| Objective:To prepare a highly efficient nonviral vector loading CRISPR/Cas9 plasmids which could actively target tumor cells and delivers the payload into nuclei of cancer cells.With the disruption of MTH1,an essential protein to tumor,the growth of non-small cell lung cancer was expected to be inhibited.Broaden the application of antitumor gene target with "cancer phenotypic lethality" in cancer treatment.Methods:(1)The material of lipid-ligand type was synthesized and confirmed by 1H-NMR and FT-IR spectrum.PS@HA-Lip was prepared and characterized by zetasizer and TEM.The stability and safety of vector was evaluated by the determination of relative turbidity after co-incubation with fetal bovine serum and cell viability in the vector-containing medium.(2)Tracked by fluorescent probe,the localization of nonviral vector was captured by CLSM and the amount was quantified by FCM.EGFP-coding plasmids were used to determine the transfection efficiency of the prepared vector.The function of pCas9/MTH1 was verified by the expression of MTH1 and Sanger sequencing of target gene.The genome editing efficiency was also quantified.(3)Tumor targeting ability of the multifunctional nonviral vector was investigated by IVIS spectrum in orthotopic non-small cell lung cancer model.(4)Antitumor efficacy of PS@HA-Lip/pMTH1 was evaluated in tumor model with the body weight,bioluminescence intensity and survival monitored.The proliferation and apoptosis were analyzed by TUNEL and IHC assay.Results:(1)HA was successfully connected with DSPE-PEG with the validation of 1H-NMR and FT-IR spectrum.PS@HA-Lip showed a moderate size and spheroid morphology observed by TEM.Relative turbidity of the mixture of PS@HA-Lip and FBS remains low which indicated the good stability of vector.Besides,cell viability showed no difference when cells were cultured with or without the existence of PS@HA-Lip.That is to say,PS@HA-Lip could stayed stable in the blood circulation and caused little cytotoxic effect to normal cells.(2)HA could increase the amount of vector that entering tumor cells through HA-CD44 receptor interaction.PS@HA-Lip escaped from endosomes and delivered pMTH1 into nuclei with the assistance of protamine which containing nuclear localization signal.The significantly elevated cellular uptake and transportation into nuclei brought about greatly high transfection efficiency.MTH1 level was downregulated.(4)Loaded with pMTH1,PS@HA-Lip could significantly downregulate MTH1 protein in tumor tissue,promote tumor cell apoptosis,inhibit tumor growth and metastasis,prolong the survival time of tumor bearing nude mice and improve the quality of life of tumor bearing nude mice.Conclusions:The highly effective nonviral vector could load the gene editing plasmid and deliver it to the nuclei of tumor cells to exert gene editing effect and disrupt the expression of MTH1 protein.After treatment,tumor growth and metastasis were inhibited and tumor cell apoptosis was promoted,which confirming that PS@HA-Lip is a safe and efficient targeted nano delivery system. |
