| In clinical treatment,cancer seriously affects human life and health,and clinical chemotherapy drugs have toxic side effects on normal cells.Anticancer peptides have attracted extensive attention due to their broad-spectrum,rapid-acting,and non-resistance to drug resistance tumor cells.In our previous work the anticancer peptide chensinin-1b was obtained,but it’s activity to kill MCF-7 cells was low.To improve the anticancer activity,the17th lysine residue in chensinin-1b was conjugated with the fatty acids with different carbon chain lengths including octanoic acid（OA）,lauric acid（LA）and palmitic acid（PA）,which caused the hydrophobicity of the lipo-chensinin-1b increased.Then the anticancer activity and mechanism of these lipo-chensinin-1b analogs was investigated.First,the CCK-8 cell viability assay and LDH release assay showed that the anticancer activity of lipo-chensinin-1bwas significantly enhanced,and it showed specific anticancer activity against MCF-7 cells compared to MCF-10A cells.The IC50s of OA-C1b,LA-C1b,and PA-C1b are 115.8±3.1μM,59.09±10.11μM,46.09±3.82μM,and 30.95±1.84μM,respectively.From the above results,the hydrophobicity of chensinin-1b lipopeptide enhanced as the lipophilic alkyl chain length increased,and its anticancer activity is also gradually enhanced.The anticancer mechanism of lipo-chensinin-1b on MCF-7 cells was examined by various assay,including Annexin V-FITC/PI,cell content release experiment,and MCF-7 cell uptake assay.The results showed that lipo-chensinin-1b did not significantly induce Annexin V-FITC single positive.Meanwhile,chensinin-1b,OA-C1b,LA-C1b and PA-C1b can beinternalized by MCF-7 cells have an effect on the integrity of the plasma membrane,indicating that the lipo-chensinin-1b pass through the cells.Lipo-chensinin-1b induced MCF-7 cell death is associated with cytoplasmic membrane action.To investigate the anticancer mechanism of lipo-chensinin-1b againist MCF-7,the uptake of PI by MCF-7 cells was detected by flow cytometry.The results showed that OA-C1b,LA-C1b and PA-C1b can enhance the ability of the uptake on PI.The concentration increasing of OA-C1b,LA-C1b,and PA-C1b can enhance the permeability of the plasma membrane.The surface morphology of PA-C1b-treated MCF-7 cells was examined by scanning electron microscopy.The results showed that PA-C1b destroyed the surface integrity of MCF-7 cells and the pores on the surface of the plasma membrane were formed,and cell membrane was collapsed.The interaction between the peptides and mimetic cell membrane was studied by tryptophan blue shift assay,which used phospholipids to mimic tumor cell plasma membrane and eukaryotic plasma membrane.The interaction between chensinin-1b,OA-C1b,LA-C1b and PA-C1b and liposome was detected by tryptophan blue shift assay using phospholipids to simulate the plasma membrane and eukaryotic plasma membrane of tumor cells,combined with CCK-8 assay.This indicates that chensinin-1b lipopeptide has a certain selectivity for cancer cell membranes.In vivo anti-tumor activity of PA-C1b was investigated by in vivo experiments with nude mice,and tumor-bearing mice were injected with PA-C1b regularly to observe the inhibition of tumor growth by PA-C1b.The results showed that PA-C1b inhibited tumor growth in mice,and PA-C1b has antitumor activity in vivo.Finally,the MSNs-NH2@Cy7-PA-C1b@FA-GO nano-targeted drug delivery system was constructed to improve the stability of lipo-chensinin-1b and target to tumor,in which folic acid is used as a tumor-targeting molecule,and graphene oxide is used as a"molecular switch"for stimulus-response controlled release of drug delivery systems.The results showed that MSNs-NH2@Cy7-PA-C1b@FA-GO could be taken up by tumor cells,and under the irradiation of near-infrared at 808 nm,Cy-7-PA-C1b could be released in vivo in mice tumor models and showed the specific targeting ability on tumor.In general,the OA-C1b,LA-C1b,and PA-C1b obtained by the lipid-modified chensinin-1b can cause cell death of human breast cancerMCF-7cellsthroughthemembraneactivitymechanism,andthe stimulation-responsive nano drug delivery system MSNs-NH2@Cy7-PA-C1b@FA-GO can successfully achieve controlled release and has certain targeting to tumors in vivo. |
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