Abstract
Ischemic reperfusion injury after a stroke is a leading cause of mortality and disability due to neuronal loss and tissue damage. Mitochondrial dysfunction plays a major role in the reperfusion-injury sequelae, and offers an attractive drug target. Mitochondrial derived reactive oxygen species (ROS) and resultant apoptotic cascade are among the primary mechanisms of neuronal death following ischemia and reperfusion injury. Here we optimized a nanoparticle formulation for the mitoNEET ligand NL-1, to target mitochondrial dysfunction post ischemic reperfusion (IR) injury. NL-1, a hydrophobic drug, was formulated using PLGA polymers with a particle size and entrapment efficiency of 123.9 ± 17.1 nm and 59.7 ± 10.1%, respectively. The formulation was characterized for physical state of NL-1, in vitro release, uptake and nanoparticle localization. A near complete uptake of nanoparticles was found to occur by three hours, with the process being energy-dependent and occurring via caveolar mediated endocytosis. The fluorescent nanoparticles were found to localize in the cytoplasm of the endothelial cells. An in vitro oxygen glucose deprivation (OGD) model to mimic IR was employed for in vitro efficacy testing in murine brain vascular endothelium cells (bEND.3 cells). Efficacy studies showed that both NL-1 and the nanoparticles loaded with NL-1 had a protective activity against peroxide generation, and displayed improved cellular viability, as seen via reduction in cellular apoptosis. Finally, PLGA nanoparticles were found to have a non-toxic profile in vitro, and were found to be safe for intravenous administration. This study lays the preliminary work for potential use of mitoNEET as a target and NL-1 as a therapeutic for the treatment of cerebral ischemia and reperfusion injury.