Biological Systems Engineering

 

Date of this Version

7-2013

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Agricultural and Biological Systems Engineering, Under the Supervision of Professor Angela K. Pannier. Lincoln, Nebraska: July 2013

Copyright (c) 2013 Jessica D. Taylor

Abstract

Gene delivery, the introduction of DNA into cells, is applicable to gene therapy, DNA vaccination, functional genomics and diagnostics, tissue engineering, and drug-eluting medical devices. Particulates incorporating DNA are promising vehicles for gene delivery and overcome some of the barriers that hinder successful gene transfer, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Furthermore, innovative new gene delivery strategies that incorporate DNA particulates or complexes within films or coatings for devices and scaffolds could further provide for controlled and sustained transfection at the site of implant. Zein, a hydrophobic protein from corn, has many unique properties that make it a promising candidate material for both particulate- and film-mediated gene delivery, including its ability to easily form both nanospheres and films. Zein/DNA nanospheres, formed through a simple coacervation process, demonstrated the ability to protect DNA against nucleases, displayed robust biocompatibility, and were able to be internalized by cells. In addition, zein films were developed for substrate-mediated gene delivery. Zein films were formed by evaporation- induced self-assembly and were comprised of particles that increased in size with zein concentration, up to 1.8 0.11 nm in diameter. Films formed at different pH values (2-12) resulted in morphological differences, from a smooth surface at pH 2 to films composed of spheres that decreased in size as the pH of the zein solution used to form the film increased. Zein films degraded minimally in PBS, and were able to adsorb DNA complexes formed with cationic lipids or cationic polymers, with 0.4 µg and 1.12 µg of total DNA on film surface for lipid/DNA and polymer/DNA complexes, respectively, which was independent of pH. However, transfection levels increased on films with increasing pH and highest gene expression was achieved on pH 9 films with lipid/DNA complexes. This increase in transfection on pH 9 films could be attributed to the decreased surface roughness and increased hydrophilicity of the film. Both the zein spheres and films investigated in this thesis display great potential for gene delivery applications, in particular for oral and intramuscular gene delivery and as a surface-coating for biomedical devices.

Advisor: Angela K. Pannier