Evaluation of Polymeric- and Gold-Nanoparticles for Gene Delivery in the Cornea

Abstract

Purpose:: Recent reports showed transgene delivery into various cells/tissues using biocompatible and biodegradable nanoparticles. The goals of current study are to evaluate toxicity of poly-lactide-co-glycolide (PLGA), N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), and gold (AuNP) nanopaticles for human corneal cells and identify transfection mixture(s) suitable for delivering therapeutic genes in the cornea.

Methods:: Human corneal fibroblasts (HSF) and human corneal endothelial (HCN) cells were generated from human corneas procured from eye banks. Cultures were prepared by seeding 15000cells/cm² using DMEM containing 10% serum and maintained at 37^oC in humidified atmosphere with 5% CO₂. The gold-nanoparticles (12nm), stabilized with glycoprotein rich gum arabic material, were obtained from Nanoparticle Core Production Facility, University of Missouri-Columbia, and PLGA and DOTAP were purchased from Sigma. The pTRUF11-EGFP plasmid vector expressing EGFP under control of hybrid CMV+chicken beta actin promoter (UF11gfp) and pTR-UF5-ßgal plasmid vector expressing ßgal under control of CMV promoter (UF5ßgal) were used. Transfection mixtures were prepared by condensing different concentration of plasmid (2-10µg/ml) and nanoparticles (3-30µg/ml). Cultures were exposed to various concentrations (0-10µg/ml) of PLGA, DOTAP or AuNP for 0-72 hours or DNA-nanoparticle formulations for 1-4 hours. Live/dead cell assay, immunocytochemistry, bright/fluorescent and electron microscopy were used to evaluate toxicity and transfection efficiency.

Results:: Tested nanoparticles showed concentration-dependent toxicity to the HSF and HCN cells. PLGA (≤5ug/ml), DOTAP (≤6ug/ml) and AuNP (≤20ug/ml) induced 3 to 8% (±0.5, n=5) cell death at 24hours, 9 to18% (±1.1, n=5) at 48hours and 11 to 21% (±1.4, n=5) at 72 hours in HSF and HCN cultures. The electron microscopy showed substantial uptake of gold particles in HSF (27%±4.3) and HCN (21%±3.2) cells. The transfection mixture prepared with DOTAP (5µg/ml) and UF11gfp (4.0µg) showed significant transgene expression in HSF (19.3% ±2.6; p=<0.01) and HCN (13.6%±1.9, p=<0.1) cells. The gene transfer experiments using PLGA and AuNP are underway.

Conclusions:: Selected nanoparticles are safe and have potential for expressing therapeutic genes in the cornea in vivo.

Keywords: gene transfer/gene therapy • cornea: stroma and keratocytes • cornea: basic science