Polymeric Nanoparticles: A promising platform for gene delivery to RPE monolayers

Abstract

Purpose : Efficient non-viral gene delivery into hard-to-transfect cells is still a challenge despite significant progress in the development of various gene delivery tools. Here we demonstrate the use of a high-throughput platform to screen for biodegradable polymeric nanoparticles (NPs) that can transfect retinal cells with high efficiency and low toxicity. From this screen we identified a NP formulation that can transfect Retinal Pigment Epithelium (RPE) monolayer cells at efficiencies of up to 40 ± 5% in complete media with relatively low toxicity.

Methods : RPE was differentiated (Maruotti et al., 2013 and 2015) from a human iPS cell line that contains H2B-nuclear-GFP. Cells were differentiated into monolayers for 25 days in 384-well tissue culture plates. A biodegradable polymer library was synthesized from small molecule diacrylate and primary amine monomers. NPs were created by mixing polymers from a library of >50 poly(beta-amino esters) (PBAEs) with plasmid DNA containing the chicken beta-actin (CAG) promoter upstream of the gene for mCherry fluorescent protein. NPs were added to RPE monolayers at doses between 25-200 ng/well and incubated at 37°C for 2 hours, then media was exchanged. After 48 hours, nuclei were stained with Hoechst and images acquired using an automated fluorescence-based imaging system (Cellomics VTI). Transfected cells were identified as those expressing both the endogenous nuclear GFP and mCherry and the percent of transfected cells, as well as cell viability, was determined for each NP and condition.

Results : Using a High Content Screening (HCS) platform, we identified a NP formulation that can transfect RPE monolayers at an efficacy of up to 40 ± 5%.

Conclusions : We have established a high-throughput platform to screen NPs created from a wide variety of polymers for their ability to transfect human stem cell-derived retinal cells. Using this system, we have identified synthetic polymers that can be used for high efficacy non-viral gene delivery to human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling and developmental pathways. This platform can be used to identify the optimum polymer, weight-to-weight ratio of polymer to DNA, and dose of NP for various retinal cell types.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

hiPSC-RPE monolayers transfected with polymeric NP containing plasmid DNA (lower panel: active NP, upper panel: non-active NP).