PEGylated Nano Lipopolyplexes with Synergistic Lipid Combinations Demonstrate High-Efficiency Nonviral Cellular Delivery of CRISPR/Cas9 Plasmids

Lipopolyplexes (LPPs), a hybrid nonviral vector that combines lipids and polymers with RNA or DNA, are emerging as a key platform for nucleic acid therapeutics such as mRNA vaccines, gene therapy, and CRISPR/Cas9. However, the influence of various lipid structures and their physicochemical characteristics on LPP formation is less well-understood. Here, we systematically evaluated how different lipid types affect cationic polyethylenimine (PEI)-based LPPs for delivering plasmid DNA (pDNA) encoding Cas9 endonuclease and single-guide RNA (sgRNA). LPPs formulated with four lipid types, cationic, ionizable, anionic, and neutral/fusogenic, and their combinations were evaluated for their influence on transfection efficiency and biocompatibility. Among the tested formulations, LPPs composed of anionic lipid Lecithin and fusogenic lipid DOPE exhibited excellent transfection efficiency and biocompatibility compared to those containing cationic lipid DOTAP and ionizable lipid DODMA. Further optimization with a small percentage of PEG lipid (DMG-PEG) resulted in a lead LPP formulation (Lecithin:Chol:DOPE:DMG-PEG) that achieved transfection efficiencies of 62.3%, 54.4%, and 52.6% in HEK293T, PANC1, and A549 cells, respectively, with significantly improved biocompatibility in vitro. The performance of this optimized LPP was significantly higher than that of the commercially available pDNA transfection reagent Lipofectamine 3000. By targeting the KRAS oncogene, the Lecithin:Chol:DOPE:DMG-PEG LPP system achieved gene-editing efficiencies of 18.3% and 15.6% in PANC1 and A549 cells, respectively, highlighting its therapeutic potential as a safe and effective nonviral delivery vehicle for CRISPR/Cas9-based gene editing and other nucleic acid delivery applications.