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Eric T. Hansen, Ajay Sharma, Ashish Tandon, Rangan Gupta, Jennifer A. Fortune, Alexander M. Klibanov, Jonathan C. Tovey, Yasaman J. Hemmat, Rajiv R. Mohan; Nitrogen To Phosphate Ratio In Nanoparticle-plasmid Transfection Reagent Modulates Gene Delivery And Toxicity In The Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):483.
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Recently we demonstrated that PEI2-GNP nanoparticles (2-12nm) generated from polyethylenimine (PEI) conjugation to gold nanoparticles are capable of delivering genes in rabbit cornea in vivo with mild-to-moderate side effects. We sought to test whether PEI nitrogen (N) and plasmid DNA phosphate (P) molar ratio in PEI2-GNP transfection reagent modulates gene delivery and affects cytotoxicity in the cornea using in vitro model.
Human corneal fibroblasts (HSF) cultures were established from donor human corneas. Transfection reagents having N-P molar ratio 60, 120, 150, 180, 210 and 240 were prepared incubating appropriate volume of 150 mM PEI2-GNP nanoparticles and 1-5µg plasmid DNA (pTRUF11 expressing green fluorescent protein under control of CMV+chicken-β-actin promoter) using 10% glucose and balanced salt solution at room temperature for 30 minutes. Cultures (60-70% confluent) were exposed to transfection reagent for various time points (30 minutes to 4hrs). Phase-contrast microscopy, trypan blue and TUNEL assays evaluated phenotype, cellular viability and toxicity. Delivered transgene expression in HSF was analyzed with immunocytochemistry, real-time PCR and immunoblotting. DNA complexation to nanoparticles was confirmed by gel-retardation assay.
Tested transfection reagents showed effective complexation between PEI2-GNP nanoparticles and plasmid DNA. Transfection reagent of N-P ratio ≤180 did not alter HSF viability or phenotype whereas transfection reagents of N-P ratio ≥210 significantly compromised phenotype and cellular viability (13-20%, P < 0.01). Highest transgene delivery was observed with transfection reagent with N-P ratio 180 (53-58%, p <0.001) followed by 150, 120 and 60 (23-41%, p <0.01). A maximum 3-fold increase in the expression of delivered-gene mRNA and protein was detected with transfection reagent N-P 180 vs. N-P ≤ 150. More comparative studies are underway.
Nanoparticle-based nonviral gene transfer approaches for the cornea requires testing of N-P molar ratio in transfection reagent.
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