Elastin-like polypeptide mediated proangiogenic and anti-inflammatory gene therapy for critical limb ischemia

Abstract

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been modest success in reducing the rate of amputations in these patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this thesis was to develop a controlled delivery system capable of delivering multiple therapeutic genes in an extended manner. Initially, a Chitosan/Polyglutamic acid hollow sphere system was tested in vitro as a model system to elucidate the combinatorial effects of physicochemical properties such as size and surface charge on cell viability and, most importantly, on cellular internalization. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and in situ scaffold of elastin-like polypeptide. The hollow spheres and in situ scaffold were independently capable of carrying gene complexes and released the gene complexes in an extended manner. Furthermore, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. Initially, a subcutaneous dose study was performed in the mouse model to determine a therapeutic dose of hIL-10 and heNOS. In the injectable ELP system, hIL-10 was loaded inside the scaffold and heNOS inside the ELP hollow spheres. Human eNOS(20 µg) and hIL-10(10 µg)/heNOS(20 µg) showed comparatively more blood vessel density than others and hIL-10(10 µg) showed comparatively reduced the amount of inflammatory cells. These groups were then selected for the hind limb ischemic study including control groups: saline, injectable system only. The treatment groups that showed higher blood perfusion measured using laser doppler perfusion imaging were the groups with heNOS treatment groups. Saline group showed signs of sever necrosis. Human IL-10 treatment groups showed reduction in the level of inflammation cells. Furthermore, a mechanistic study showed proangiogenic activity of eNOS by up-regulating major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B and fibroblast growth factor 1. These factors help in formation of a stable vascular network. Thus, ELP injectable system mediated non-viral delivery of IL10-eNOS is a promising therapy towards treating limb ischemia.