Design,sSynthesis And Mechanism Of Lysosomal-Targeted Anticancer Peptides

The incidence and mortality of tumors are increasing year by year,and they have become a major killer threatening human health.At present,the commonly used tumor treatment methods are surgical resection and drug radiotherapy.However,due to poor cell selectivity,obvious side effects,and induction of drug resistance in tumor cells,radiotherapy and chemotherapy drugs are not suitable for continuous treatment of tumors.Therefore,we urgently need to find targeted drugs with low toxicity,targeted positioning and targeting advantages.Anti-tumor polypeptides have the advantages of high cell selectivity,good biocompatibility,small toxic and side effects,high biological activity,and are not easy to cause multidrug resistance and non-specific immune reactions.They can improve the therapeutic effect and show good application prospects.However,common anti-tumor peptides have problems such as unclear mechanism of action in vivo and poor stability,and their performance needs to be further improved.Based on this,according to the characteristic that the activity of cathepsin L in the lysosome of tumor cells is higher than that of normal cells,this study used peptide molecules can self-assembly to form special under the specific conditions and the nature of the orderly nanostructure,design a series of cathepsin L responsiveness and the ability to target lysosome peptide,peptide molecular self-assembly properties were studied and antitumor activity,and,from the perspective of the lysosome death approaches,explore its antitumor mechanism.Through research,we found that（1）Cathepsin L can accurately cleave Fmoc-FFRIKFERQ-OH,Ac-QRFFE-NH₂,Ac-DLLRQ-NH₂ polypeptide molecules under acidic conditions in vitro,and produce the corresponding enzyme digestion products:Fmoc-FFR-OH,RFFE-NH₂,Ac-DLLR-OH.For the peptides Fmoc-FFRIKFERQ-OH and Ac-QRFFE-NH₂,they can be digested with cathepsin L for 2 h to produce the peptides Fmoc-FFR-OH and RFFE-NH₂,for the peptides Ac-DLLRQ-NH₂,treated with cathepsin L for 24 h,can be completely digested to produce the digested product peptide Ac-DLLR-OH.（2）AFM and TEM results show that Fmoc-FFRIKFERQ-OH can self-assemble to form short rod-shaped nanostructures,and Fmoc-FFR-OH can assemble long-fiber nanostructures.Ac-QRFFE-NH₂,Ac-DLLRQ-NH₂,RFFE-NH₂,Ac-DLLR-OH are not assembled.Combined with MTT data,Fmoc-FFRIKFERQ-OH and Fmoc-FFR-OH have better anti-tumor effects than Ac-QRFFE-NH₂,Ac-DLLRQ-NH₂,RFFE-NH₂,Ac-DLLR-OH.The assembly structure formed by the polypeptide can improve its killing effect on tumor cells.（3）MTT data shows that Fmoc-FFRIKFERQ-OH,Ac-QRFFE-NH₂,Ac-DLLRQ-NH₂ have good effects on tumor cells A375,B16,SH-SY5Y,Hela,MCF-7,Hep G2,and PC-12.Inhibition,and less toxic to normal cells（L929,293E）,with good cell selectivity.（4）An inverted fluorescence microscope and flow cytometer were used to explore the anti-tumor mechanism of peptides.The data shows that peptides can enter the lysosome.Peptides can induce the increase of lysosomal membrane permeability and mitochondrial membrane permeability,that is,cause the occurrence of lysosomal death pathway and mitochondrial death pathway.The experimental data of E-64d inhibitors show that pretreatment of SH-SY5Y and B16 cells with E-64d can significantly inhibit the increase in mitochondrial permeability.This proves that the increase in mitochondrial permeability is caused by the leakage of lysosomal enzymes.In other words,polypeptides promote the release of various proteolytic enzymes in the lysosome by inducing an increase in the permeability of the lysosomal membrane.The leakage of enzymes induces an increase in the permeability of the mitochondrial membrane,triggers the mitochondrial death pathway,and ultimately leads to the death.