Open Access

Daniel Keim, Michaela Dehne, Patricia Miller, Valérie Jérôme, Janina Bahnemann, Ruth Freitag

• Nonviral gene delivery offers promise for treating age-related macular degeneration (AMD), a major cause of blindness. Genetic modification of retinal pigment epithelium (RPE) cells is a potential therapeutic strategy for AMD. This study presents a multiparametric approach to enhance nonviral transfection of human ARPE-19 cells using linear poly(ethylenimine) (l-PEI, 25 kDa) as a delivery agent for plasmid DNA (pDNA). The transfection protocol was optimized by adjusting the N/P ratio through nucleic acid concentration, varying polymer density, reducing transfection volume, and minimizing contact time between cells and polyplexes. Under optimized conditions, transfection efficiency (TE) reached 88% with similar to 85% viability. A semi-automated method for polyplex formation was developed using a 3D-printed microfluidic system, thereby enabling standardized production. This optimized protocol was successfully adapted to the microfluidic system without compromising TE or viability. ThisNonviral gene delivery offers promise for treating age-related macular degeneration (AMD), a major cause of blindness. Genetic modification of retinal pigment epithelium (RPE) cells is a potential therapeutic strategy for AMD. This study presents a multiparametric approach to enhance nonviral transfection of human ARPE-19 cells using linear poly(ethylenimine) (l-PEI, 25 kDa) as a delivery agent for plasmid DNA (pDNA). The transfection protocol was optimized by adjusting the N/P ratio through nucleic acid concentration, varying polymer density, reducing transfection volume, and minimizing contact time between cells and polyplexes. Under optimized conditions, transfection efficiency (TE) reached 88% with similar to 85% viability. A semi-automated method for polyplex formation was developed using a 3D-printed microfluidic system, thereby enabling standardized production. This optimized protocol was successfully adapted to the microfluidic system without compromising TE or viability. This semi-automated approach represents a step toward the scalable and reproducible application of l-PEI-based transfection technologies for future therapeutic use.…