Biological Systems Engineering
Date of this Version
Controlling Nonviral Gene Delivery through the Cell-Biomaterial Interface
Beth Ann Duensing, M.S. University of Nebraska, 2010
Adviser: Angela Pannier
Cell-biomaterial interactions and the corresponding cellular behaviors are poorly understood. Therefore, in this study, the ability of biomaterial surface properties to control nonviral gene delivery was investigated through surface chemistry and protein adsorption and subsequently correlated to cellular behaviors controlled by cell-biomaterial interactions. Self-assembled monolayers (SAMs) of alkanethiols on gold were used as model biomaterials to investigate the effect of surface properties on nonviral gene transfer to cells adhered to these surfaces. SAMs presenting terminal CH3, OH, COO-, and NH3+ functionalities were adsorbed with varying concentrations of FN to determine the amount of FN needed to form an adsorbed monolayer on the surface of SAMs, onto which cells were then seeded and plasmid DNA was delivered via a bolus approach using polymer- and lipid-mediated delivery techniques. SAMs without adsorbed FN were used as a comparison, in addition to traditional polystyrene (PS) culture surfaces. The results from these preliminary studies were used to select conditions for subsequent studies.
For the final studies, SAMs were adsorbed with either a monolayer or multilayer of fibronectin (FN), onto which cells were then seeded and plasmid DNA was delivered via a bolus. SAMs without adsorbed FN were used as a comparison, in addition to traditional polystyrene (PS) culture surfaces. FN dose response and underlying surface properties together contributed to transfection profiles, which, for Lipofectamine 2000 (LF2000), exceeded levels on traditional PS surfaces, even with the addition of FN. These results indicate not simply the presence of FN, but also its interaction with the underlying surface and resulting cell behaviors, enhance transfection. After FN multilayer adsorption to SAMs, the CH3-terminated surface had the greatest transfection in comparison to all other SAMs and the PS control. It is proposed that the increase can be correlated to cytoskeleton reorganization and FN fibrillogenesis. These studies provided initial understanding of the relationships between biomaterial surface properties, adsorbed proteins, and nonviral gene transfer to cells interacting with these surfaces, in order to design optimal material surfaces that promote gene delivery for use in therapeutic and diagnostic applications, including biomaterials-based delivery strategies. A greater understanding of the cells’ interactions with their microenvironment will allow better design of tissue engineering scaffolds for gene delivery as well as promote efficient gene transfer for gene therapy.
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: March, 2010
Copyright 2010 Beth Duensing