342. Water Soluble and Bio-Friendly Nanoceria Reduces Laser-Induced CNV Mouse Model of Wet AMD
Authors: Rajendra N. Mitra, Min Zheng, Zongchao Han
Journal: Molecular Therapy (2016)

Purpose: Recently Nanoceria has been widely explored in various disciplines including biomedical science. Nanoceria has intrinsic antioxidant property, which is restricted by its poor aqueous solubility, and therefore possesses less residual time in the blood stream. For biological applications, nanoceria requires sufficient surface shield to prevent aggregation. Use of toxic solvents during its synthesis are also creating additional detrimental effects in its clinical applications. To avoid these issues and to make more biocompatible nanoceria, we have synthesized a water soluble and stable nanoceria formulation using chitosan derivative in plain water. Our purpose is to utilize this water soluble nanoformulation to scavenge free radicals generated in the Age Related Macular Degeneration (AMD) mice model, a retinal degenerative dystrophy which leads to irreversible central vision loss and finally causes legal blindness in people over 50 years of age. Methods: We have used cerium (III) chloride heptahydrate and chitosan derivative to synthesize the ceria nanoparticle (NPs). These NPs are then purified and solubilized in plain water. These NPs were characterized by dynamic light scattering, FTIR, UV-Vis, and TEM. The characterized NPs were then incubated with human retinal pigmented epithelium cells (ARPE19) in presence and absence of H2O2 that mimic the in vitro AMD model. The MTT assay was carried out to determine the cellular toxicity of the NPs at different concentrations. H2O2 induces reactive oxygen species (ROS) inside the APRE19 cells, which were determined by using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The safe dose of NPs were then injected into adult wild type (C57Bl/6) mice after laser induced choroidal neovascularization to validate the therapeutic effect of this NP formulation in AMD mice model. Results: The TEM studies showed that the NPs are conjugated with the organic matrix and shows a diameter of ~4 nm and demonstrated uniform structure throughout the TEM-grid. The NPs showed considerable amount of cell viability with varying concentrations (n=3-5). The NPs (5 different concentrations) were then incubated over night with ARPE19 cells and DCF assay was carried out with 10 µM DCFH-DA and the fluorescence of DCF was determined with excitation 485 nm and emission 530 nm. The results revealed that the NPs could scavenge ROS with a significant amount (n=5, p<0.0001) compared to the uninjected and mock injected controls. Furthermore, a single intravitreal injection of the NPs significantly reduced the size of laser-induced AMD mouse model as measured by fundus fluorescence angiography. Conclusion: Our result are reflecting consistency with our idea that water soluble ceria NPs are more stable in water and can reduce the injury of the retina in laser induced CNV model. We are working to define the morphological and functional changes of the retina in this animal model. If successful, this will be a major breakthrough in AMD treatment.