Purpose: Ischemic stroke is one of the leading causes of disability and death worldwide. MitoNEET is an iron sulfur cluster containing protein present in the outer mitochondrial membrane, that is known to regulate cellular bioenergetics. In the current work, we studied the pharmacologic actions of a novel mitoNEET ligand NL-1 as a potential neuroprotective agent following cerebral ischemia and reperfusion injury. Herein, we also report development of a polymeric nanoparticle system for the delivery of NL-1 to the brain for stroke therapy.
Methods: The effect of NL-1 in reducing ROS levels was studied on N2a cells using the Amplex red fluorimetric assay. NL-1 actions on the cellular respiration of N2a cells were studied using the seahorse assay. PEG-PLGA nanoparticles for the brain delivery of NL-1 were synthesized using emulsification and solvent evaporation technique. The prepared nanoparticles were passed through a Sephadex gel filtration column to remove the unentrapped drug, and subsequently freeze dried with 5% mannitol as a cryoprotectant. The nanoparticles were characterized for particle size, zeta potential and entrapment efficiency for NL-1. Atomic Force Microscopy was used for determining the morphology of the particles. The association of NL-1 with the nanoparticles was studied using powder X-ray diffraction. The nanoparticle toxicity was studied on HepG2 cells using the MTT cell viability assay.
Results: NL-1 was found to decrease reactive oxygen species production with an IC50 of 5.95 µM in Na cells. At a concentration of 10 µM, NL-1 was found to increase cellular respiration in N2a cells, as seen from the increased oxygen consumption rate. The particle size of PEG-PLGA nanoparticles was found to be 175.63 ± 6.8 nm, and the zeta potential was found to be -26.2 ± 1.27 mV. The size was seen to remain stable over a period of 60 days. NL-1 entrapment efficiency was found to be 26 ± 2.2%. The cell viability upon treatment with blank PEG-PLGA nanoparticles was found to be 70% at the highest concentration of 10 mg/mL.
Conclusion: Targeting mitoNEET with NL-1 has a potential to serve as a neuroprotective in alleviating stroke pathology, as it reduces the levels of the noxious ROS, and improves cellular respiration. Polymeric nanoparticles for NL-1 delivery were successfully prepared by emulsification and solvent evaporation technique, and a particle size of under 200 nm was achieved. The NL-1 entrapment was found to be lower than expected. Measures to improve entrapment of NL-1 would be studied. We further plan on doing a comparative analysis of a liposomal and polymeric delivery system for NL-1, and to study its efficacy in stroke mouse models using middle cerebral artery occlusion as a stroke mimetic technique.
Jacob Boos– West Virginia University, Morgantown, West Virginia
Alexander Mdzinarishvili– University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Justin Legleiter– West Virginia University, Morgantown, West Virginia
Werner Geldenhuys– Associate Professor of Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia