Study On Antitumor Effect And Related Mechanism Of Glutathione-Regulated Self-Assembled Peptides

Glutathione(GSH),a tripeptide composed of glutamate,cysteine and glycine,is an important intracellular regulator of metabolism.The level of GSH is significantly increased in many types of tumor cells.Taking advantage of this biological characteristic,researchers designed GSH-responsive drug delivery systems,or reduced the level of GSH in tumor cells to assist other anti-tumor therapies,such as reversing chemotherapeutic drug resistance and improving the radiosensitisation of tumor cells.However,intracellular GSH is constantly compensated by biosynthesis and it is difficult to completely deplete intracellular GSH with GSH depletion strategy.Thus it is still a great challenge to use GSH depletion strategy as a single tumor therapy.Self-assembled peptides have been widely used in drug delivery,disease diagnosis and treatment and tissue engineering because of easy chemical modification,good biocompatibility and efficient biological activity.Studies have been conducted to design GSH-responsive self-assembled peptide drug delivery systems for tumor therapy,or GSH-depleting self-assembled peptides for adjuvant tumor therapy.However,there are little reports of efficient killing of tumor cells using GSH regulation as an independent strategy and reports on the use of self-assembled peptides to design anticancer nanodrugs with both GSH depletion and GSH biosynthesis inhibition.In this paper,an amphiphilic peptide derivative Nap-DFDFY-CS-DEVD-BSO(NSBSO)was designed by introducing a GSH-responsive disulfide bond and a GSH biosynthesis inhibitor L-buthionine-sulfoximine(BSO)into the self-assembled peptide sequence.NSBSO was assembled into nanoparticles in vitro by hydrophobic interaction,when the nanoparticles were taken up by tumor cells,numerus GSH reacted with disulfide bonds and were largely consumed,this reaction broke NSBSO into two parts,which could co-assembly form nanofibers by π-π stacking interaction.This morphology transformation also increased the retention of BSO in tumor cells.Finally,disulfidedependent reduction and BSO-dependent biosynthesis inhibition orchestrated a great decline of intracellular GSH level.In this scenario,the tumor cells would undergo programmed death such as ferroptosis or pyroptosis.In this paper,the peptide derivative NSBSO was prepared by liquid phase and solid peptide phase synthesis.It was observed that NSBSO self-assembled into nanoparticles with the average diameter of 48.26 ± 6.17 nm at pH 7.4.In the presence of 1 mM GSH,part of NSBSO molecules were reduced,and the morphology transformation from nanoparticles to nanofibers took place at 0.5 h.When the concentration of GSH was increased to 10 mM,more NSBSO molecules were reduced and more nanofibers were formed.The results of in vitro cytotoxicity assay showed that NSBSO had GSHdependent cytotoxicity,that is,the higher the GSH level of tumor cells,the stronger the killing effect of NSBSO.After 24 h treatment,the IC50 values were 0.844 μM for B16 cells with high GSH levels and 4.741 μM for 4T1 cells with medium GSH levels(about half the GSH concentration of B16 cells),which was higher than that of many chemotherapeutic drugs,but it showed almost no toxicity to normal cells.The results of GSH level detection and cell uptake showed that NSBSO had a dual functions of GSH depletion and GSH biosynthesis inhibition.In the event of intracellular GSH responsive bond-breaking disassembly,NSBSO depleted intracellular GSH and was accompanied by in situ co-assembly of NSBSO and NSBSO reduction products to form intracellular nanofibers,thus retaining more BSO in tumor cells and better exerting the inhibitory function of GSH biosynthesis,thereby exhausting intracellular GSH and killing tumor cells efficiently.Through the mechanism study,we found that the killing effect of NSBSO on tumor cells with different GSH levels originated from different death mechanisms.For 4T1 cells with medium level of GSH,intracellular GSH depletion by NSBSO would inactivate glutathione peroxidase 4,trigger the accumulation of lipid peroxides and induce ferroptosis.For B16 cells with high level of GSH,intracellular GSH depletion by NSBSO indirectly increased the level of intracellular reactive oxygen species,activated Caspase 3,followed by activation of Gasdermin E(GSDME)and pore-froming of the cell membrane,and ultimately induced pyroptosis.The results of anti-tumor experiment in vivo showed that NSBSO had good anti-tumor effects in vivo with good biocompatibility,with tumor inhibition rates of 75.10%and 54.24%in 4T1 and B16 tumor-bearing mice,respectively.In summary,a self-assembled peptide derivative with dual functions of GSH depletion and biosynthesis inhibition was successfully prepared.The self-assembled peptide underwent a GSH-responsive nanoparticles-to-nanofibers morphology transformation and played GSH-dependent cytotoxicity to tumor cells.In this paper,we achieved efficient tumor cell killing through a simple strategy of GSH regulation based on self-assembled peptides,which provides insights into the design of efficient GSHregulated tumor nanomedicines.