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Bibhudatta Mishra, David R Wilson, Yuan Rui, Mark P. Suprenant, Baranda S. Hansen, Cynthia Berlinicke, Jordan J Green, Donald J Zack; Nanoparticle based transfection of hard-to-transfect primary photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):363.
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© ARVO (1962-2015); The Authors (2016-present)
Primary retinal cells represent the main functional component of their in vivo counterparts more closely than transformed or immortalized retinal cell lines. Efficient gene transfer into primary retinal cells, however, is still a major challenge. While viral strategies can be effective at delivering nucleic acids, they provide low throughput for potential screening applications and studying genetic mechanisms. Here, we demonstrate an alternative strategy for photoreceptors (PRs) transfection, the application of biodegradable polymeric nanoparticles to introduce plasmid DNA into primary PRs cells with relatively low toxicity and transfection efficiencies of up to 30% in complete media.
Retinas from transgenic mouse expressing GFP under a PR specific promoter (QRX-GFP) were dissociated and cultured into 384-well tissue culture plates. A biodegradable polymer library was synthesized from small molecule diacrylate and primary amine monomers. Nanoparticles were created by mixing polymers from a library of >50 poly(beta-amino ester) (PBAE) with CAG driven mCherry plasmid DNA in sodium acetate buffer, pH 5.0. Nanoparticles were then added to the cultures at doses between 25-200 ng/well and the plate was incubated at 37°C for 2 hours, before the media was exchanged. After 24 hours of culture, nuclei were stained with Hoechst and images were acquired using an automated fluorescence-based imaging system (Cellomics VTI). Transfected PRs were identified as GFP expressing cells also expressed mCherry.
PR Transfection efficiencies of up to 26 ± 4.3% were achieved using a subset of nanoparticle formulations. (A) compared to <5% transfection efficiency using the commercial reagent Lipofectamine (B).
We have established a high-throughput platform to screen nanoparticles created from a wide variety of polymers for their ability to transfect retinal cells. Using this system, we have identified synthetic polymers that can be used for high efficacy non-viral gene delivery to primary murine PRs, enabling cell signaling and developmental pathways to be more thoroughly studied at the molecular level. This platform can be used to identify the optimum polymer, w/w ratio of polymer to DNA and dose of nanoparticle for different retinal cell types.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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