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J. C. K. Tovey, A. Sharma, J. W. Cowden, D. Robertson, J. Fortune, A. Kleibanov, R. R. Mohan; Does Gold Nanoparticle-Mediated Non-Viral Gene Delivery in the Cornea Depend on Disease Condition. Invest. Ophthalmol. Vis. Sci. 2010;51(13):434.
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© ARVO (1962-2015); The Authors (2016-present)
We previously showed that gold nanoparticles (GNP) stabilized in polyethyleneimine (GNP-PEI) successfully transport genes into normal and neovascularized rabbit corneas. The aim of this study was to examine whether GNP-PEI vector could deliver therapeutic genes into hazy (scarred) rabbit cornea in vivo with similar efficiency.
New Zealand White rabbits were used. Haze was produced with -9D photorefractive keratectomy surgery with excimer laser. Transfection solutions contained 5µg plasmid DNA expressing green fluorescent protein, 18µl 150mM GNP-PEI, and 10% glucose. Corneas were collected 8h and 72h after vector application. Neutron activation analysis, transmission electron microscopy (TEM), and silver staining determined gold uptake. Slit-lamp microscopy and TUNEL assay tested toxicity. Stereomicroscopy, immunocytochemistry, and fluorescent microscopy quantified gene delivery in normal, neovascularized, and hazy corneas.
Laser treated hazy corneas (96-139ppm) showed gold uptake similar to normal rabbit corneas (63-159ppm) but significantly less than neovascularized corneas (72-377ppm, p<0.05). TEM did not show significant differences in intracellular trafficking or clearance of GNP in normal, hazy, and neovascularized corneas. Interestingly, silver stained normal and hazy corneal tissue sections demonstrated GNP predominantly in the anterior stroma contrary to neovascularized corneas where 60-70% GNP were detected in the anterior stroma and 30-40% in the mid-to-posterior stroma. Quantification of TUNEL assay, CD11b, F4/80 immunocytochemistry, and slit lamp biomicroscopy data did not show major differences in the levels of GNP-PEI toxicity to the cornea in all three groups at two tested time points. The levels of foreign gene delivery in normal, hazy, and neovascularized corneas with GNP-PEI vector were dissimilar. Normal and laser-treated hazy rabbit corneas showed 5-11% differences in transgene expression whereas neovascularized corneas showed up to 31% (p<0.05) more transgene expression compared to these tissues.
Disease condition affects gold nanoparticle-mediated gene delivery in the cornea. Optimization of gene delivery in various corneal disorders is thus necessary for successful treatment of corneal diseases with gene therapy.
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