R. K. Fisher1, S. I. Mattern-Schain2, M. D. Best2, S. S. Kirkpatrick1, M. B. Freeman1, O. H. Grandas1, D. J. Mountain1 1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA 2University Of Tennessee,Department Of Chemistry,Knoxville, TN, USA
Introduction: Molecular gene therapy shows promise in the management of vascular disease, and the development of biocompatible nanocarriers provides alternative non-viral delivery methods with translational potential. Cationic liposomes (CLs) have been shown to be effective nanocarriers for gene therapeutics in vitro. However, in vivo success has been hampered by CL-induced cytotoxicity resulting from their electrostatic interactions with mammalian cell membranes. Recently, neutral liposomes (NLs) formed with natural lipid constituents have been modified with cell-penetrating peptides (CPPs) with the goal of enhancing cellular uptake. Here we aim to establish CPP-modified neutral liposomes (CPP-NLs) as efficient molecular nanocarriers in vascular smooth muscle cells (VSMCs), with reduced cytotoxicity and enhanced siRNA delivery.
Methods: Unmodified NLs, CPP-NLs, and CLs were formed with 30% cholesterol +/- PEGylation for enhanced membrane stability. Using phosphatidylcholine (PC) as the bulk lipid, CPP-NLs were formed by anchoring stearylated-octaarginine (STR-R8) in the liposome bilayer, confirmed by MALDI-TOF. Liposomes were loaded with GAPDH siRNA via ethanol injection, extruded at 100nm, and purified via dialysis. Narrow size distribution at ~100nm and neutral/positive charge was confirmed with dynamic light scattering and electrophoretic mobility, respectively. Encapsulation was measured via RiboGreen assay, and VSMCs were transfected at 100nM or 200nM siRNA. Non-transfected VSMCs served as the control. Live/dead dual stain was used to measure cytotoxicity. Cy3 labeled siRNA was used to quantify liposomal:cell interaction via fluorometry. GAPDH gene silencing was measured by qPCR.
Results:VSMC cytotoxicity was increased by CL-mediated transfection (12±2 and 13±4%; P<0.05 vs. control; A). VSMCs transfected with CLs exhibited higher fluorescence units (FAU; B), but effective GAPDH silencing was not significantly different among any group (P=NS; C).
Conclusion:NLs and CPP-NLs demonstrated virtually no cytotoxic effect in VSMCs, while CLs were significantly more cytotoxic. While CLs induced greater liposomal:cell interaction, NL and CPP-NL-mediated delivery demonstrated equivalent gene knockdown. The cytotoxic nature of CLs compromises its future as a translatable drug delivery system. Considering the efficacy demonstrated here, NLs and CPP-NLs show promise as alternative non-viral, non-toxic nanocarriers in the delivery of vascular gene therapeutics. Future studies will aim to optimize transfection capacity of CPP-NLs in vascular cells via further lipid/surface modifications, with the goal of developing a molecular drug delivery platform for future in vivo application in vascular injury models of disease.
