ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia

With critical limb ischemia (CLI) being a multi-factorial disease, it is becoming evident that gene therapy with a multiple bio-functional growth factor could achieve better therapeutic outcomes. Cytochrome P450 epoxygenase2J2 (CYP2J2) and its catalytic products epoxyeicosatrienoic acids (EETs) exhibit pleiotropic biological activities, including pro-angiogenic, anti-inflammatory and cardiovascular protective effects, which are considerably beneficial for reversing ischemia and restoring local blood flow in CLI. Here, we designed a nanoparticle-based pcDNA3.1-CYP2J2 plasmid DNA (pDNA) delivery system (nanoparticle/pDNA complex) composed of a novel three-arm star block copolymer (3S-PLGA-po-PEG), which was achieved by conjugating three-armed PLGA to PEG via the peroxalate ester bond. Considering the multiple bio-functions of CYP2J2-EETs and the sensitivity of the peroxalate ester bond to H2O2, this nanoparticle-based gene delivery system is expected to exhibit excellent proangiogenic effects while improving the high oxidative stress and inflammatory micro-environment in ischemic hindlimb. Our study reports the first application of CYP2J2 in the field of therapeutic angiogenesis for CLI treatment and our findings demonstrated good biocompatibility, stability and sustained release properties of the CYP2J2 nano-delivery system. In addition, this nanoparticle-based gene delivery system showed high transfection efficiency and efficient VEGF expression in vitro and in vivo. Intramuscular injection of nanoparticle/pDNA complexes into mice with hindlimb ischemia resulted in significant rapid blood flow recovery and improved muscle repair compared to mice treated with naked pDNA. In summary, 3S-PLGA-po-PEG/CYP2J2-pDNA complexes have tremendous potential and provide a practical strategy for the treatment of limb ischemia. Moreover, 3S-PLGA-po-PEG nanoparticles might be useful as a potential non-viral carrier for other gene delivery applications.