Vascular oxidant stress plays a key role in many pulmonary pathologic states including hyperoxia, acute lung injury and inflammation. Current antioxidant means are only marginally effective, in part due to sub-optimal delivery to endothelial cells (EC) and limited duration. We found that the antioxidant enzymes (AOE, e.g., catalase that detoxifies freely diffusing H202) conjugated with EC Cell Adhesion Molecules (CAM) antibodies are delivered into EC in vivo and protect animals from acute oxidant lung injury. However, anti-CAM/AOE conjugates afford only transient effects due to rapid elimination from the blood and degradation in EC. In order to achieve higher therapeutic duration and utility, we propose to load catalase into H2O2-permeable, anti-CAM targeted all-block PEG-PLGA Polymer Nano-Carders (PNC) that will prolong the AOE circulation and protect from lysosomal proteolysis. We also will deliver Prx-VI, a candidate AOE detoxifying diverse organic and hydroperoxydes, into EC interior. Pilot data show that catalase loaded inside stealth anti-CAM/PNC degrades H202, binds to and protects EC from oxidant injury, circulates in animals for a prolonged time, and targets the pulmonary vasculature. Our goal is to explore, test and optimize this new strategy based on polymer nanotechnology, pursuing the following Specific Aims: 1. Optimize the design of PNC for AOE delivery to EC. PEG-PLA PNC formulation, composition, size, AOE loading, activity and protease resistance, rate of degradation, coupling of anti-CAM, binding, uptake and metabolism by EC, and protection of EC will be studied and optimized in vitro. 2. Characterize the behavior and targeting of anti- CAM/PNC/AOE in vivo. Their blood clearance, systemic effects, biodistdbution, EC binding, pulmonary targeting and persistence in the pulmonary vasculature will be tested in naive animals and animals with oxidant vascular stress. 3. Evaluate the effects of anti-CAM/PNC/AOE in animals. Mouse models of acute H2O2 generation in the pulmonary vasculature and sub-acute oxidant stress in the lungs (hyperoxia) will be employed to evaluate the degree and duration of protection, and refine regimens of AOE targeting. Results of this study will provide a basis for translation of this new technology platform into the clinical domain.
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