Study And Application Of Renewable Oxidation-Reducing Nano-Enzyme Cerium Dioxide In Ocular Neovascularization

Objective:Cerium dioxide（CeO₂）nanomases with reactive oxygen species（ROS）scavenging performance were prepared,while platinum（Pt）was loaded to increase the antioxidant performance of the nanomaterials,and its effect on pathological neovasculation was studied.Methods:In this study,CeO₂with nanozyme properties was prepared.At the same time,Pt was loaded to increase the oxygen production performance of the material.The material was coated with bovine serum albumin（BSA）modification material.The material not only has the catalytic properties of natural enzymes,but also has the uniqueness of nanomaterials.They have stable structure,adjustable activity and diversified functions.The material only catalyzes the reaction,but does not participate in the reaction,It can be recycled and has the effect of continuously inhibiting the formation of neovascularization.The research and application of nanozymes with regenerative redox properties in ocular anti-neovascularization are described in the following two parts:1.Preparation of CeO₂-Pt-BSA:The ceric ammonium nitrate（CAN）and oleylamine were heated under intense stirring,then cooled to room temperature and oleic acid was added.The above solution was slowly added to sodium dodecyl sulfate（SDS）dissolved in ultra-pure water.The solution was fully stirred,the upper oil phase product was centrifuged and washed with cyclohexane and ethanol for three times.The above materials were ultrasonic dispersed in dimethyl sulfoxide（DMSO）,heated and mixed with Polyethylenimide（PEI）dissolved by DMSO,and washed in ultra-pure water after heating.The above materials were dispersed in ultra-pure water and ethylene glycol,then dissolved by ultrasonic dispersion,then hydro chloroplatinic acid（H₂Pt Cl₆）was added,and sodium hydroxide（Na OH）solution was added after further ultrasonic dispersion dissolution.The product obtained by reaction under the condition of heating reflux was mixed with BSA in equal proportion at room temperature.2.Application of CeO₂-Pt-BSA in ocular neovascularization:Mouse retinal pigment epithelial cells（RPE）were selected for in vitro experiments.CeO₂-Pt-BSA of different concentrations were prepared by DMEM high-glucose medium solution and cultured in 96-well plates.CCK-8 solution was added to detect material toxicity.At the same time,cells were incubated with different concentrations of CeO₂-Pt-BSA in DMEM medium for 12 h,then treated with H₂O₂（1m M） medium and incubated for another 24 h.Cell viability was assessed by CCK-8 assay.After 24 h incubation of RPE cells,CeO₂-Pt-BSA labeled with fluorescein isothiocyanate（FITC）was added to 96-well plates with 100μg/ml and incubated for 2,4,and 6 h,respectively.After adding DAPI staining,the intracellular fluorescence was detected by fluorescence microscope after fixation with 4%tissue cell fixating solution.After incubating RPE cells for 12 h,the blank group was replaced with fluid,and the rest were treated with lipopolysaccharide（LPS）in each well.Then,CeO₂-Pt-BSA with different concentrations were added,further incubated for 4h,and co-incubated with reactive oxygen species fluorescence probe（DCFH-DA）.Fluorescence microscopy was used to detect intracellular fluorescence.Results:1.Excellent dispersion and uniform morphology of CeO₂-Pt nanoparticles were observed under transmission electron microscopy（TEM）.2.The synthesis of CeO₂,the loading of Pt,the loading of amino group and the packing of BSA were demonstrated by Fourier transform infrared spectrophotometer（FTIR）,Zeta potential and X-ray diffraction（XRD）,respectively.3.Through the superoxide dismutase（SOD）activity detection kit and hydroxyl radical（·OH）assay kit,it was found that CeO₂-Pt BSA nanoparticles have SOD-like activity,and have the ability to remove superoxide（·O₂^-）and hydroxyl free radical（·OH）.The SOD activity and hydroxyl radical inhibition ability of CeO₂-Pt BSA nanoparticles increased with the increase of material concentration.The oxygen generation capacity of CeO₂cube and CeO₂-Pt BSA nanoparticles after decomposition of hydrogen peroxide（H₂O₂）was tested by oxygen dissolution instrument respectively.It was found that compared with the blank group and the control group,the decomposition capacity of H₂O₂of CeO₂-Pt BSA nanoparticles increased significantly with the increase of time.4.CeO₂-Pt BSA nanoparticles have good biocompatibility at cell level.5.The ROS scavenging ability of CeO₂-Pt BSA nanoparticles was observed at the cell level,and the cell vitality induced by H₂O₂decreased significantly.The cell vitality increased significantly after the addition of CeO₂-Pt BSA nanoparticles,and the cell vitality increased more obviously with the increase of material concentration.6.At the cellular level,the uptake effect of CeO₂-Pt BSA nanoparticles by RPE cells at different time periods was observed using fluorescence microscopy.In mouse RPE cells,the fluorescence intensity of CeO₂-Pt BSA nanoparticles gradually increased with the extension of incubation time within a certain time limit.7.Fluorescent staining of CeO₂-Pt BSA nanoparticles was performed on RPE cells treated with CeO₂-Pt BSA nanoparticles by LPS at the cellular level,and the green fluorescence staining of cells was significantly weakened after the addition of CeO₂-Pt BSA nanoparticles.Conclusion:CeO₂-Pt BSA nanoparticles have excellent dispersion,uniform morphology and good biocompatibility.Moreover,it has SOD-like properties,inhibition of·OH and decomposition ability of H₂O₂.The nanozyme has good biocompatibility,and can reduce the production of ROS,improve the hypoxic environment and inhibit the generation of VEGF on the premise of ensuring safety.