Date of Award
Doctor of Philosophy (PhD)
Vyavahare , Naren R.
Burg , Karen J.L.
Metters , Andrew T.
The incorporation of nonviral vectors into biomaterial matrices has been employed to improve localization at the implant site and to protect from loss by clearance or extracellular barriers. However, several limitations such as detrimental crosslinking mechanisms, uncontrolled burst release require improved design of matrix-based gene delivery systems that provides sustained and controlled vector release as well as overcomes extracellular barriers to gene transfer in proximity to target cells. The long-term objective of this dissertation project is to provide the basis for the eventual creation of tissue engineering scaffolds that combine structural and biological activity through the creation of composite materials consisting of polymeric fibers with hydrogel coatings encapsulating nonviral vectors for localized gene delivery. The primary focus of this dissertation was to develop hydrogels with suitable physical/chemical properties for the localized, sustained gene delivery application; including variable degradation rate for controlled release, mild crosslinking conditions compatible with vector encapsulation, and minimal swelling after crosslinking to maintain stability as a fiber coating. The hypothesis was that vector release from hydrogel coatings in intimate proximity with adherent cells will overcome extracellular barriers to gene transfer and increase transfection efficiency relative to conventional bolus delivery methods.
In order to concentrate nonviral vectors for efficient hydrogel loading, several approaches to vector lyophilization were investigated. The inclusion of 10% sucrose as a cryoprotectant was shown to preserve DNA structural integrity and biological activity. In addition, incubation of nonviral vectors with sodium tripolyphosphate was shown to effectively release DNA from nonviral gene complexes, which provides a basis for accurate measurement of vector/DNA release. To develop polymer-based gene encapsulation system for gene delivery, hydrolytically degradable hydrogels with various degradation-kinetics were developed and characterized. First, linear poly(ethylene glycol)(PEG) polymers were modified with ester bonds of varying susceptibility to hydrolytic degradation and crosslinkable acrylate groups. Hydrogels were prepared by photopolymerization and shown to variable degradation rates and controlled release of a model macromolecule. Secondly, hydrogels crosslinked by Michael-type addition from various acrylate-terminated four-armed amphiphilic poloxamine (Tetronic®) were evaluated. It was shown that the physical properties of these gels are attributable to both temperature-dependent noncovalent interactions and covalent crosslinking. Hydrogels prepared from Tetronic T904 did not experience significant swelling after crosslinking and were shown to form stable coatings on polymer fibers. Finally, lyophilized nonviral-gene complexes were incorporated within T904-based hydrogels. DNA integrity and sustained release was observed although the kinetics was not considered optimal due to delayed release. Preliminary transfection experiments using an in vitro model with serum-containing medium demonstrated that released vectors could transfect surrounding cells, although the efficiency of transfection was limited and further improvements to the gene delivery system are required.
Cho, Eunhee, "HYDROGEL COMPOSITIONS FOR NONVIRAL GENE DELIVERY" (2009). All Dissertations. 447.