene in vivo.

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