Production of Extracellular Vesicles expressing Serpin-B3 and evaluation of their antioxidant activity in-vitro

Abstract

In heart transplantation, DCD organs (Donation after Circulatory Death) undergo a warm ischemia. The process of reperfusion can induce an inflammatory response and the release of free radicals with cardiomyocyte death and a tissue injury. The aim of this study was developing an in-vitro model based on Extracellular Vesicles (EV), a type of bio-nanoparticles, enriched in Serpin-B3 and evaluate their biological activity as antioxidant in ischemic damage. EVs are formed during the invagination of multivesicular bodies, from previous studies EVs mediate cardioprotection in murine model. Another aspect in this work is given by Serpins, a group of serine protease inhibitors. SB3 induces deregulation of adhesion processes and increases the invasiveness potential by epithelial–mesenchymal transition. EVs were obtained through ultrafiltration from HepG2, these cells were engineered to overexpress SB3 by transfection with a plasmid expression vector containing the human SB3 gene. Physicochemical characterization of the EV-SB3 was observed through TRPS technique SB3 over-expressing cells secrete 32% of EVs-SB3 more than control. Both types of EVs have a diameter of around 110nm as evidenced by the TEM analysis. Western blot and ELISA assays confirmed the presence of SB3 only in the EV-SB3 group. The biological activity of EV-SB3 was tested by an in-vitro assay assessing the protection against oxidative damage, and through MTT assay EV-SB3 improved cell survival after oxidative injury.