Thursday, November 12, 2015: 1:06 PM
251A (Salt Palace Convention Center)
Gene delivery alters the genetic expression of cells and can be used in various applications including tissue engineering, medical devices, and diagnostics. Nonviral gene delivery is a method to deliver DNA to cells which uses lipid or polymer vectors to electrostatically complex DNA, packaging it for delivery to cells. The complex delivers DNA in a relatively nontoxic and nonimmunogenic manner, but its efficiency is low compared to viral vectors. Many previous studies have attempted to improve nonviral gene delivery systems by engineering different polymer or lipid carriers, but clinical efficacy has yet to be achieved. Previous research has shown that cell-surface interactions can affect cellular response to nonviral gene delivery. In this work we expand on those studies, focusing on the role of nanotopography on cellular response to nonviral delivery vectors. Previous studies have shown that nanotopography improves cell attachment, adhesion, proliferation, and filopodia production, all vital processes implicated in successful gene delivery. Sculptured thin film (STF) substrates are a type of nanostructured surface, produced by glancing angle deposition, a physical vapor method that produces columns on the nano- to microscale that are highly ordered and reproducible. We have demonstrated that titanium (Ti) STFs significantly improve cellular adhesion and proliferation in multiple cell types. Therefore, the objective of this project was to investigate whether STFs could improve nonviral gene delivery in fibroblasts via enhancement of proliferation, adhesion, and filopodia production. Initial studies demonstrated that varying column orientation (slanted, vertical, spiral) and intercolumnar spacing (30 or 100 nm) yielded no statistical differences in resulting transfection to cells adhered on those surfaces, in comparison to the flat Ti control. Subsequent studies were performed on structures with a slanted columnar orientation, and 100 nm intercolumnar spacing, but with the height of nanocolumns varied from 25-100 nm. These studies indicated that Ti STFs at a height of 50 nm improve transfection compared to flat Ti and this effect may be mediated by filopodia production. SEM imaging of cells on flat Ti revealed long filamentous protrusions from the adhered cells, while cells cultured on STF substrates had lamellopodia and shorter protrusions, potentially indicating better adhesion and maturation of focal adhesions after probing, which may improve transfection. Inhibiting filopodia generation, achieved through addition of pharmacological agents Jasplankinolide and Blebbistatin, resulted in statistically reduced transfection levels, further implicating filopodia production in cellular transfection efficacy. Therefore, altering the columnar parameters has a variable effect on transfection efficacy in fibroblasts, which may be mediated through cell-surface interactions that affect filopodia production. Future studies are focused on optimization of STF parameters to improve transfection and inhibition studies to validate that the mechanism by which nanotopography influences transgene expression is related to filopodia. These studies demonstrate that the efficacy of established transfection vectors can be improved by nanotopographical cues with possible applications in drug and gene delivery such as a stent coating to decrease stenosis.