【Angew.Chem.】西南交大田原|7倍靶向积聚！利用铂(IV)金属钉合策略打造肿瘤特异性前药，实现高效化学-免疫代谢联合治疗
文章标题：A Platinum(IV) Metallo‐Stapling Approach to Tumor‐Specific Prodrugs for Targeted Chemo‐Immunometabolic Cancer Therapy
通讯作者：Yuan Tian, Shaobing Zhou
文章概要
传统金属抗肿瘤药物因缺乏特异性常导致严重的全身毒副作用。针对这一瓶颈，研究团队创新性地提出了一种铂(IV)介导的金属钉合策略，成功构建了兼具高稳定性和高靶向性的宏环金属前药模型。该策略巧妙地将铂(IV)配合物同时作为细胞毒性弹头与结构钉合骨架，从而对表皮生长因子受体（EGFR）靶向肽进行构象锁定。该前药在肿瘤微环境中能够特异性响应并释放双重有效载荷，在诱导强烈的免疫细胞死亡（ICD）的同时逆转免疫抑制，为实现精准的化学-免疫代谢联合治疗开辟了新途径。
引言
现代肿瘤临床治疗中，以奥沙利铂为代表的铂类化疗药占有举足轻重的地位，但它们“伤敌一千，自损八百”的非选择性分布极大限制了临床疗效。尽管学术界尝试将铂(IV)前药与肿瘤定向肽进行线性偶联以提高靶向性，但线性肽因构象灵活、在体内易被蛋白酶降解且药代动力学表现不佳，导致整体递送效率低下。受天然金属蛋白中金属配位稳定蛋白质结构的启发，如何利用具有双重功能的金属中心去“钉合”约束定向肽，在提升构象稳定性的同时实现精准靶向释放，成为了当前金属药物研发领域的关键科学问题。
SCHEME 1 (a) Synthetic scheme of platinum(IV) stapled metalloprodrug sEBP-NLG-Oxal(IV). (b) Illustration of sEBP-NLG-Oxal(IV)-mediated immunometabolic cancer therapy.
主要实验及结论
研究人员首先通过模块化合成路径，将免疫调节剂NLG919与修饰了双半胱氨酸的EGFR靶向肽偶联，随后利用双功能铂(IV)前体进行溶液反应，使其同时作为治疗弹头和结构钉合剂，成功锁定了肽段构象，制备出钉合前药。
FIGURE 1 Preparation and characterization of linear conjugate EBP-NLG-Oxal(IV) and stapled conjugate sEBP-NLG-Oxal(IV). (a) Synthetic scheme of EBP-NLG-Oxal(IV) and sEBP-NLG-Oxal(IV) conjugates. (b) HPLC traces of EBP-NLG-Oxal(IV) and sEBP-NLG-Oxal(IV) conjugates. (c) Intact peptide percentages of sEBP-NLG-Oxal(IV) and EBP-NLG-Oxal(IV) after incubation with FBS (20% v/v) at indicated time intervals as determined by HPLC. (d) Release of NLG919 from sEBP‑NLG‑Oxal(IV) at pH 7.4 or pH 5.5 in the presence of esterase using HPLC. (e) Release of platinum from sEBP‑NLG‑Oxal(IV) at pH 5.5 with or without glutathione (GSH) using dialysis followed by ICP‑MS detection. All data are presented as mean ± SD (n = 3).
体外理化性质测试表明，这种独特的宏环结构赋予了前药极佳的生物学稳定性。在血清稳定性实验中，如图1所示，钉合前药在胎牛血清中孵育16小时后的完整残留率高达51.7%，其循环半衰期达到了14.4小时，相较于线性对照组提升了近3倍。同时，该前药还表现出优异的血液相容性，且能在模拟溶酶体及高谷胱甘肽的肿瘤微环境下，特异性响应并高效释放出铂类药物和NLG919。
FIGURE 2 Evaluations of EGFR-dependent cellular uptake and cytotoxicity. (a) Quantitative assessment of EGFR expression levels in different cell lines. (b), (c), and (d) Cell viability of MCF-7, SW480, and HCT116 cells. Cells were co-incubated with different compounds for 48 h. (e) ICP-MS analysis of Pt in MCF-7 and HCT116 cells. (f) The inhibitory effect of the sEBP-NLG-Oxal(IV) on IDO-1. All data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.
在细胞层面的靶向性与毒性评估中，如图2所示，前药对EGFR阳性的结直肠癌细胞表现出强烈的选择性细胞毒性，而对EGFR阴性细胞的杀伤作用微乎其微。细胞内铂元素定量分析证实，钉合前药在靶向细胞内的积累量显著高于普通奥沙利铂和线性对照。此外，释放的NLG919能有效阻止色氨酸向犬尿氨酸的转化，展现出强大的ID0-1酶活性抑制能力。
FIGURE 3 In vitro immunogenic cell death induction. (a) and (b) Fluorescence microscopic imaging of the surface exposure of CRT (green) of the EGFR-overexpressed HCT116 cells and the low EGFR-expressed MCF-7 cells. The cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm. (c) and (d) Fluorescence microscopic imaging of HMGB1 after different treatments in HCT116 cells and MCF-7 cells. (e) Quantification of HMGB1 release by ELISA kit. (f) Quantification of extracellular ATP secretion by enhanced ATP assay kits. (g) Schematic illustration of Pt-induced ICD in tumor cells, characterized by CRT exposure, ATP secretion, and HMGB1 release. All data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.
为进一步验证其激活免疫系统的潜力，团队检测了肿瘤细胞免疫原性死亡的核心生物标志物。如图3所示，在EGFR阳性细胞中，前药处理诱导了明显的钙网织蛋白（CRT）细胞表面转位，并大幅促进了高迁移率族蛋白B1（HMGB1）的释放以及三磷酸腺苷（ATP）的向外分泌，成功拉响了免疫系统的“警报”。
FIGURE 4 In Vivo Biodistribution of the metalloprodrug. (a) Scheme of experimental approach for HCT116 tumor establishment in naïve nude mice, intravenous administration of different compounds, and organ collection for platinum quantification by ICP-MS (195Pt). (b) and (c) Platinum levels in different organs after 6 h of treatment with various platinum compounds. (d) Fold-change of sEBP-Oxal(IV)/Oxal(II) from the experiment shown in (b). All data are presented as mean ± SD (n = 3). ***p < 0.001.
最令人振奋的是体内的精准靶向与治疗效果。在荷瘤小鼠的生物分布实验中，如图4所示，钉合前药组在肿瘤组织中的铂富集量达到了母体铂(II)配合物的7倍，同时也是线性前药的5倍，证实宏环约束能显著延长体内循环并增强受体介导的胞吞作用。
FIGURE 5 In vivo antitumor efficacy and immune activation. (a) Therapeutic schedule of sEBP-NLG-Oxal(IV) in a CT26 tumor-bearing mouse model. (b) Representative images of tumor samples from mice taken post-treatment. Scale bar: 2 cm. (c) and (d) Volume curves of tumors in mice (mean ± SD, n = 5, ***p < 0.001). (e) Tumor growth inhibition rates (mean ± SD, n = 5, **p < 0.01, ***p < 0.001). (f) Percentage of matured DC cells (CD11c+ CD80+CD86+) in the tumors of the mice post different treatments as indicated (mean ± SD, n = 3, **p < 0.01, ***p < 0.001). (g) The population of CD4+ T cells (CD3+CD4+) in the tumors of the mice post different treatments as indicated (Mean ± SD, n = 3, *p < 0.05, **p < 0.01). (h) The population of CD8+ T cells (CD3+CD8+) in the tumors of the mice post different treatments as indicated (mean ± SD, n = 3, **p < 0.01, ***p < 0.001). (i) Quantification of Treg cells (CD4+FOXP3+CD25+) in the tumors of the mice post different treatments as indicated (mean ± SD, n = 3, *p < 0.05). (j–l) Secretion of IL-6, TNF-α, and IFN-γ in the cell supernatant by ELISA kits (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). (m) H&E and TUNEL analyses of tumor slices after different treatments. Scale bar, 200 µm.
在体内抗肿瘤疗效评价中，如图5所示，该前药单药治疗即可显著抑制结直肠肿瘤生长。而当其与抗PD-L1抗体联合使用时，抑瘤率更是高达90.9%。流式细胞术分析表明，该疗法显著提高了肿瘤浸润CD4+和CD8+ T细胞的比例，降低了调节性T细胞（Tregs）的浸润。最后，如图6所示的肿瘤疫苗接种实验进一步证实，经前药处理的死亡肿瘤细胞进入小鼠体内后具有极佳的免疫保护屏障效应，能有效阻止活肿瘤细胞的二次侵袭。
FIGURE 6 In vivo immunogenic cell death induction. (a) Vaccination schedule in a syngeneic CT26 tumor‑bearing mouse model. CT26 cells were exposed ex vivo to PBS, Oxal(II), EBP‑NLG‑Oxal(IV), or sEBP‑NLG‑Oxal(IV) at 50 µM for 48 h to generate dying tumor cells. After removal of the compounds, the cell suspensions were injected subcutaneously into the left flank of BALB/c mice as a prophylactic vaccine. Seven days later, the same mice were re‑challenged with live CT26 cells on the contralateral flank. Tumor incidence and growth were monitored for 50 days post‑challenge. (b) Percentage of tumor‑free mice after re‑challenge with live CT26 cells. (c) Tumor volume curves of the contralateral tumors that developed after re‑challenge (n = 9).
总结及展望
该研究成功搭建了基于铂(IV)中心介导的金属钉合药物研发平台，不仅实现了化疗载荷在病灶部位高达7倍的精准富集，还通过重塑肿瘤免疫微环境构建了高效的化学-免疫代谢协同治疗模式。这种将“金属配合物”与“结构稳定剂”合二为一的设计理念具有极高的普适性。展望未来，该策略可进一步拓展至其他肿瘤定向配体甚至复杂的生物大分子和功能蛋白质的结构约束中，为开发下一代多功能、精准靶向的金属抗癌药物提供了崭新的思路与技术支撑。