Biological Systems Engineering


Date of this Version

Summer 7-21-2011


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, 2011

Copyright 2011 Mary C. Regier


Particulates incorporating DNA provide for protection and sustained release of DNA, and thus are promising candidates for DNA delivery systems. Among the routes of administration for gene delivery, the oral route is perhaps the most appealing as it is associated with patient comfort and compliance and allows for targeting to intestinal targets for therapeutic and vaccination applications. With the goal of realizing the potential of an oral DNA delivery system, zein, a hydrophobic protein from corn that is biocompatible and degraded enzymatically, was investigated. This thesis describes the formulation of zein nanospheres encapsulating DNA by a coacervation technique and their characterization. Zein/DNA nanospheres ranged from 57.8 ± 3.9 nm to 396.8 ± 16.1 nm and from -21.8 ± 4.2 mV to -46.6 ± 1.6 mV for hydrodynamic diameter and zeta potential measured in water, respectively. Spheres formed at all ratios aggregated to some degree in PBS, with 20:1 and 40:1 zein:DNA spheres flocculating; aggregation was found to be dependent on salt concentration. DNA encapsulation efficiency was as high as 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. DNA that was encapsulated and released retained its integrity. Release studies indicated that zein was degraded primary enzymatically with slow release of DNA in PBS, and faster release in pepsin containing media, and nearly instantaneous release in simulated intestinal fluid. Spheres demonstrated similar biocompatibility at high and low concentrations, and showed cellular association and evidence of internalization. Possible improvements to this delivery system include increasing loading, improving stability against aggregation, increasing resistance to enzymatic degradation, and improving internalization, nuclear localization, and transfection. The spheres formed and characterized as described in this thesis show great potential for oral gene delivery, and with judicious modification should be rendered even more likely to be applied clinically in oral gene delivery as well as tissue engineering and intramuscular injection applications.