Purchase this article with an account.
R. R. Mohan, S. Sinha, K. V. Katti, R. Kannan, W. M. Stapleton, G. S. Schultz; Evaluation of Polymeric- and Gold-Nanoparticles for Gene Delivery in the Cornea. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2733.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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 37oC 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.
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.
Selected nanoparticles are safe and have potential for expressing therapeutic genes in the cornea in vivo.
This PDF is available to Subscribers Only