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Methods: : Plasmid-loaded nile red PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. KNV was induced in BALB/C mice by mechanical-alkali injury using 2 ul of 0.15M NaOH for 10 seconds followed by scraping of the corneal epithelium with a Tooke corneal knife. Vessels were allowed to mature over 4 weeks after which the mice were randomly divided into 4 groups, each of which received one of the following interventions: pSEC.siRNA.VEGFA NR PLGA NPs (2ug plasmid), naked pSEC.siRNA.VEGFA plasmid (2ug plasmid), blank NR PLGA NPs, and DMSO. The plasmid-loaded NPs were prepared in sterile DMSO to a concentration of 1 ug/ul and 2 ul were injected intracorneally using a 33 gauge needle. 4 weeks after intervention, the mice were sacrificed and the corneas were harvested for flatmounts, RT-PCR, and VEGF-A ELISA. Flatmounted corneas were immunostained for CD31 (endothelial cell marker) and the neovascular area was quantitated using Scion Image. VEGF-A gene expression was evaluated using RT-PCR. Protein levels were determined using VEGF-A ELISA.
Results: : siRNA.VEGFA loaded PLGA NPs showed significant regression of KNV compared to naked plasmid and controls. siRNA.VEGFA loaded PLGA NPs regressed KNV to 12.5%. Naked plasmid treatment resulted in a KNV area of 28%. The two control groups had highly vascular corneas with 53% KNV for DMSO and 55% KNV for blank NPs. VEGF-A protein and RNA expression were reduced significantly in siRNA.VEGFA loaded PLGA NP-treated corneas. 781b155fdc