【JACS】苏州大学钟志远|精准释放“叫醒”冷肿瘤：光控纳米光笼联合免疫治疗实现80%肿瘤根除
文章标题：STING Activation by Photo-immunogenic Nanophotocages for Synergistic Photoimmunotherapy of Cold Tumor
通讯作者：Wenhai Lin, Shengliang Li, Zhiyuan Zhong
文章概要
苏州大学钟志远教授团队在《美国化学会志》上发表了最新研究成果。研究团队巧妙设计了一种基于BODIPY的光敏纳米光笼，用于精准递送STING激动剂DMXAA。该系统在530纳米绿光照射下，不仅能高效产生化学活性氧诱导免疫原性细胞死亡，还能实现激动剂的实时时空可控释放。这种“光动力治疗+STING激活”的双重协同策略，成功逆转了冷肿瘤的免疫抑制微环境，联合CTLA4免疫检查点阻断疗法，最终在小鼠模型中实现了高达80%的肿瘤完全根除。
引言
肿瘤免疫治疗在近年来取得了突破性进展，然而以三阴性乳腺癌为代表的“冷肿瘤”，由于内部缺乏足够活化的T细胞浸润、肿瘤免疫原性极低，往往对常规的免疫检查点抑制剂毫无反应。激活干扰素基因刺激因子，即STING信号通路，已被证实能够强效促进树突状细胞成熟、增强抗原递呈并诱导T细胞向肿瘤内部浸润，是逆转冷肿瘤免疫抑制微环境的有力武器。然而，STING在全身各种组织细胞中广泛表达，直接全身给药会导致致命的“免疫风暴”和自身免疫疾病。为了打破这一临床应用瓶颈，如何实现STING激动剂在肿瘤部位的精准、安全释放，成为了学术界亟待解决的科学难题。
Scheme 1. Schematic of STING Activation by Photo-immunogenic Nanophotocages for Synergistic Photoimmunotherapy of Cold Tumor
主要实验及结论
研究团队首先合成了四种不同卤素取代的BODIPY光笼衍生物。密度泛函理论计算与光谱学表征显示，随着氯、溴、碘等重原子的引入，其吸收光谱和发射光谱均发生了明显的红移。如图1所示，在这四种光笼分子中，二碘取代的衍生物呈现出最低的分子轨道能级差，并表现出最强的高效产生单线态氧与超氧阴离子的能力，这表明其具有最佳的光动力治疗潜能，从而被确立为后续实验的核心载体。
Figure 1. (A) Normalized absorption spectra and (B) fluorescence spectra of BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM in dichloromethane (DCM). (C) Calculated HOMO and LUMO distributions of BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM. (D) Absorption spectra of DPBF with IBDP-DM under green light irradiation (530 nm, 10 mW cm–2). (E) Decay profiles of the absorption peak from DPBF with BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM. (F) Fluorescence spectra of DHR123 in the presence of IBDP-DM under green light irradiation (530 nm, 10 mW cm–2). (G) Intensities of the fluorescence peak at 525 nm from DHR123 with BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM.
接下来，研究团队对光控药物释放行为进行了细致的验证。如图2所示，在530纳米绿光的激发下，二碘取代的光笼表现出最高的催化光解效率，在45分钟内DMXAA的释放率高达49.4%，其释放动力学与光笼的降解速率完美契合，而未引入卤素或引入其他卤素的对照组则表现出不稳定性或较低的释放效率。瞬态吸收光谱和清除剂捕获实验共同证实，该释放机制遵循光诱导的一级亲核取代反应路径，通过形成碳正离子中间体来实现药物的精准切割。更重要的是，通过反复切换光源的对冲实验，证实了该系统具有极佳的时空可控性，即“有光释放，无光停止”。
Figure 2. (A) Percentages of DMXAA released from BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM under light irradiation (530 nm, 10 mW cm–2, 45 min). (B) Photolysis rates of BDP-DM, CBDP-DM, BBDP-DM, and IBDP-DM under green irradiation (530 nm, 10 mW cm–2, 45 min). (C) The absorbance spectra of BBDP under light irradiation (530 nm, 50 mW cm–2, 4 min). (D) Postulated photorelease mechanism of BODIPY photocages. (E) Percentage of DMXAA released from IBDP-DM in N2-saturated methanol under light irradiation (solution: N2-saturated methanol) (530 nm, 10 mW cm–2, 10 min). (F) Nanosecond transient absorption spectra of IBDP-DM in methanol measured at 30–1500 μs after excitation. (G) Decay trace of lifetime: IBDP-DM in methanol. (H) MADLI-TOF MS of IBDP-DM under green light irradiation (530 nm, 10 mW cm–2, 5 min). (I) Percentage of DMXAA released from IBDP-DM upon repeated light switching.
为了克服疏水性药物在生物体内的应用限制，研究团队利用两亲性脂质材料，将该光笼自组装制备成水色分散性良好的纳米颗粒。如图3所示，该纳米颗粒的平均粒径约为149.4纳米，呈现出规则的球形，且在一周内保持了优异的胶体稳定性。细胞实验表明，三阴性乳腺癌细胞能够高效内吞该纳米颗粒。在无光照时，纳米颗粒几乎没有细胞毒性；而在绿光照射下，细胞内迅速激发出强烈的绿色荧光产生大量活性氧，引发了显著的细胞毒性，这也表明其光毒性完全由光动力效应介导。
Figure 3. (A) Size distribution of IBDP-DM NPs. Inserted: (left) TEM image of IBDP-DM NPs. Scale bar, 200 nm; (right) SEM image of IBDP-DM NPs. Scale bar, 500 nm. (B) Decline of the absorption peak at 418 nm from DPBF with IBDP-DM NPs. (C) Normalized absorption spectra of IBDP-DM in DCM and IBDP-DM NPs in water. (D) Fluorescence spectra of IBDP-DM in DCM and IBDP-DM NPs in water. (E) DLS-measured colloidal stability of IBDP-DM NPs in water over a week. (F) Percentage of DMXAA released from IBDP-DM NPs upon green light irradiation (530 nm, 10 mW/cm2). (G) Flow cytometry analysis of intracellular fluorescence intensity in 4T1 cells following incubation with IBDP-DM NPs for 2, 4, and 6 h. (H) Intracellular ROS generation in 4T1 cells induced by IBDP NPs and IBDP-DM NPs under green light irradiation (10 mW/cm2, 10 min). Scale bar, 200 μm. (I) Phototoxicity of IBDP-DM NPs under green light irradiation (530 nm, 10 mW/cm2, 30 min). (J) Live/dead fluorescence imaging of 4T1 cells following treatment with IBDP NPs and IBDP-DM NPs under green light irradiation (530 nm, 10 mW/cm2, 30 min). Scale bar, 200 μm. Data are presented as the mean ± SD, and statistical significance was calculated via a one-way ANOVA test. ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.
这种强效的光动力效应能够进一步引发免疫原性细胞死亡。如图4所示，绿光照射后，肿瘤细胞表面的钙网蛋白表达量显著上调，细胞外腺苷三磷酸的释放量也大幅增加。研究团队进一步将处理后的肿瘤细胞与未成熟的骨髓源性树突状细胞进行共孵育，流激细胞术结果显示，光控释放的DMXAA与光动力引发的免疫原性细胞死亡产生了强烈的协同效应，促使成熟树突状细胞的比例飙升至33.23%。蛋白质印迹分析也确证，该纳米系统能够强烈激活肿瘤细胞内STING、TBK1及IRF3等核心蛋白的磷酸化，成功启动了内在的抗肿瘤免疫机制。
Figure 4. (A) Quantitative assessment of CRT expression after 4T1 cells were subjected to distinct treatments (n = 3). (B) Released ATP after 4T1 cells were subjected to distinct treatments (n = 3). (C) Schematic illustration of the coculture system between BMDCs and 4T1 cells. (D) Representative flow cytometry plots depicting CD80 and CD86 expression on BMDCs. (E) Percentage of mature BMDCs (CD11c+CD80+CD86+) (n = 3). (F) Western blot analysis of STING, TBK1, IRF3, p-STING, p-TBK1, and p-IRF3 phosphorylation of related proteins, which were extracted from 4T1 cells with different treatments. Data are presented as the mean ± SD, and statistical significance was calculated via a one-way ANOVA test. ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.
在体内抗肿瘤药效学研究中，研究团队利用荷瘤小鼠模型评估了这一光免疫疗法的实际效果。如图5所示，单纯的免疫检查点抑制剂无法抑制冷肿瘤的生长，而普通的纳米光敏剂由于光穿透深度的限制，其治疗效果也相当有限。相比之下，注射了光笼纳米颗粒并接受绿光照射的治疗组表现出优异的肿瘤抑制效果。当进一步联合抗CTLA4抗体时，不仅小鼠的免疫抑制微环境被彻底逆转，外周血和肿瘤浸润的CD4阳性与CD8阳性T细胞比例显著增加，脾脏中分泌干扰素的免疫细胞数量也达到顶峰。最终，该联合治疗组中80%的小鼠原发性肿瘤被完全根除，且全治疗周期内小鼠体重未见异常，表现出极高的高效能与安全性。
Figure 5. (A) Changes in tumor volume of mice with distinct treatments (n = 5). (B) Average tumor weights of mice with different treatments (n = 5). (C) Body weight change curves of mice with different treatments. (D) CRT and CD8 staining of 4T1 subcutaneous tumors after different treatments. (E) Representative flow cytometry analysis of CD80 and CD86 in the lymph nodes of mice with different treatments. (F) Representative relative quantification results of matured DCs (CD11c+CD80+CD86+) in the lymph nodes of mice with different treatments. Representative relative quantification results of (G) CD4+ T cells and (H) CD8+ T cells in the peripheral blood of mice. (I) Representative relative quantification results of TFN-α+CD8+ T cells in the spleen of mice. (J) Representative flow cytometry graphs and (K) Quantification of IFN-γ + CD8+ T cells in the spleen of mice. Scale bar, 200 μm. Data are presented as the mean ± SD, and statistical significance was calculated via a one-way ANOVA test. ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.
为了进一步模拟临床上肿瘤的远端转移，研究团队建立了双侧双部位肿瘤模型。如图6所示，仅对一侧原发瘤进行局部绿光照射治疗，在光免疫协同效应下，不仅受照侧的肿瘤迅速消退，未接受光照的远端转移瘤生长也受到了显著的抑制。免疫组织化学染色证实，远端肿瘤组织中形成了大量活化的T细胞浸润，这强有力地证明了该疗法成功诱导了系统性的抗肿瘤免疫记忆，实现了冷肿瘤向“热肿瘤”的根本性转变。
Figure 6. (A and B) Distant tumor volume change curves of mice with various treatments (n = 5). (C) Average distant tumor weights after mice were subjected to distinct treatments (n = 5). (D) Images of the CD4/CD8 staining of 4T1 subcutaneous tumors after mice were subjected to distinct treatments. Scale bar, 100 μm. Data are presented as the mean ± SD, and statistical significance was calculated via a one-way ANOVA test. ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.
总结及展望
综上所述，该研究成功构建了一种具有光免疫原性的新型纳米光笼平台，通过可见光调控，完美实现了光动力治疗介导的免疫原性细胞死亡与STING信号通路的局部精准激活。这一策略不仅在体内外表现出强大的协同抗肿瘤活性，还为攻克临床上冷肿瘤对抗体药物不敏感的医学难题提供了全新思路。展望未来，通过进一步分子结构改造，如引入长波长近红外光响应的化学骨架，并嫁接临床相关的针对人类的STING激动剂，该平台技术有望实现更深层组织的光控精准递送，发展成为下一代更具临床转化前景的精准癌症光免疫治疗系统。