谷胱甘肽（Glutathione，GSH）是由谷氨酸、半胱氨酸和甘氨酸组成的三肽，是细胞内重要的代谢调节物质。同时，在多种类型的肿瘤细胞中GSH水平显著升高，利用这一生物学特点，研究者可以设计GSH响应型药物递送系统，也可以通过降低肿瘤细胞内的GSH水平来辅助其他抗肿瘤疗法，如逆转肿瘤细胞化疗药物耐受、提高肿瘤细胞对放射治疗的敏感性以及联合光动力治疗进行肿瘤高效杀伤等。但与此同时，细胞内GSH不断地通过生物合成进行补偿，GSH消耗策略难以完全耗尽细胞内的GSH，因此利用GSH消耗策略作为单一肿瘤疗法仍然具有很大的挑战。

自组装多肽因具有合成简单、易化学修饰、生物相容性好、具有高效生物活性等特点被广泛用于药物递送、疾病诊疗以及组织工程等领域。目前已有研究设计GSH响应型自组装肽药物递送系统用于肿瘤治疗，或设计GSH消耗型自组装肽，通过消耗细胞内的存量GSH来实现肿瘤的辅助治疗。但是，目前鲜有利用GSH调节作为独立策略高效杀伤肿瘤细胞的报道，也无利用自组装多肽设计兼具存量GSH消耗和GSH生物合成抑制的抗癌纳米药物的研究报道。本论文将具有GSH响应性的二硫键和GSH生物合成抑制剂l-丁硫氨酸亚砜亚胺（L-buthionine-sulfoximine，BSO）引入到自组装多肽序列中，设计了一种两亲性多肽衍生物Nap-^DF^DFY-CS-DEVD-BSO（简称NSBSO）。NSBSO在体外通过疏水作用组装形成纳米球，被肿瘤细胞胞吞后部分NSBSO被GSH还原断键并消耗胞内GSH，疏水片段Nap-^DF^DFY-thiol与剩余的NSBSO通过π-π堆积作用组装形成纳米纤维，让BSO在细胞内更好的驻留，最终通过对细胞内GSH的消耗和GSH生物合成抑制的双重作用发挥了GSH依赖的细胞毒性。

本论文利用液相合成和多肽固相合成相结合的方法制备了多肽衍生物NSBSO。通过透射电镜观察以及液相-质谱仪检测发现，NSBSO在pH 7.4条件下自组装成粒径为48.26 ± 6.17 nm的纳米球。在1 mM GSH存在时，部分NSBSO分子被还原，0.5 h即可发生纳米球到纳米纤维的形貌转变。当GSH浓度为10 mM时，更多的NSBSO分子被还原，形成更多的纳米纤维。体外细胞毒性实验表明，NSBSO的细胞毒性具有胞内GSH依赖性，即肿瘤细胞GSH水平越高，NSBSO的杀伤效果越强。作用24 h后对GSH高水平的B16细胞的IC₅₀值为0.844 μM，对GSH中等水平（约为B16细胞GSH浓度的一半）的4T1细胞的IC₅₀值为4.741 μM，毒性均高于多种肿瘤化疗药物，但对GSH低水平的正常细胞几乎不表现出毒性。GSH水平检测和细胞摄取结果显示，NSBSO具有GSH消耗和GSH生物合成抑制双重功能。在发生胞内GSH响应性断键解组装时，NSBSO消耗胞内GSH，并伴随着NSBSO和NSBSO还原产物的原位共组装，在胞内形成纳米纤维，从而将BSO更多地滞留在肿瘤细胞内，更好地发挥GSH生物合成的抑制功能，从而耗竭胞内GSH，对肿瘤细胞进行高效杀伤。通过机制研究我们发现，NSBSO对不同GSH水平肿瘤细胞的杀伤作用源于不同的死亡机制。对于GSH中等水平的4T1细胞，NSBSO消耗胞内GSH后会使谷胱甘肽过氧化物酶4失活，引发脂质过氧化物的累积，诱导铁死亡。对于GSH高等水平的B16细胞，NSBSO耗竭胞内GSH后，间接引起胞内活性氧水平提高，激活Caspase 3，随后激活消皮素E（Gasdermin E，GSDME）并在细胞膜上打孔，最终诱导细胞焦亡。体内抗肿瘤实验结果显示，NSBSO对4T1和B16荷瘤小鼠的抑瘤率分别为75.10%和54.24%，具有较好的体内抗肿瘤效果，并具有良好的生物安全性。

综上所述，本论文成功制备了一种具有GSH消耗和生物合成抑制双功能的自组装肽衍生物，该自组装肽在肿瘤细胞内发生GSH响应的纳米球到纳米纤维的形貌转变，对肿瘤细胞发挥了GSH水平依赖的杀伤作用。本论文通过基于自组装肽的GSH调控这一简单策略实现了高效的肿瘤细胞杀伤，这为设计高效GSH调控肿瘤纳米药物提供了启示。

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-^DF^DFY-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, disulfide-dependent 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 GSH-dependent 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 GSH-regulated tumor nanomedicines.