L. M. Bools1, D. J. Mountain1, R. K. Fisher1, S. S. Kirkpatrick1, J. D. Arnold1, S. L. Stevens1, M. H. Goldman1, M. B. Freeman1, O. H. Grandas1 1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA
Introduction: RNA interference (RNAi), using short interfering RNA (siRNA) as one of the strategies to degrade mRNA in the cytoplasm and transiently attenuate intracellular proteins, shows promise in the inhibition of vascular pathogenesis. A critical obstacle for its therapeutic application is a safe and effective delivery system, as unfavorable physiochemical properties limit intracellular siRNA delivery. Synthetic polymers are promising alternative molecular carriers due to their ability to deliver genetic material to cells in a predictable, non-toxic way. Here we aim to establish polymeric transfection as a feasible non-viral, less-toxic method for gene therapy in cells of vascular origin.
Methods: Human aortic smooth muscle cells (HASMC) were transfected in vitro with polyethylenimine and poly(B-amino ester) polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA:polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability and GAPDH gene silencing was measured by qPCR normalized to 18S.
Results:The highest rate of polyethylenimine-mediated silencing was achieved with a 9µl polymer:220pmol/ml siRNA conjugate (16±2% expression vs. NC; n=6; Fig1A). Comparable poly(B-amino ester)-mediated silencing could be achieved with a 1.95µl polymer:100pmol/ml siRNA conjugate (10±1% expression vs. NC; n=5; Fig1B). Transfection using polyethylenimines resulted in silencing equivalent to other methods, but with less efficiency and increased cell toxicity at 24h polymer exposure (Fig1C,D). Limiting polyethylenimine exposure to 4hr resulted in similar silencing efficacy (25±9% – 33±8% expression vs. NC, n=3) with decreased toxicity (Fig 1E,F).
Conclusion:Polymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. Polyethylenimine bioconjugates demonstrated silencing equivalent to poly(B-amino ester) bioconjugates, though less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and polyethylenimine’s ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that polyethylenimine bioconjugates are a viable transfection agent for vascular tissue. Future studies will expand on this method of gene therapy for in vivo transfection in animal models of vascular disease. Our long term goal is to deliver molecular inhibitors in vivo, targeting the regulation of genes thought to play a significant role in intimal hyperplasia and restenosis development.
