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S. Sinha, D. McKnight, K. V. Katti, R. Kannan, D. J. Robertson, J. W. Cowden, R. R. Mohan; Gold Nanoparticles Stabilized in Gum Arabic for Corneal Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4787.
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Nanoparticles can efficiently transport DNA into the cells. Gold nanoparticles stabilized in naturally occurring non-toxic gum arabic (GA-AuNP) were developed recently at the University of Missouri-Columbia, bind to DNA efficiently and have shown exceptional in vivo stability, uptake and clearance in pigs and rodents. The aims of this study were to (i) evaluate GA-AuNPs cytotoxicity to the cornea, (ii) examine the effects of GA-AuNPs on corneal morphology and function and (iii) determine their suitability for corneal gene therapy.
Donor human corneas and cultured human corneal fibroblasts (HSF) were used. Cultures were maintained at 37oC in humidified atmosphere with 5% CO2. GA-AuNPs (size 16-20 nm) were obtained from the Nanoparticle Production Core, University of Missouri-Columbia. Cultures were exposed to GA-AuNP (0-60µg/ml) for various durations (0-72hrs). Trypan blue exclusion, MTT, MultiTox-Fluor, glutathione and TUNEL assays were used to evaluate cytotoxicity. Immunocytochemistry, bright/fluorescent microscopy and transmission electron microscopy (TEM) were used to analyze morphological changes, relative populations of live and dead cells and/or intracellular trafficking of GA-AuNP in the human cornea and HSF. Inductive coupled plasma enhanced absorption spectroscopy (ICP-EAS) and neutron activation atomic absorption (NAA) were used to determine cellular uptake of gold in human cornea in vitro and ex vivo.
None of the tested doses of GA-AuNP (≤30µg/ml) induced morphological changes, apoptosis, cytotoxicity or oxidative damage to the human cornea or HSF at examined time points. Significant uptake of gold was detected in HSF at 4 hours (22.5±0.85-30.85±1.20 ng/ml) by ICP-EAS and in ex vivo human cornea at 4 hours (2.3-22.5 ppm) and 24 hours (6.6-47.0 ppm) by the NAA analysis. TEM analysis demonstrated that GA-AuNPs can enter the human corneal epithelial, stromal and endothelial cells freely. Furthermore, TEM confirmed the presence of GA-AuNPs in the cytoplasm and revealed that GA-AuNPs did not cause damage to the vital cell organelles.
Gum arabic-stabilized gold nanoparticles are nontoxic, safe and biocompatible for the use in the cornea and may serve as vehicles for delivering drugs or genes to the cornea. Studies are underway to assess their suitability for delivering therapeutic genes in the cornea in vivo.
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