Charge-neutralized polyethylenimine-lipid nanoparticles for gene transfer to human embryonic stem cells.

[Display omitted] • Developed a novel library of charge-neutralized polyethylenimine (PEI)-lipid nanoparticles tailored for efficient gene delivery into stem cells. • Achieved superior transfection efficiency, exceeding that of Lipofectamine 2000 and FuGENE HD, across diverse cell types, including hard-to-transfect human embryonic stem cells (hESCs). • Identified critical structure–activity relationships, highlighting the significance of hydrophobic alkyl chain length and substitution ratios for optimizing gene delivery. • Demonstrated the ability of the modified PEI-lipid nanoparticles to co-deliver multiple plasmids, facilitating optogenetic manipulation and enabling precise spatiotemporal control in stem cell differentiation studies. • Established a rapid, scalable synthesis protocol for parallel production of PEI-lipid nanoparticles, presenting a cost-effective platform for future nucleic acid delivery system development in stem cell research. Gene delivery is fundamentally crucial for the genetic manipulation of stem cells. Here, we present a straightforward approach to create a library of charge-neutralized polyethylenimine (PEI)-lipid nanoparticles designed for stem cell transfection. These lipid nanoparticles were formulated using small, branched PEI and lipidic anhydrides. Remarkably, over 15% of the lipid nanoparticles demonstrated high transfection efficiency across various cell types, surpassing the efficiency of both Lipofectamine 2000 and FuGENE HD. A structure–activity analysis indicated that the length and ratio of hydrophobic alkyl substitutions were critical parameters for efficient gene delivery. Notably, the transfection efficiency was higher than that of the original cation PEI. Our optimized PEI-lipid system enabled highly effective plasmid DNA delivery and successfully co-transferred two plasmid DNAs into difficult-to-transfect human embryonic stem cells (hESCs), facilitating optogenetic manipulation within these cells. [ABSTRACT FROM AUTHOR]