目前，基于免疫检查点阻断（Immune checkpoint blockade，ICB）的免疫疗法对多种类型的癌症尤其是高免疫原性的黑色素瘤治疗已经取得了显著成效。然而，仍有一半以上的黑色素瘤患者对ICB治疗不敏感。研究表明，黑色素瘤细胞可通过代谢重编程来满足自身的生物合成和能量生产需求，并利用代谢物构建和维持免疫抑制肿瘤微环境（Tumor microenvironment，TME），以逃避免疫监视。因此，破坏黑色素瘤细胞的代谢稳态以减少免疫抑制性代谢物的分泌，有望逆转免疫抑制的TME，从而提高黑色素瘤对ICB治疗的敏感性。

过氧亚硝酸盐（Peroxynitrite，ONOO⁻）是一种具有强氧化性和硝化性的活性氮物种，其能够对生物体内的蛋白质，包括多种参与代谢的酶等进行结构修饰和功能改变。因此，我们预测ONOO⁻可能会通过降低关键代谢酶的活性引起肿瘤细胞代谢稳态失衡，从而实现TME的免疫调节。本研究以自组装多肽作为骨架结构，制备了一种对乙酰氨基酚（4-Acetamidophenol，APAP）和一氧化氮（Nitric oxide，NO）共递送的黑色素瘤特异性ONOO⁻纳米发生器，其可通过破坏肿瘤细胞代谢稳态精准调控免疫抑制TME，从而增强黑色素瘤免疫治疗。

本论文成功设计并合成了APAP和NO偶联的多肽衍生物APAP-SA-FFFpY-Pen-NO，并采用核磁共振氢谱、高分辨质谱以及高效液相色谱仪确证了化合物的结构和NO的接枝率。在碱性磷酸酶作用下，多肽衍生物可在pH 7.4的磷酸盐缓冲液中发生自组装，形成直径为8 nm左右的纳米纤维材料APAP-P-NO。APAP-P-NO在pH 6.5和GSH（10 mM）条件下分别展现出快速的APAP和NO释放行为。与APAP-P-NO共孵育后，黑色素瘤B16F10细胞中的NO、O₂^•−及ONOO⁻水平均显著升高。蛋白质印迹结果显示ONOO⁻可通过对半胱氨酸上巯基的亚硝基化修饰来抑制代谢关键酶甘油醛-3-磷酸脱氢酶的活性。代谢组学分析显示ONOO⁻导致细胞内参与三羧酸循环的代谢物大量减少。同时，在ONOO⁻应激下，有氧糖酵解产生的乳酸急剧下降。经APAP-P-NO作用后，黑色素瘤细胞的代谢改变有效逆转了免疫抑制TME，引起了强大的抗肿瘤免疫反应，包括M2型巨噬细胞向M1型的重极化，髓源抑制性细胞和调节性T细胞的减少以及CD8⁺ T细胞浸润增加。最终的体内抑瘤实验显示，APAP-P-NO和α-PD-L1联用对小鼠黑色素皮下移植瘤和肺转移瘤模型均具有显著的肿瘤抑制效果，且未对机体造成明显的毒副作用。

综上所述，我们成功制备了一种黑色素瘤特异性的ONOO⁻纳米发生器，其可通过破坏肿瘤细胞代谢稳态来调节肿瘤免疫微环境，实现“冷”肿瘤到“热”肿瘤的转变，进一步将其与α-PD-L1联用可获得极好的黑色素瘤抑制效果，为多数黑色素瘤患者对ICB治疗应答率不高的问题提供了新的解决策略。

Over the past decade, immune checkpoint blockade (ICB) based immunotherapy has achieved unprecedented clinical benefits toward diverse types of cancer, especially the most immunogenic melanoma. However, roughly half of melanoma patients could not receive clinical benefits from ICB. Melanoma cells elicit metabolic reprogramming to fulfill their increased biosynthetic and bioenergetic demands as well as establish an immunosuppressive tumor microenvironment (TME) to escape from immunosurveillance. Representatively, lactate, the terminal product of aerobic glycolysis, has been validated to drive the polarization of tumor associate macrophage to an immunosuppressive phenotype. Hence, strategies that disrupt the metabolic homeostasis of melanoma cells to reduce the secretion of immunosuppressive metabolites may offer promising opportunities to correct the dysregulated immunological state for improving the efficacy of ICB.

Peroxynitrite (ONOO⁻), a powerful oxidizing and nitrating reactive nitrogen species, comes into our sight due to its structure modification and function alteration capabilities toward numerous proteins, some of which are indispensable enzymes involved in metabolic pathways，which is predicted to disrupt metabolic homeostasis for achieving TME immunomodulation. In this study, we prepared a melanoma-specific ONOO⁻ nanogenerator with acetaminophen (APAP) and nitric oxide (NO) co-delivery using self-assembled peptides as a backbone to precisely modulate tumor immunosuppression TME and thus enhance melanoma immunotherapy.

In this thesis, we developed an APAP-conjugated and NO-grafted self-assembling peptide derivate, the structure and NO grafting rate of the compounds were confirmed by ¹H NMR, HRMS, and HPLC spectra. The peptide derivate could be dephosphorylated under the catalysis of alkaline phosphatase and then self-assemble into supramolecular nanofibers (APAP-P-NO). The APAP-P-NO was observed by TEM as nanofibers with a diameter of about 8 nm. APAP-P-NO showed excellent drug release behavior at pH 5.5 and GSH (10 mM) for the release of APAP and NO, respectively. NO/O₂^•−/ONOO⁻ levels in melanoma cells were significantly increased after incubation with APAP-P-NO. WB results showed that ONOO⁻ inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (a key metabolic enzyme) through S-nitrosylation of cysteine. Metabolomics profiling revealed that the overproduced ONOO⁻ induces a great decrease in metabolites involved in the TCA cycle. Meanwhile, the aerobic glycolysis-produced lactate dropped sharply under ONOO⁻ stress. The metabolic alterations in melanoma cells effectively reversed the immunosuppressive TME to evoke potent antitumor immune responses, including polarization of M2-like macrophages to M1-phenotype and restoration of CD8⁺ T cell infiltration. Combining α-PD-L1 therapy with APAP-P-NO pretreatment achieved a significant tumor inhibition effect against both primary and metastatic melanoma models without eliciting systemic toxicity.

In summary, we have successfully prepared a melanoma-specific ONOO⁻ nanogenerator, which regulated the tumor immune microenvironment by disrupting the tumor metabolic homeostasis to achieve the transition from a "cold" to a "hot" tumor. The combination of APAP-P-NO with α-PD-L1 resulted in excellent melanoma inhibition, which provides a new strategy to overcome the challenge of melanoma insensitivity to ICB.