Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing

CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both...