RESUMO
PURPOSE: This study investigated the efficiency and potential toxicity of a linear 22-kDa polyethylenimine (PEI)-DNA nanoconstruct for delivering genes to corneal cells and the effects of PEI nitrogen-to-DNA phosphate (N:P) ratio on gene transfer efficiency in vitro and in vivo. METHODS: A gel retardation assay, zeta potential measurement, bright-field microscopy, transfection with green fluorescent protein (GFP), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to characterize the physicochemical and biological properties and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assay for cytotoxicity of the linear PEI-DNA nanoconstruct using in vitro cultured primary human corneal fibroblast and in vivo mouse models. RESULTS: Of the several evaluated N:P ratios, the highest gene transfection efficiency achieved without any notable cytotoxicity was observed at an N:P ratio of 30:1 (N:P 30). In vivo gene transfer studies revealed substantial GFP gene delivery into the corneas of mice 3 days after a single 5-min topical application without any significant adverse ocular effects. Slit-lamp biomicroscope ophthalmic examination of the mouse exposed to the linear PEI-DNA nanoconstruct showed no evidence of hyperemia (redness), corneal edema, ocular inflammation, or epiphora (excessive tearing). CONCLUSIONS: The 22-kDa linear PEI-DNA nanoconstruct is an efficient and well-tolerated vector for corneal gene therapy in vitro and in vivo and could be used as a platform for developing novel gene-based nanomedicine approaches for corneal diseases.
