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Joel C. Sunshine, Sarah B. Sunshine, James T. Handa, Jordan J. Green; Transfection Of Retinal Pigment Epithelial Cells With A Combinatorial Library Of poly(beta-amino ester)s. Invest. Ophthalmol. Vis. Sci. 2011;52(14):465.
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A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, as well as macular degeneration, diabetic retinopathy, and retinopathy of prematurity, have utilized viral-based gene delivery as a potential treatment strategy. Non-viral systems for gene delivery offer a host of potential advantages compared to viruses, including reduced toxicity, immunogenicity, and increased ease of production. Poly(beta-amino) esters (PBAEs) have shown great potential as gene delivery reagents and are easily synthesized, rapidly screened, and transfect cells with high efficacy in vitro. As a result, we synthesized a combinatorial library of PBAEs and tested them for transfection efficacy and toxicity in ARPE-19 cells to identify lead polymer structures and transfection formulations.
The library of PBAEs was synthesized by adding primary amines to diacrylate compounds (1:1.2 molar ratio of amine:diacrylate) at 90oC for 24h, and then the base polymers were end-capped by end-capping amines (at 10-fold molar excess of amine to diacrylate termini) at room temperature for 24h. Polymers were complexed with CMV-Luc plasmid through electrostatic self-assembly and added to cells. The medium was changed 4h post transfection. Viability was assessed by the MTS assay at 24h post transfection, and luciferase expression was assessed using the luciferase assay (BrightGlo) 48h after transfection.
The screen identified 2-methylpentane-1,5-diamine terminated poly(1,4- butanediol diacrylate-co-5-amino-1-pentanol) and (PEO)4-bisamine terminated poly(1,4- butanediol diacrylate-co-5-amino-1-pentanol) (454 and 455) as lead polymers for transfection of ARPE-19 cells. Optimized formulation of each polymer showed transfection efficiencies comparable to Lipofectamine 2000 or FuGeneHD, two of the lead commercially available alternatives for non-viral gene delivery.
Poly(beta-amino) esters show great promise for delivery of plasmids to retinal pigment epithelial cells.
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