近期，吉林大学林权教授和史册教授在科爱创办的期刊Bioactive Materials（IF：18）上联合发表研究文章：该团队制备了一种“原位纳米疫苗”用于增强TNBC的免疫治疗。一方面，通过发挥Au/CuNDs-R848的光热治疗和光热增强的化学动力学治疗作用，有效抑制了原发肿瘤的进展。同时，释放的抗原和损伤相关分子模式促进了树突状细胞的成熟和T淋巴细胞的浸润。因此，Au/CuNDs-R848作为一种“原位纳米疫苗”，通过诱导免疫原性细胞死亡来增强TNBC的免疫原性。另一方面，该纳米疫苗减少了髓源性抑制细胞的数量，从而逆转了免疫抑制微环境。通过双重免疫调节作用，“冷肿瘤”被成功转变为“热肿瘤”，实现了“化敌为友”的治疗策略，增强了对原发性和转移性TNBC的免疫治疗效果。此外，Au/CuNDs-R848作为一种优异的纳米探针，可通过近红外荧光成像和CT成像准确定位TNBC，实现诊断与治疗的一体化。
三阴性乳腺癌（TNBC）具有高度的侵袭性且易发生转移，导致患者的复发率和死亡率逐年上升。作为一种“冷肿瘤”，TNBC对目前的免疫治疗反应有限。为了增强TNBC的免疫原性和逆转免疫抑制微环境，我们设计了一种兼具双模式成像引导能力和PTT/CDT协同治疗的“原位纳米疫苗”Au/CuNDs-R848（Scheme 1）。首先，我们采用共还原法构建了一种具有光热剂和纳米酶功能的金铜纳米点（Au/CuNDs）。随后，通过超分子相互作用负载免疫佐剂R848，实现免疫增强。PTT和光热增强的CDT有效抑制了原发肿瘤的进展，并使原发肿瘤自身作为TAAs的来源，促进发生强烈的抗原特异性免疫反应。此外，由于抗原成分来源于自身，因而不会引起与传统肿瘤疫苗类似的伦理问题。这一过程还伴随高迁移率族蛋白B（HMGB1）、钙网蛋白（CRT）和三磷酸腺苷（ATP）等DAMPs的释放。在免疫佐剂的刺激下，TAAs和DAMPs被DCs更有效地内化。然后，DCs分化成熟，有效地发挥抗原提呈作用并最终激活细胞毒性T淋巴细胞的生成。因此，Au/CuNDs-R848通过诱导ICD增强TNBC的免疫原性，发挥了“原位纳米疫苗”的作用。此外，ICD通过分泌干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）等与肿瘤抑制相关的炎症因子，从而抑制了转移瘤的生长。同时，具有免疫抑制作用的MDSCs在这一过程中被有效抑制，从而逆转了免疫抑制微环境。最终，“冷肿瘤”转变为“热肿瘤”，实现了“化敌为友”的治疗策略。总的来说，作为具有双重免疫调节的“原位纳米疫苗”Au/CuNDs-R848，可以增强对转移性TNBC的免疫治疗。此外，Au/CuNDs-R848作为一种优异的纳米探针，可通过近红外荧光成像和CT成像准确定位TNBC，实现诊断与治疗的一体化。
Scheme 1. An advanced “in situ nanovaccine” Au/CuNDs-R848 for dual-mode imaging and photothermal-chemodynamic therapy, triggering dual immunoregulation to enhance immunotherapy against metastatic triple-negative breast cancer.
Figure 1. Structure, morphology, and composition characterization of Au/CuNDs-R848. (a) 1HNMR spectrum and (b) FTIR spectra of Au/CuNDs, R848, and Au/CuNDs-R848. (c) Zeta potential of Au/CuNDs and Au/CuNDs-R848. (d) TEM image and (e) size distribution histogram of the Au/CuNDs-R848. (f) UV-vis absorption spectrum of Au/CuNDs-R848. Inset, photographs of Au/CuNDs-R848 solution. (g) XPS survey spectrum of Au/CuNDs-R848. High-resolution XPS spectra of (h) Au 4f and (i) Cu 2p of Au/CuNDs-R848.
Figure 2. Evaluation of the photothermal and chemodynamic properties of Au/CuNDs-R848. (a) Temperature elevation and (b) infrared thermal images of PBS (used as control) and Au/CuNDs-R848 under 808 nm laser irradiation as a function of irradiation time. (c) Temperature profiles of Au/CuNDs-R848 under 808 nm laser irradiation at different power densities (1.0, 1.5, 2.0, and 2.5 W/cm2). (d) Thermal stability of Au/CuNDs-R848. (e) Absorbance of MB after the addition of Au/CuNDs-R848 and H2O2 at different pH levels (5.4, 6.5, and 7.4). (f) Absorbance of MB after the addition of Au/CuNDs-R848 and H2O2 under different laser power irradiations (0.5, 1.0, 1.5, 2.0 W/cm2). (g) ESR spectra under various reaction conditions with DMPO as a spin trap agent. (h) GSH depletion by Au/CuNDs-R848
Figure 3. In vitro NIR-II fluorescence and CT imaging properties of Au/CuNDs-R848. (a) Emission spectrum of Au/CuNDs-R848. (b) Photostability and (c) fluorescence intensity of Au/CuNDs-R848 and ICG. Trends in (d) FL intensity and (e) CT intensity of Au/CuNDs-R848 across varying concentrations. Inset, FL images and CT images at specific Au/CuNDs-R848 concentrations.
Figure 4. In vitro detection of synergistic therapeutic effects and intracellular ROS. (a) Cell viability of 4T1 and HC11 cells after 24-hour exposure to graded concentrations of Au/CuNDs-R848 (0, 50, 100, 200, 300, and 400 μg/mL). (b) Cell viability of 4T1 cells after incubation with Au/CuNDs-R848 in the absence or presence of 808 nm laser irradiation at different power densities (1.0, 1.5, and 2.0 W/cm2). (c) Fluorescence images displaying ROS levels in 4T1 cells stained with DCFH-DA, subsequent to different treatments (scale bar: 200 µm). (d) Quantification of intracellular ROS levels across different treatments (n = 3). (e) Apoptosis quantification in 4T1 cells treated with Au/CuNDs or Au/CuNDs-R848, with and without 808 nm laser irradiation at 2.0 W/cm2, as analyzed by flow cytometry. (Q2: late apoptotic cells; Q3: early apoptotic cells), with percentages indicating the overall apoptosis rate (Q2 + Q3). (f) Apoptosis rate of 4T1 cells treated with Au/CuNDs or Au/CuNDs-R848, with and without 808 nm laser irradiation at 2.0 W/cm2. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5. In vitro induction of ICD in tumor cells by Au/CuNDs-R848-mediated PTT/CDT. (a) Cell surface CRT expression was determined by flow cytometry 12 h post-treatment. (b) Quantitative analysis of CRT expression on cell surfaces across different treatment groups (n = 3). (c) Detection of extracellular ATP levels in 4T1 cells 2 h after treatment, quantified by luminescence (n = 3). (d) Quantification of HMGB1 release into the medium 24 h post-treatment, measured by enzyme‐linked immunosorbent assay (ELISA). (e) Translocation of HMGB1 (red) using confocal laser scanning microscope (CLSM), with cell nuclei counterstained with DAPI (blue). (scale bar: 20 μm) (f) Schematic illustration of the in vitro maturation of DCs induced by Au/CuNDs-R848. (g) Gating strategy of flow cytometry employed to identify matured DCs based on the expression of co-stimulatory molecules CD11c, CD80, and CD86. (h) Flow cytometry analysis of mature DCs (CD11c+, CD80+, and CD86+) in different treatment groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6. In vivo NIR-II FL and CT imaging performance of Au/CuNDs-R848. (a) A comparative fluorescence imaging analysis between ICG and Au/CuNDs-R848. (b) CT imaging showcasing the capabilities of Au/CuNDs-R848. The red dashed box in each panel highlights the location of the tumor. (c) Quantitative evaluation based on the NIR-II fluorescence imaging data for ICG and Au/CuNDs-R848. (d) Trend analysis of CT intensity over time following the administration of Au/CuNDs-R848.
Figure 7. In vivo induction of DAMPs by Au/CuNDs-R848-mediated PTT/CDT. (a) Scheme of the strategies employed to detect the release of DAMPs in mice bearing bilateral 4T1 tumors. (b) Measurement of extracellular ATP levels in the primary tumors. (c) Quantification of HMGB1 in the primary tumors as detected by ELISA. (d) Localization of HMGB1in the primary tumors using CLSM, with cell nuclei counterstained with DAPI (blue). (scale bar: top, 200 μm; bottom, 20 μm). (e) Evaluation of cell surface CRT (red) using CLSM. (scale bar: 200 μm). (f) TUNEL staining (red) of the primary tumors to identify apoptotic cells (scale bar: 100 μm) (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 8. In vivo treatment of primary and metastatic TNBC. (a) In vivo temperature changes under laser irradiation. (b) Treatment schedule for mice with bilateral 4T1 tumors. (c) Photographs, (d) weights, and (e) growth curves of primary tumors. (f) Photographs, (g) weights, and (h) growth curves of distant tumors. Plasma levels of (i) IL-6, (j) IFN-γ, and (k) TNF-α determined by ELISA (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 9. In vivo immune regulatory effects of Au/CuNDs-R848-mediated PTT/CDT against TNBC. (a) Flow cytometry analysis of the maturation of DCs. (b) Relative quantification of the maturation of DCs. (c) Flow cytometry analysis of CD8+ T cells (CD3+ and CD8+), CD4+ T cells (CD3+ and CD4+), and MDSCs (CD11b+ and Gr1+) in the spleen. (d) Relative quantification of CD8+ T cells, (e) CD4+ T cells, and (f) MDSCs in the spleen. (g) Flow cytometry analysis of CD8+ T cells (CD3+ and CD8+), CD4+ T cells (CD3+ and CD4+), and MDSCs (CD11b+ and Gr1+) in distant tumors. (h) Relative quantification of CD8+ T cells, (i) CD4+ T cells, and (j) MDSCs in distant tumors (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.
综上，该团队成功开发了一种“原位纳米疫苗”Au/CuNDs-R848，用于双模式成像引导的PTT和光热增强的CDT，以增强原发性和转移性TNBC的免疫治疗。Au/CuNDs-R848可以充分发挥光热剂和纳米酶的作用，实现PTT和CDT协同治疗，有效抑制原发肿瘤的生长。此外，原发肿瘤细胞死亡后释放的“自身”TAAs和DAMPs促进了DCs的成熟和T淋巴细胞的浸润，从而激发了全身抗肿瘤免疫反应。同时，MDSCs被抑制，从而逆转免疫抑制微环境。我们通过双重免疫调节效应，成功将“冷肿瘤”转化为具有更高免疫原性的“热肿瘤”，实现“化敌为友”的治疗策略，有效地治疗了转移肿瘤。此外，Au/CuNDs-R848作为一种纳米探针表现出优异的双模式成像性能，显示出其在临床肿瘤检测和手术指导中的潜在应用价值。综上所述，制备的“原位纳米疫苗”有效地结合了PTT、光热增强CDT和免疫佐剂R848，增强了免疫应答，并使肿瘤细胞对基于ICD的免疫治疗敏感，为未来的免疫治疗提供了新的策略。
第一作者：王泽
吉林大学化学学院&超分子结构与材料国家重点实验室2021级博士研究生。从事多功能金属基纳米材料的设计制备及其在生物医学方面的应用研究。
第一作者：沙桐
吉林大学白求恩口腔医学院2022级博士研究生。从事肿瘤免疫以及纳米生物材料的相关研究。
通讯作者：林权教授
吉林大学化学学院&超分子结构与材料国家重点实验室教授，博士生导师，吉林大学“唐敖庆学者”英才教授。曾主持承担国自然、国家重点研发计划等10余项省部级项目，获吉林省自然科学学术成果等奖项，在Angew. Chem. , Adv. Funct. Mater, ACS nano等国际权威期刊发表科技论文100余篇。获授权中国发明专利近30余项。
通讯作者：史册教授
教授，主任医师，博士生导师。入选 “教育部长江学者奖励计划——青年学者”。吉林大学超分子结构与材料国家重点实验室博士后；美国密西根大学牙学院访问学者。中华口腔医学会口腔病理学专业委员会常委；国际牙医师学院Fellow。主持国家自然科学基金项目3项、中国博士后科学基金特别资助项目1项、其他省级项目5项。共发表SCI收录论文31篇，其中第一作者/通讯作者16篇。曾获口腔医学青年科学家论坛最具潜力奖、口腔病理杰出青年研究论坛奖、口腔生物医学新锐奖、“超星杯”第二届吉林省本科高校智慧课堂教学创新大赛一等奖。作为负责人，获批第二批国家级一流本科课程。作为主要参加人，获省科技进步一等奖1项、省教学成果一等奖1项。
This work was supported by the National Key Research and Development Program of China (2022YFC2504200), the National Natural Science Foundation of China (Nos. 82270959 and 81970903), the Natural Science Foundation of Jilin Province (No. SKL202302002), the Key Research and Development Project of Jilin Provincial Science and Technology Department (Nos. 20210204142YY), the Jilin University Norman Bethune Program (No. 2023B28), the Fundamental Research Funds for the Central Universities, the Natural Science Foundation of Liaoning Province (No. 2022-BS-123), the Science and Technology Project of Shenyang (No. 21-173-9-34), and “Medical + X” Interdisciplinary Innovation Team “Announcement and Leadership” Construction Project (2022JBGS08).
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Ze Wang, Tong Sha, Jinwei Li, Huanyu Luo, Annan Liu, Hao Liang, Jinbiao Qiang, Lei Li, Andrew K. Whittaker, Bai Yang, Hongchen Sun, Ce Shi* and Quan Lin*. Turning Foes to Friends: Advanced “In Situ Nanovaccine” with Dual Immunoregulation for Enhanced Immunotherapy of Metastatic Triple-Negative Breast Cancer. Bioactive Materials, 39 (2024) 612-629. DOI: 10.1016/j.bioactmat.2024.04.023
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Bioactive Materials 创建于2016年，自2019年被SCIE检索收录以来影响因子实现跳跃式增长（IF 2019: 8.724；IF 2020: 14.593；IF 2021: 16.874；IF 2022:18.9; IF 2023: 18）；JCR materials science, biomaterials 领域国际排名连续四年第一。此外， 2020年到2023年连续四年入选中国科学院文献情报中心期刊分区表一区，Top期刊；入选材料科学综合类高质量科技期刊分级目录T1区。
