研究背景与科学问题#

以PD-1/PD-L1阻断为代表的免疫疗法已成为现代肿瘤治疗的重要支柱，但在许多实体瘤中作为单药疗法时疗效依然受限。这主要归因于肿瘤免疫原性偏低、肿瘤微环境具有强抑制性以及T细胞耗竭等因素。光动力疗法作为一种无创治疗手段，能够诱导肿瘤细胞发生免疫原性细胞死亡，进而促进树突状细胞成熟并增强T细胞增殖，具有重塑肿瘤微环境的潜力。

然而，传统光敏剂往往面临系统间跨越效率低和活性氧生成不足的问题。TADF材料因其极小的单重态-三重态能隙和高效的系统间跨越能力，被视作提升光动力疗法性能的理想分子。此外，细胞焦亡作为一种由气解蛋白介导的程序性细胞坏死，在释放促炎因子的同时可激活cGAS-STING通路，从而形成强大的免疫激活循环。因此，开发能够同时引发细胞焦亡并激活cGAS-STING通路的TADF光敏剂具有重要的临床前研究价值。

Scheme.1 Schematic illustration of the mechanism of TA2O in photodynamic therapy (PDT), which activates both pyroptosis and the cGAS-STING pathway to potentiate immuno-phototherapeutic efficacy.

核心实验过程与发现#

研究团队首先设计合成为了将电子受体羰基与电子供体氮原子融为一体的融合环光敏剂分子TA2O，如图1A所示。量子化学计算表明，TA2O的前线轨道在空间上有效分离，其单重态-三重态能隙仅为0.19 eV，且旋光耦合矩阵元高达2.96 cm⁻¹，如图1B所示。这种结构特征显著促进了系统间跨越过程，赋予其明显的延迟荧光特性，如图1E所示。在水相聚集态下，限制分子运动使得TA2O的荧光强度显著增强，如图1D所示。

Fig.1 The characterization of TA2O. (A) Molecular structure of TA2O. (B) Calculated HOMO and LUMO distributions and spin-orbit coupling (SOC) matrix element, the tested energies for the HOMO and LUMO, and the singlet-triplet energy gap (ΔEST) for TA2O. (C) UV-Vis absorption and (D) photoluminescence (PL) spectra of TA2O in pure DMSO and DMSO/water (1<99>, v/v) mixtures (10 µM). (E) Transient PL decay spectrum of TA2O in DMSO/water (1<99>, v/v) mixtures (10 µM). Time-course analysis of ROS generation by TA2O and commercial Ce6 in DMSO/water (1<99>, v/v) mixtures (10 µM) under white light irradiation (20 mW/cm2): (F) Fluorescence enhancement of DCFH; (G) 1O2 production tracked by ABDA decomposition; (H) Fluorescence enhancement of DHR 123; (I) Fluorescence enhancement of HPF.

在活性氧生成测试中，聚集态TA2O展现出优异的活性氧响应能力，如图1F所示。热力学分析确认TA2O具备双重I型与II型光动力反应机制，其单线态氧生成量子产率达到了26.0%，如图1G所示。同时，自旋共振与荧光探针实验证实TA2O在光照下能大量产生超氧阴离子自由基，这为其在低氧肿瘤微环境下的有效作用提供了理论依据，如图1H与图1I所示。

在细胞层面，共聚焦显微成像显示TA2O能够高效进入细胞并优先蓄积于内质网，如图2A和2B所示。在白光照射下，TA2O在多种肿瘤细胞系中诱导了剧烈的细胞毒性效应，而暗光条件下则保持高度安全，如图2E和2F所示。活死细胞染色直观证实了光照组细胞的广泛死亡，如图2G和2H所示。即使在1%氧浓度的模拟缺氧环境中，TA2O依然维持了良好的光毒性。

Fig.2 Intracellular localization, ROS generation, and cytotoxicity of TA2O. (A, B) Colocalization of TA2O with organelle-specific trackers (ER-Tracker Green, MitoTracker Green, or LysoTracker Green) in YTN16 (A) and HGC27 (B) cells. (C, D) Representative Confocal Light Scanning Microscopy (CLSM) showing intracellular ROS levels (detected by CellROX Deep Red) in YTN16 (C) and HGC27 (D) cells after 24 h of TA2O treatment, with or without white light irradiation. (E-F) Viability of CT2A, MC38, AGS, HGC27, YTN16, U118MG, and MKN28 cells treated with increasing concentrations of TA2O under dark conditions (E) or following a 10 min light exposure (100 mW/cm2) (F) under normoxia. (G, H) Live/dead cell staining (Calcein-AM, green; PI, red) of YTN16 (G) and HGC27 (H) cells subjected to the indicated treatments.

机制研究进一步揭示，受处理的肿瘤细胞在光照3小时内即表现出胞体膨胀和气泡化等典型的焦亡形态特征，如图3A所示。印迹实验证实TA2O促使caspase-1与GSDME发生特异性剪切，生成具有膜穿孔活性的GSDME-N片段，如图3B和3C所示。功能抑制实验表明使用caspase-1抑制剂可显著挽救细胞死亡，证实了该过程依赖于焦亡通路。伴随焦亡发生，细胞大量释放三磷酸腺苷、暴露钙网织蛋白并转移高移动性组蛋白B1，如图3D至3I所示，全面激活了免疫原性细胞死亡。

Fig.3 TA2O induces pyroptosis and immunogenic cell death in vitro. (A) Morphological progression of pyroptosis in YTN16 and HGC27 cells at the indicated time points (0, 1, 3, 6, 12, and 24 h) after treatment. (B, C) Western blot analysis of pyroptosis-related proteins, showing the expression of full-length and cleaved GSDME and caspase-1. (D, E) Extracellular ATP release from YTN16 (D) and HGC27 (E) cells treated with TA2O, with or without light irradiation (100 mW/cm2, 10 min). (F, G) CRT exposure on the surface of YTN16 (F) and HGC27 (G) cells visualized by confocal microscopy following treatment with TA2O and light. (H, I) HMGB1 translocation in YTN16 (H) and HGC27 (I) cells under the same treatment conditions, as detected by immunofluorescence.

体外共培养实验表明，经过TA2O光动力处理的肿瘤细胞能显著促使骨髓来源树突状细胞发生成熟，并诱导巨噬细胞向促炎的M1型分化。在活体肿瘤模型中，TA2O结合光照不仅显著抑制了肿瘤生长，还成功触发了小鼠体内的cGAS-STING通路激活。免疫组化分析显示肿瘤组织内细胞毒性T细胞与M1巨噬细胞浸润量明显增加，而抑制性的M2巨噬细胞比例显著降低。当将TA2O介导的光动力疗法与抗PD-1抗体联合使用时，协同方案实现了针对实体瘤的深层清除，疗效显著优于任何单一治疗组。

Fig.4 TA2O promotes dendritic cell maturation and M1‑macrophage polarization via STING pathway activation. (A) Schematic of the transwell co-culture system in which BMDCs/BMDMs are stimulated by DAMPs released from ICD-induced tumor cells. (B) Flow cytometric analysis of DC maturation (gated on CD45+CD11c+MHC-II+ cells). (C) Western blot detection of key proteins in the STING signaling pathway. (D) Flow cytometric identification of M1-polarized macrophages (gated on CD45+CD11b+F4/80+CD86+ cells). (E-F) Quantitative summary of the percentages of matured DCs (E) and M1 macrophages (F) from panels (B) and (D), respectively.

Fig.5 In vivo antitumor efficacy of TA2O in a YTN16 subcutaneous tumor model. (A) In vivo fluorescence imaging of tumor‑bearing mice at indicated time points after intratumoral injection of TA2O. (B) Schematic of the treatment regimen. (C) Tumor growth curves for each treatment group (n = 6). (D, E) Tumor weights (D) and representative photographs of excised tumors (E) at the experimental endpoint (n = 6). (F) Histopathological evaluation of tumor sections by H&E staining, Ki-67 immunohistochemistry (IHC), and TUNEL assay.

Fig.6 Immunofluorescence analysis of tumor‑infiltrating immune cells. (A, B) Representative immunofluorescence images showing the infiltration of CD4+ and CD8+ T cells (A), as well as macrophages (F4/80+) and dendritic cells (CD11c+) (B) in tumor sections from each treatment group. (C–F) Quantitative summary of the fluorescence intensities or positive cell counts for CD4+ T cells (C), CD8+ T cells (D), macrophages (E), and dendritic cells (F).

Fig.7 Flow cytometric profiling and quantification of tumor-infiltrating immune cells. (A–C) Representative flow cytometry plots showing the proportions of total immune cells (CD45+) (A), CD4 T cells (CD45+CD3+CD4+) (B), and IFN-γ+CD8 T cells (CD45+CD3+CD8+IFN-γ+) (C) in tumor single-cell suspensions. (D-F) Quantitative summary of the percentages of total immune cells (D), CD4 T cells (E), and IFN-γ+CD8 T cells (F) across treatment groups.

研究意义与未来展望#

本研究成功构建了一种基于TADF机制的高效单分子光敏剂TA2O，为破解肿瘤免疫治疗响应率低提供了新的分子工具。TA2O通过特有的分子设计将细胞焦亡与cGAS-STING通路精准结合，在光照触发下实现了先天免疫与后天适应性免疫的双重激活。这种将新型光敏材料与免疫检查点阻断剂相融合的联合策略，不仅重塑了肿瘤微环境的免疫抑状态，也为未来开发多功能精密光药物及临床转化提供了重要的实验依据。