Fibrin Mediated Proangiogenic and Secretory Control Gene Therapy for Compromised Wound Healing

Abstract

Chronic or compromised healing is a major clinical problem. Particularly, in diabetes where the disease process hinders the capacity to repair the tissue damage, the patients are more susceptible to chronic ulcers, especially on the lower extremity. Despite rigorous treatment regimes, there has been modest success in reducing the rate of amputations in these patients. Hyperglycemia, the defining biochemical phenomenon of diabetes, is by far the most important predisposing factor for chronic wound healing complications. The objective of this thesis was to unleash the pathological disarray by studying the effects of hyperglycemia at molecular level on wounded keratinocytes and subsequently to develop a controlled delivery system capable of delivering therapeutic genes in an extended manner. A fibrin lipoplex system capable of simultaneous delivery of multiple genes was tested in vitro and in vivo. As a step to improve this system for controlled release and increase the capacity of the system, fibrin microspheres loaded with gene complexes were successfully developed and investigated for functional gene delivery in vivo using proangiogenic gene - eNOS. From the microarray data analysis on the wounded keratinocytes under hyperglycemic culture conditions, a profound differential gene regulation was revealed with a number of up- and down-regulated genes. The secretory control molecule Rab18, found to be significantly down-regulated, was chosen as target therapeutic gene considering the hypersecretory state of proinflammatory cytokines and proteolytic enzymes in diabetic wound healing. eNOS was chosen as other therapeutic gene, considering its proangiogenic action and reduced angiogenesis in diabetic wound healing. With eNOS gene complexes in fibrin gel and Rab18 gene complexes in fibrin microspheres embedded in fibrin gel, the Rab18-eNOS loaded fibrin-in-fibrin system was investigated in alloxan induced hyperglycemic rabbit ear ulcer model of compromised wound healing. Rab18-eNOS treated group showed significantly higher percent wound closure at day 14 post-wounding with reduced inflammatory cell infiltrate and more functional angiogenesis. Thus, fibrin mediated non-viral delivery of Rab18-eNOS is a promising therapy towards normalization of diabetic wound healing.