Upregulation of VEGF in Adult Leptin Deficient Diabetic Mouse Adipose Derived Stromal Cells Using Biodegradable Nanoparticulate Polymeric Vectors Can Be Used to Accelerate Wound Closure in a Diabetic Mouse Excisional Wound Healing Model

Abstract

Introduction: Angiogenesis is essential to wound repair, and adipose-derived stromal cells (ASCs) are attractive vehicles for affecting angiogenesis. However, diabetic ASCs are known to express VEGF to a lesser degree than wild type ASCs, and angiogenic growth factor expression in both cell types is inadequate to have an appreciable effect on wound healing. We have previously demonstrated both in vitro and in vivo efficacy of VEGF DNA plasmid delivery to wild type ASCs using a novel, non-viral biodegradable nanoparticulate polymeric vector. Diabetes is known to interfere with the stages of wound repair. Therefore, the focus of this study is to apply this novel technology to diabetic ASCs to enhance VEGF expression and improve wound healing. Methods: Mouse diabetic and wild type ASCs were transfected with DNA plasmid pEGFP-N1 or pBLAST49-hVEGF using biodegradable poly(β-amino ester). Control cells were transfected using Lipofectamine 2000, and transfection efficiency was determined by flow cytometry. VEGF concentration in conditioned media was measured by ELISA, and cell survival was measured using a viability assay. One million transfected diabetic cells were injected into excisional wounds in diabetic mice, and wound healing measurements were assessed every other day until closure. These measurements were compared to positive ASC-injected and vehicle-treated controls. Results: VEGF ELISA confirmed decreased VEGF concentration in conditioned media of diabetic ASCs compared to wild type ASCs. Polymer-mediated gene delivery to wild type and diabetic ASCs led to approximately two-fold increase in transfection efficiency compared to positive controls (9.91% and 8.70%, versus 3.55% and 3.61%, respectively, *p<0.05). VEGF ELISA showed the diabetic ASCs transfected with polymer/VEGF resulted in VEGF concentration almost three-fold that of positive controls. (*p<0.05). Cell viability on day 2 post-transfection was 60-70% and comparable to positive controls. Diabetic mice injected with VEGF/polymer transfected ASCs healed wounds in 10 days after the procedure and treatment, versus ASC-injected and vehicle controls that healed wounds at an average of 12-14 days (*p<0.05). Conclusions: the use of polymeric nanoparticles is a safe and effective means of upregulating VEGF in both wild type and diabetic ASCs, addressing the limitations of traditional gene delivery methods. Furthermore, VEGF transfected cells injected into excisional wounds of syngeneic mice accelerate wound closure. Therefore, this technology could be used in the development of novel gene-therapy based wound healing strategies for both healthy and diabetic subjects.