Date of Award
Doctor of Philosophy
Comparative and Experimental Medicine
Oscar Grandas, Michael Best, Stephen Kania
Percutaneous transluminal angioplasty (PTA) is a common endovascular procedure that restores blood flow in peripheral vascular disease. Unfortunately, endovascular procedures inherently cause injury to intimal layer. This exposing the medial layer and vascular smooth muscle cells (VSMCs) to hemodynamic flow. The injury response induces dysfunctional VSMC phenotypes, leading to thickening of the vessel wall known as intimal hyperplasia (IH). Eventually, IH leads to restenosis, a common complication of PTA.Most therapeutic strategies for IH are aimed at reducing VSMC migration and proliferation. However, recent studies have shown healing of the VEC layer is crucial mitigating factor in IH. We postulate that an optimal intervention could be achieved by employing both therapeutic strategies simultaneously in a cell-type specific manner at the site of PTA-induced injury. Gene therapy techniques provide an opportunity to accomplish this goal. Many IH-associated genetic targets have been successfully modulated to improve reendothelialization of VECs and inhibit the proliferation of VSMCs. However, clinical success of vascular gene therapy is limited due to the lack of a translational delivery vehicle.Liposomes have been shown to be effective gene vectors with translational efficacy. The addition of polyethylene glycol (PEG) to liposomes (PLP) can improve in vivo pharmacokinetics, but reduces cellular uptake of liposomal cargo. Ligand-modified liposomes provide an opportunity to enhance transfection of neutral PLPs and target specific cell types for gene delivery. Cell-penetrating peptides (CPPs) containing arginine-rich motifs have the ability to enhance membrane translocation of their conjugated cargo. Cell-targeting peptides (CTP) can also be used to decorate the liposome surface, providing cell-type specificity. In this in vitro proof-of-concept study, we aim to develop a modified PLP comprised of neutral lipids and capable of enhanced transfection and cell-type specific delivery to VSMCs and VECs, respectively.Using a Ca2+-mediated ethanol injection technique, a novel method for the self-assembly of CPP-modified PLPs with enhanced transfection, optimized siRNA loading efficiency, minimal cytotoxicity, and cell-targeting capabilities was developed. These nanocarriers convey chemical stability to siRNA in the presence of nuclease activity. This liposomal delivery system could provide the foundation necessary to increase the bench-to-bedside success of systemically administered vascular gene therapy.
Fisher, Richard Kern III, "The Development of a Dual-ligand PEGylated Liposome Nanotechnology for Cell-selective Targeted Vascular Gene Therapy. " PhD diss., University of Tennessee, 2019.