(542c) Controlled Release of N-Acetylcysteine Positively Affects the Viability, Redox State and Morphology of Oligodendrocyte Progenitor Cells Under Oxidative Stress

Introduction: Overproduction of reactive oxygen species (ROS) is a major factor in the pathogenesis of multiple sclerosis. Localized antioxidant delivery is therapeutically relevant to counteract and neutralize ROS in such oxidant-stressed systems. Poly(lactic-co-glycolic acid) (PLGA) microparticles can provide a localized, controlled release of encapsulant, though this has been limited primarily to large proteins or small hydrophobic molecules. Here we investigate the therapeutic efficacy of controlled release of N-acetylcysteine (NAC) on oligodendrocyte progenitor cells (OPCs) under oxidative stress by examining viability, redox state and morphology. We aim to rescue OPCs from oxidative stress by using highly loaded PLGA microparticles encapsulating the hydrophilic, small molecule NAC.

Materials and Methods: NAC-loaded PLGA microparticles were prepared via the double emulsion, solvent evaporation technique including doping of all external water phases with NAC to maximize drug load (yielding between 40 and 133 ug NAC/mg particle). For all cell experiments, OPCs were grown in proliferation media for 24 hours. Experimental media was free of growth factors and antioxidants. For the 24-hour rescue experiment, OPCs were incubated for 30 minutes with 1mM H2O2. Concentrated free NAC, empty particles, or NAC-loaded particles were then added to the wells containing H₂O₂ to yield 1mM NAC. Particle dose was determined by cumulative NAC released within 24 hours. For the prolonged treatment experiment, OPCs were incubated for 30 minutes with 0.5 mM NAC or 48-hour equivalents of empty or NAC-loaded particles. Concentrated H₂O₂ was then added to the wells to yield 0.2mM in the well. After 24 hours of incubation, H₂O₂ was added again to yield 0.2mM in the well and incubated for an additional 24 hours. At the end of each experiment, wells were imaged with a Zeiss fluorescent microscope under brightfield and GFP channels. OPCs were lysed and ATP (CellGlo), DNA (PicoGreen) and glutathione (GSH-Glo) were measured.

Results and Discussion: Added 30 minutes after the onset of oxidative stress, controlled release of NAC for 24 hours rescues the viability of OPCs to 80% of healthy control, from 30% without treatment. There is an insignificant increase in viability relative to the free, soluble drug. Controlled released of NAC protects OPCs from multiple administrations of H₂O₂ over the course of 48 hours, yielding a 2-fold increase in reduced glutathione per cell which indicates a more reduced redox state. Controlled release of NAC also enhances OPC morphology, yielding 0.34 processes/cell with particle treatment compared to 0.29 processes/cell without treatment. Ongoing work aims to fabricate core-shell microparticles with a subdued initial burst release. Preliminary confocal images suggest that we have fabricated core-shell PLGA microparticles with two distinct polymer phases.

Conclusions: Highly loaded PLGA microparticles encapsulating the small-molecule antioxidant NAC rescued OPCs from oxidative stress, yielding a viability near control levels. NAC-loaded particles were shown to protect OPCs from multiple administrations of oxidants over 48 hours by increasing the levels of reduced glutathione above control levels. NAC-loaded particles also prevented degradation of OPCs. This work shows that efficiently loaded antioxidant microparticles capable of controlled and localized delivery could have therapeutic potential in oxidant-stressed systems by rescuing and protecting cells from harmful oxidants.