【Angew.Chem.】中山大学夏炜|细菌介导的四价铂前药靶向递送与肿瘤深层渗透实现协同化疗-免疫治疗：肿瘤体积缩小72.2%#

文章标题：Bacteria‐Mediated Targeted Delivery and Deep Tumor Penetration of a Pt(IV)‐Drug Enables Synergistic Chemo‐Immunotherapy 文章作者：He Meng, Jinhui Wang, Bing Shi, Tao Ouyang, Kai Lu, Wenkai Lin, Yuxiang Lu, Jing Wang, Xiyu Huang, Hailong Zhao, Yufang Xiong, Zilong Xu, Zong-Wan Mao, Wei Xia 文章链接：https://doi.org/10.1002/anie.1710587

一、研究背景与核心痛点#

铂类药物（如顺铂）是临床抗癌化疗的基石，主要通过与肿瘤细胞核内的DNA结合并造成破坏来杀伤癌细胞。然而，传统二价铂药物在临床应用中存在两大严重瓶颈：一是缺乏靶向性，进入体内的绝大部分药物会游离至正常组织，引发严重的全身毒副作用（如肾毒性、神经毒性）；二是由于实体瘤内部致密的细胞结构和高间质压，常规药物难以向肿瘤核心深层渗透，导致疗效大打折扣。

为了降低毒性，科学家开发了惰性更高的四价铂前药 Pt(IV)，它在血液循环中性质稳定不发生反应，只有进入肿瘤微环境被还原后才会释放出具有杀伤力的顺铂。但四价铂依然面临“进不去肿瘤深部”和“在复杂肿瘤免疫微环境中单兵作战易耐药”的难题。

Scheme.1 (a) Schematic illustration of the sequential construction of the Pt(IV) drug-bacteria conjugate. (b) Schematic illustration of tumor-targeted chemo-immunotherapy using the injected Pt(IV)-bacteria conjugate. The Pt(IV)-bacteria conjugate achieves deep penetration into solid tumors by leveraging inherent bacterial hypoxic tropism. Elevated H2O2 and GSH in tumor microenvironment activate the conjugate, simultaneously releasing cytotoxic cisplatin and an A2AR antagonist. This dual action induces chemotherapeutic tumor cell death and activates dendritic cells and T lymphocytes. This synergistic chemo-immunotherapy achieves highly potent solid tumor eradication.#

二、核心创新设计：“生物活体导弹” Pt-EcN-HER2#

研究团队巧妙利用了厌氧益生菌大肠杆菌 Nissle 1917（EcN）天生喜欢趋向肿瘤内部缺氧、坏死区域（缺氧趋化性）且具备自主游动能力的特性，构建了一种结合了活体细菌与多功能前药的复合递送系统：

代谢标记与生物正交偶联：通过给益生菌喂食非天然叠氮糖（Ac4GalNAz），使细菌表面布满叠氮化学基团，随后利用生物正交“点击化学”反应，将修饰有环辛炔基团（DBCO）的多功能四价铂前药（Pt-A-D）精准锚定在细菌表面。实验测得每个细菌的药物结合量可达 198.2 ± 1.2 nmol / 10⁸ CFU，且完全不影响细菌的生长活力。
表面精准导航工程：研究人员在细菌表面展示了抗 HER2 靶向分子（Affibody），制备成 Pt-EcN-HER2，使其不仅能依靠细菌趋氧性深入肿瘤，还能特异性紧锁 HER2 阳性肿瘤细胞。
微环境双重响应释放机制：所设计的四价铂前药带有两个功能轴向配体——一端通过过氧化氢响应的硫缩酮键连接在细菌上，另一端装载了高亲和力的腺苷 A2A 受体（A2AR）拮抗剂。当细菌抵达肿瘤微环境（TME）后，肿瘤内高浓度的过氧化氢（H₂O₂）会切断连接键释放前药，随后肿瘤细胞内高浓度的谷胱甘肽（GSH）进一步将四价铂还原为两部分：一部分是高杀伤力顺铂，另一部分是解除免疫抑制的 A2AR 拮抗剂。

Fig.1 (a) Schematic illustration of the activation mechanism of Pt(IV) drug Pt-A-D. The Pt-A-D is oxidized by hydrogen peroxide (H2O2) to release Pt-A and reduced by glutathione (GSH) to release cisplatin and the axial ligand A-COOH. (b) Reduction kinetics of Pt-A-D by GSH. Time-dependent HPLC quantification of Pt(IV)-to-Pt(II) conversion for Pt-A-D (10 µM in PBS) incubated at 37°C with or without 80 µM GSH. (c) H2O2-mediated thioketal cleavage kinetics in Pt-A-D. Time-resolved 1H NMR spectra for Pt-A-D (1 mM in DMSO-d6) treated with 0.5 mM H2O2. (d) Time-dependent quantitation analysis of residual thioketal in the presence of H2O2. (e) Schematic illustration of the procedure for Pt(IV)-bacteria conjugate construction. E. coli Nissle 1917 (EcN) is metabolic engineering with Ac4GalNAz for surface azide display. DBCO-functionalized Pt(IV) drug (Pt-A-D) is bioorthogonal coupled to the bacterial azides to form the Pt(IV)-bacteria conjugate. (f) CLSM images comparing EcN pre-incubated with natural galactose (Gal) or unnatural azido-galactose (Ac4GalNAz) followed by treatment with FITC-DBCO click chemistry probe. Scale bar: 10 µM. (g) Quantification of Pt-A-D loading capacity on EcN. EcN is treated with various concentrations of natural galactose (Gal) or unnatural azido-galactose (Ac4GalNAz) followed by treatment with excess DBCO-functionalized Pt(IV) drug (Pt-A-D). Bacterial platinum contents are determined by inductively coupled plasma mass spectrometry (ICP-MS). All experiments are performed in triplicate (n = 3), and data are presented as mean ± SD.#

三、体外机制验证：多途径细胞杀伤与免疫激活#

研究团队利用三维肿瘤球模型与共培养体系对该系统的抗癌与免疫调节机制进行了全面解析：

深度渗透与高效杀伤：在直径约 600 µm 的 4T1-HER2 肿瘤球模型中，常规小分子四价铂主要滞留在外周，而 Pt-EcN-HER2 在 8 小时内深层渗透至球体核心，铂富集量达到 89.51 ± 0.58 nmol，显著高于游离小分子对照组（65.0 nmol），并在 H₂O₂ 触发下引起肿瘤核心区大规模坏死。
触发多重细胞死亡机制：在与乳腺癌细胞 MDA-MB-231 的反应中，微环境激活的 Pt-EcN 在 48 小时内导致 癌细胞存活率下降 92.4%。机制研究证明，四价铂还原过程不仅造成 DNA 损伤诱导凋亡，还剧烈消耗了癌细胞内部的抗氧化物质 GSH（下调 GPX4 表达），引发强烈的铁依赖性脂质过氧化（铁死亡）；同时激活了 GSDMD 裂解，诱发了典型的细胞破裂胀亡（焦亡）。
逆转免疫抑制环境：肿瘤内高水平的腺苷通常会使免疫系统“瘫痪”。释放出的 A2AR 拮抗剂成功阻断了腺苷信号：促使树突状细胞（DC）的成熟标志物 CD80⁺/CD86⁺ 表达显著上调；同时逆转了 T 细胞的耗竭状态，大幅下调了免疫检查点分子 PD-1 和 TIM-3 的转录表达，使得 CD25⁺ 活化 T 细胞对肿瘤细胞的杀伤能力得到全面恢复。

Fig.2 (a) Schematic illustration of Transwell co-culture assay design for validating tumor cell cytotoxicity mediated by Pt(IV) drug-bacteria conjugate in vitro. (b) HPLC-MS profiles of Pt-A, A-COOH in MDA-MB-231 cell lysate after Pt-EcN treatment using selective ion monitoring (SIM) mode. (c) Time-dependent quantitation analysis Pt-A, A-COOH in MDA-MB-231 cell lysate after Pt-EcN treatment. (d) Cell viability of MDA-MB-231 after treatment with EcN, H2O2, EcN + A (A-COOH) + C (CDDP) + H2O2, EcN + D (DBCO-COOH) + A (A-COOH) + C (CDDP) + H2O2, Pt-EcN, EcN + Pt-A + H2O2, and Pt-EcN + H2O2 for 12, 24, and 48 h. (e) Effects of different inhibitors on cell death induced by Pt-EcN + H2O2. MDA-MB-231 cells are treated with Pt-EcN + H2O2 in a Transwell for 24 h with or without pretreatment of 3-MA (500 µM), z-VAD-fmk (50 µM), Ferr-1 (10 µM), Lip (0.4 µM), NSA (10 µM), Nec-1 (100 µM), or disulfiram (4 µM) for 1 h. Cell viability is measured by MTT assay. (f) TEM images of MDA-MB-231 cells treated with or without Pt-EcN+H2O2. Red rectangles represent the region enlarged. Red arrows indicate mitochondria. Quantitation of intracellular (g) malondialdehyde (MDA) levels and (h) GSSG ratios in MDA-MB-231 cells treated with EcN, Pt-A-D, and Pt-EcN + H2O2 for 24 h. (i) Western blot analysis of GPX4 and β-actin expression in MDA-MB-231 cells treated with EcN, Pt-A-D, and Pt-EcN + H2O2 for 24 h. Uncropped blot images are displayed in Figure S30. (j) SEM images of MDA-MB-231 cells treated with or without Pt-EcN + H2O2 for 24 h. Pt-EcN + H2O2 treatment results in pyroptosis of tumor cells. (k) Volcano plots showing differentially expressed genes (DEGs) in MDA-MB-231 cells treated with Pt-EcN + H2O2 versus control. Representative genes involved in ferroptosis and pyroptosis are highlighted. (l) Heatmap of differentially expressed genes in ferroptosis and pyroptosis pathways in MDA-MB-231 cells after Pt-EcN+H2O2 treatment. All quantitation experiments are performed in triplicate (n = 3), and data are presented as mean ± SD. Statistical significance is determined by one-way ANOVA (* p < 0.05, p < 0.01, * p < 0.001).#

Fig.3 (a) Schematic illustration of Transwell assay design for validating dendritic cells (DCs) maturation mediated by Pt(IV) drug-bacteria conjugate in vitro. Flow cytometry analysis of CD86+ and CD80+ biomarker expression levels on (b) DC2.4 cells and (c) bone marrow-derived dendritic cells (BMDCs) after treatment with indicated conditions for 48 h. (d) Schematic illustration of Transwell assay for assessing T-cell activation after indicated treatments. For (e–h), Jurkat T cells are pre-activated with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (IONO) for 24 h. After further incubation with 2 mM adenosine (Ado) for 5 h, the Jurkat T cells are treated with the indicated conditions for 24 h. The gene transcription levels of PD-1 (e) and TIM-3 (f) are determined by quantitative PCR (qPCR). (g) Flow cytometry analysis of CD25 biomarker expression levels on treated Jurkat T cells. (h) Fluorescent images of co-culture of treated Jurkat T cells and MDA-MB-231 cells. The cells are stained with Calcein-AM for live cells (green) and propidium iodide (PI) for dead cells (red). Scale bar: 50 µm. All quantitation experiments are performed in triplicate (n = 3), and data are presented as mean ± SD. Statistical significance is determined by one-way ANOVA (* p < 0.05, p < 0.01, * p < 0.001).#

四、体内抗肿瘤与免疫重塑表现#

在荷瘤小鼠（BALB/c 小鼠 4T1-HER2 乳腺癌模型）实验中，系统展现了卓越的协同化疗-免疫治疗效果：

高度特异的体内靶向性：活体生物发光与组织分布（ICP-MS）显示，Pt-EcN-HER2 注射后 48 小时在肿瘤组织内达到定植峰值，细菌与铂信号严格富集于肿瘤核心，心脏、肝脏、肺脏等主要器官几乎未见异常蓄积，解决了传统铂类药物的器官毒性难题。
显著抑制实体瘤生长：经过每 4 天一次、共 4 次给药后，Pt-EcN-HER2 治疗组实现了 72.2% 的最终肿瘤体积缩减，抑瘤效果显著超越单纯顺铂组、游离药物组合组以及无靶向修饰组；治疗期间小鼠体重保持稳定，主要器官切片未见病理损伤。
重塑肿瘤抗肿瘤免疫微环境：
促使促炎型 M1 巨噬细胞与抑制型 M2 巨噬细胞的比例激增至对照组的 5.5 倍；
肿瘤浸润自然杀伤细胞（NK 细胞）比例较对照组提升 11.5 倍；
肿瘤内成熟树突状细胞比例由对照组的 3.11% 大幅跃升至 11.61%；
肿瘤内部具有杀伤作用的 CD8⁺ 细胞毒性 T 细胞浸润增加 3.1 倍，CD4⁺ 辅助 T 细胞增加 11.9 倍；
小鼠血清中促炎性细胞因子（IFN-γ、TNF-α、IL-6、IL-12p70）表达全线显著上调，系统性激活了适应性抗肿瘤免疫。

Fig.4 (a) Schematic illustration of tumor-targeting and deep penetration of multicellular spheroids of HER2-expressing 4T1 cells (4T1-HER2) by Pt-EcN-HER2. (b) CLSM images of 4T1-HER2 tumor cells after incubation of Pt(IV) drug Pt-A-D, bacteria conjugates Pt-EcN and Pt-EcN-HER2. Bacteria conjugates Pt-EcN and Pt-EcN-HER2 harbor a plasmid encoding eGFP (green). Cell nuclei are stained with Hoechst 33342 (blue) and cell membranes are stained with Cy5-labeled Sambucus nigra lectin (SNA-Cy5, red). Scale bars: 20 µm. (c) Quantitation of platinum contents in 4T1-HER2 cells after treatments with Pt-A-D, Pt-EcN, and Pt-EcN-HER2. (d) CLSM imaging of 4T1-HER2 multicellular spheroids. The spheroids are incubated with Pt-A-D (14.6 µM), Pt-EcN (1.25 × 107 CFU), and Pt-EcN-HER2 (1.25 × 107 CFU) for 8 h. Bacteria conjugate Pt-EcN and Pt-EcN-HER2 harbor a plasmid encoding eGFP (green). Scale bar: 50 µm. (e) 3D Z-stack phosphorescence images are taken every 60 µm from the top to the bottom of the spheroids treated with Pt-A-D, Pt-EcN, or Pt-EcN-HER2. (f) Quantitation of platinum contents in 4T1-HER2 multicellular spheroids after incubation with Pt-A-D, Pt-EcN, or Pt-EcN-HER2 for 2, 4, and 8 h, respectively. All quantitation experiments are performed in triplicate (n = 3), and data are presented as mean ± SD.#

Fig.5 (a) Bioluminescence imaging of Pt-EcN-HER2 in the subcutaneous tumors of mice after intravenous administration over 4 days. A total of 3 × 108 CFU of Pt-EcN-HER2 are administered intravenously and the bioluminescence images of mice are recorded every 24 h. Mice administered with Pt-A-D are used as controls. (b) Quantitative analysis of platinum contents in the heart, liver, spleen, lungs, kidneys and tumor of mice treated with Pt-A-D and Pt-EcN-HER2. (c) Analysis of bacterial colonization in the heart, liver, spleen, lungs, kidneys, and tumor tissues of mice treated with Pt-A-D and Pt-EcN-HER2 using the spread plate method. (d) Quantitation of bacteria in various organs of mice treated with Pt-A-D and Pt-EcN-HER2. All quantitation experiments are performed in triplicate (n = 3), and data are presented as mean ± SD. Statistical significance is determined by one-way ANOVA (* p < 0.05).#

Fig.6 (a) Schematic illustration of Pt-EcN-HER2-mediated chemo-immunotherapy in a 4T1-HER2 tumor-bearing mouse model. (b) Volume changes of 4T1-HER2 tumors after different treatments are plotted (n = 5). (c) The weight of the mice during the treatments (n = 5). (d) Photographs of the tumors collected from the mice after the treatments. (e) Weights of dissected tumors from mice after treatment are plotted (n = 5). (f) Quantification of pro-inflammatory M1 to immunosuppressive M2 macrophage ratios in mice tumors post different treatments as indicated (n = 3). (g) Percentage of natural killer (NK) cells (CD45+CD335+) in the tumors of the mice post different treatments as indicated (n = 3). Percentages of (h) matured dendritic cells (CD11c+CD80+CD86+), (i) CD4+ helper T cells (CD45+CD3+CD4+), and (j) cytotoxic CD8+ T cells (CD45+CD3+CD8+) in mice tumors post different treatments as indicated (n = 3). Secreted cytokine levels of (k) INF-γ, (l) TNF-α, (m) IL6, and (n) IL12-p70 in murine serum in different experimental groups (n = 4). All quantitation experiment data are presented as mean ± SD. Statistical significance is determined by one-way ANOVA (* p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001).#

五、局限性与应用展望#

该项研究开创性地将工程化活菌的自主趋化深潜能力与双响应前药的化疗-免疫协同功能相结合，为突破实体瘤物理渗透屏障和克服铂类耐药提供了全新的解决范式。

不过，研究仍存在一定的局限性与待探索空间：由于细菌体内清除机制和活菌制剂的免疫原性差异，活菌递送系统在更复杂的大动物模型中的长期安全性、潜在全身感染风险控制以及规模化活菌药物偶联工艺的标准稳定性，仍需在未来的临床转化研究中进一步优化和评估。

【Angew.Chem.】中山大学夏炜|细菌介导的四价铂前药靶向递送与肿瘤深层渗透实现协同化疗-免疫治疗：肿瘤体积缩小72.2%