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Chaitasi Naik, Jason Rodier, Ajay Sharma, Audra Stallard, Ashish Tandon, Alexander Klibanov, Rajiv Mohan; Nanoparticle polyethylenimine-BMP7 Transfection Dose, Anti-fibrotic Efficacy and Toxicity for the Rabbit Cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4701. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The goals of study were to characterize polyethylenimine-DNA nanoparticles (NP) transfection dose to achieve high transgene delivery at low toxicity in the cornea manipulating polyethylenimine nitrogen (N) and plasmid phosphate (P) molar ratio using an in vitro model; and, test NP-BMP7 (bone morphogenic protein 7) gene therapy potency and safety to treat corneal scarring in vivo using a rabbit model.
New Zealand white rabbits and human corneal fibroblasts (HCF) generated from donor human cornea were used. NP polyplexes with N/P molar ratio 4, 8, 15, 30 and 60 were prepared using 22kD linear polyethylenimine and plasmid encoding GFP or BMP7 gene. Time- and dose-dependent transfection assays were performed for optimization studies. Corneal fibrosis (haze) in rabbit was produced with excimer laser performing -9D photorefractive keratectomy (PRK). NP-BMP7 was topically applied on rabbit eye with custom technique for 5 minutes after PRK. Slitlamp biomicroscopy in live rabbits graded haze and ocular toxicity. Rabbits corneas were collected 4-weeks after PRK. Phase-contrast microscopy, trypan blue, MTT, TUNEL assays analyzed cellular morphology, proliferation, viability and apoptosis death. Real-time PCR, western blotting, and immunfluorescence quantified gene delivery, efficacy and toxicity.
The NP-DNA gene transfer efficacy depended on N/P ratio. Higher N/P ratio in NP-DNA solution showed higher transgene delivery (40-55%, p<0.001). The N/P molar ratios >30 showed no-to-mild phenotype, proliferation or viability loss in vitro (5-13%). The transfection with N/P 8 or 15 showed low toxicity. A single 5 minutes topical NP-BMP7 (N/P 8) treatment showed significantly less corneal haze (47-53%, p<0.01) without ocular toxicity in rabbits in vivo. Further, this treatment significantly decreased mRNA (2-5 fold; p<0.01-0.001) and protein (41-56%; p<0.05-0.001) expression of 4 pro-fibrotic genes (alpha smooth muscle action, f-actin, fibronectin and collagen-1). The toxicity investigations using CD11b, F4/80 and TUNEL immunocytochemistry in rabbit tissues are underway.
Tested nanoparticles are safe and potent for treating corneal disorders in vivo. Targeted NP-BMP7 gene delivery has potential to treat corneal scarring in vivo. More animal and mechanistic studies are warranted.
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