Core–Shell Tripeptide-Lipid/PEI Nanocarriers Enable Efficient Plasmid-Based CRISPR/Cas9 Editing of VEGFR2

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile genome editing technology that holds tremendous promise for the treatment of various diseases. Although several delivery technologies such as electroporation, viral vectors, and lipid nanoparticles have already shown promise in preclinical and clinical applications for hematological and neuromuscular genetic disorders, in vivo application is still restricted by the inefficient delivery of CRISPR/Cas9 components. Herein, by employing the tripeptide lipid N, N -ditetradecyloxyamidoethyl trimeric ornithine amide (CDO) and polyethylenimine (PEI), we constructed novel ternary systems (pDNA/PEI/CL) for the delivery of pDNA encoding Cas9 and single-guide RNAs (sgRNAs) targeting the VEGFR2 gene. The pDNA/PEI/CL delivery systems were fabricated by condensing pDNA with PEI, followed by coating with cationic liposomes composed of CDO. This system demonstrated high transfection efficiency, successfully delivering CRISPR/Cas9 to A549 and MCF-7 cells with efficiencies of up to 91.0% ( n = 3, P < 0.001), while also exhibiting lower cytotoxicity. Notably, the sgRNA1/P1/C1 complex achieved higher genome editing efficiencies than sgRNA3/P1/C1, with 38.6% vs 31.0% in A549 cells ( n = 3, P < 0.01) and 26.45% vs 20.18% in MCF-7 cells ( n = 3, P < 0.01). Western blot analysis showed that VEGFR2 expression decreased by 48.1% in A549 and 44.3% in MCF-7 cells, while PI3K levels were reduced by 39.6% and 42.8%, respectively. This suppression of the PI3K/Akt signaling pathway led to cell cycle arrest, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. Furthermore, animal experiments validated the antitumor efficacy, highlighting the translational potential of this platform in cancer therapy. Collectively, these findings highlight the potential of the ternary complex system as a robust and biocompatible CRISPR/Cas9 delivery strategy, offering a promising avenue for gene therapy in cancer and other genetic diseases.