Myrcludex B-mediated Liver-targeting Liposomes Containing An Enzyme Trigger Exposure To Cell-penetrating Peptide To Enhance The Therapeutic Effect Of Hepatocellular Carcinoma

Objective: A 10-hydroxycamptothecin(10-HCPT)-encapsulated liposome,surface-modified with PEG,cell-penetrating peptides and matrix metalloproteinase-cleavable peptides were constructed,in this experiment.To investigate the tumor targeting and antitumor effect of the new liposome drug delivery system in vivo and in vitro,the normal human liver cell LO2 and a matrix metalloproteinase 2 overexpression hepatoma cell SMMC-7721 have been chosen.Methods: The amide reaction,esterification reaction,dialysis and TLC purification were used to gain a matrix metalloproteinase 2 cleavage macromolecular material PEG-PP-PE(PP).The different targeting liposomes drug delivery systems were constructed by the thin-film dispersion method and the morphology of liposomes was investigated by transmission electron microscopy and Malvern particle size analyzer.The drug loading,encapsulation efficiency,and in vitro release effects of the liposome drug delivery system were investigated by using Shimadzu HPLC.The expression levels of matrix metalloproteinase 2 and NTCP on SMMC-7721 and LO2 cells were investigated by the Western blot(WB)experiment.The anti-tumor effect of liposomes in vitro was investigated by a 3D tumor sphere experiment,MTT and flow apoptosis.The in vitro targeting effect of DID-encapsulated liposomes was investigated by confocal laser and flow uptake experiments.A subcutaneous nude mouse model of SMMC-7721 liver tumor cells was made to investigate the in vivo tumor targeting and in vivo antitumor ability of liposomes.And the main organ toxicity and antitumor effect of liposomes in nude mice were investigated by the HE staining experiment.Finally,the apoptosis of tumor cells after multiple administrations of different liposomes was investigated by the TUNEL fluorescence staining experiment.Results: The constructed PEG2k-PP-PE has been successfully verified by hydrogen spectrometry.The particle size of liposomes showed a similar result in the electron microscope and Malvern particle sizer.On the one hand,the MMP2 expression level was highly expressed in SMMC-7721 cells instead of LO2 cells,on the other hand,the NTCP expression level was highly expressed in LO2 cells instead of SMMC-7721 cells.The highest cellular uptake effectivity has been shown in both laser confocal uptake and flow uptake experiments in the TATp-modified liposomes.A better cellular uptake effect in SMMC-7721 cells of PEG2k-PP-PE modified liposomes compared with the PEG-modified group.While a better LO2 cell-penetrating ability showed in a myrcludex B-modified liposomes group.MTT results showed that the IC50 of PEG2k-LIP,PP-LIP,PPP-LIP,and TAT-LIP on SMMC-7721 cells were 15.18 μg/ml,5.678 μg/ml,5.118 μg/ml and 2.996 μg/ml,respectively.It shows that the modification of cell-penetrating peptide and the modification of MMP2-sensitive polypeptide can significantly enhance the anti-tumor effect of 10-HCPT.The results of in vivo imaging of small animals showed that PPP-LIP had strong targeting in the liver of nude mice compared with other groups,and PP-LIP showed a better targeting effect on tumor sites compared with other groups.HE staining found that the 10-HCPT API administration group showed obvious cytotoxicity in the liver,and the other liposome administration groups did not show obvious cytotoxicity,and the results were similar to TUNEL fluorescence staining results in the PP-LIP administration group more extensive tumor tissue necrosis was induced followed by the PEG2k-LIP-administered group.Conclusions: This study presents a liposome drug delivery platform in which NTCP-mediated MMP2 induces TATp exposure to boost cellular absorption,indicating a tremendous potential for selective intracellular delivery of hydrophobic anticancer medicines in the liver tumour cells.