Scheme 1 Schematic illustration of the AIE PS TBC: in situ photodegradation of CAIX triggers ferroptosis by alleviating hypoxia and inducing intracellular acidosis.#

Fig.1 (A) Normalized fluorescence (FL) spectra of TBN and TBC in THF and DMSO. (B) Plots of the relative FL intensity (I/_I_0)) of TBN and TBC as a function of the water fraction (_f_w) in DMSO/water mixtures. _I_0 = FL intensity in pure DMSO. _λ_ex = 450 nm. (C) Peak maximum of TBC as a function of the water fraction (_f_w) in DMSO/water mixtures. (D) Plot of the relative FL intensity (I/_I_0, _λ_ex = 488 nm) of DCF versus irradiation time in DMSO/water mixtures with varied _f_w in the presence of 10 µM TBC under white light irradiation (20 mW cm−2), where _I_0 is the pre-irradiation FL intensity of DCF. (E) Detection of total ROS via DCF. (F) Detection of O2**·**− via oxidation of DHR123 (_λ_ex = 495 nm, _λ_em = 525 nm). (G) Detection of **·**OH via HPF (_λ_ex = 490 nm, _λ_em = 515 nm). (H) Detection of 1O2 via bleaching of ABDA (_λ_abs = 525 nm). (I and J) Electron density distributions of the HOMO and LUMO for TBC and TBN based on DFT calculations.#

细胞及分子水平的靶向性实验证实，TBC能够通过其苯磺酰胺尾翼精准结合CAIX蛋白（如图2所示）。分子对接与质谱分析显示，TBC能够深入CAIX的活性口袋并与锌离子发生配位，其结合能达到-7.1 kcal/mol，结合后分子的荧光寿命从3.6纳秒延长至5.2纳秒，证明分子运动受到了有效限制。酶活性抑制实验表明，TBC对CAIX的黑暗抑制半数抑制浓度为145.1 nM，而在光照10分钟后，其抑酶活性显著提升至0.68 nM，抑制效力猛增了210倍（如图3所示）。凝胶电泳与质谱结果表明，光照下的TBC通过产生活性氧诱导了CAIX蛋白发生选择性交叉联结与断裂降解。这种光降解效应不仅显著降低了MDA-MB-231细胞中CAIX和HIF-1α的表达量，还成功提升了细胞内的氧气浓度，缓解了细胞缺氧状态，并引发了显著的细胞内酸中毒。

Fig.2 (A) Colocalization of TBN (2 µm, 0.5 h) with BODIPY 493/503 or TBC (2 µm, 0.5 h) with Dil in MDA-MB-231 cells. TBN/TBC: _λ_ex = 450 nm, _λ_em = 660 ± 30 nm; BODIPY: _λ_ex = 493 nm, _λ_em = 520 ± 20 nm; Dil: _λ_ex = 561 nm, _λ_em = 590 ± 20 nm; Scale bar: 10 µm. Colocalization analysis was performed using the Coloc 2 plugin in ImageJ/Fiji. (B) Molecular docking of TBC with CAIX (PDB code: 3IAI). Zn2+ was shown as a gray sphere. (C) MALDI-TOF MS analysis of TBC upon incubation with CAIX. (D) Changes in the fluorescent lifetimes of TBN and TBC upon incubation with CAIX.#

Fig.3 (A) SDS-PAGE analysis of CAIX incubated with TBC (left) or TBN (right) under the indicated conditions (dark/light, normoxic/hypoxic). Lane 1/8/15: Ladder; Lane 2/9: CAIX (dark); Lane 3/10: CAIX under normoxia (light); Lane 4/11: CAIX under hypoxia (light); Lane 5/12: CAIX + 1 eq. TBC/TBN (dark); Lane 6/13: CAIX + 1 eq. TBC/TBN under normoxia (light); Lane 7/14: CAIX + 1 eq. TBC/TBN under hypoxia (light). (B) Inhibitory effect of TBC on the activity of CAIX isoforms measured by in vitro kit test. Data are presented as mean ± SD (n = 3). (C) Western blot analysis of CAIX and HIF-1α expression in MDA-MB-231 cells treated with TBC at indicated concentrations, under dark or light conditions. GAPDH served as the loading control. All Western blot bands were quantified by grayscale analysis using ImageJ/Fiji software, and the control group was set to 1.00. (D) Representative CLSM of intracellular hypoxia in cells treated with TBC or TBN for 24 h under hypoxia, with or without light irradiation. (E) pHi measurements in normoxic and hypoxic MDA-MB-231 cells following the indicated treatments (dark/light, 20 mW cm−2, 10 min). Data are presented as mean ± SD (n = 3). (F) Schematic illustration of TBC-mediated CAIX photodegradation, which amplifies the therapeutic effect through hypoxia alleviation and cellular acidosis.#

深入的细胞毒性机制研究表明，铁死亡抑制剂能够显著恢复TBC光照处理后的细胞活力，透射电镜观察也证实细胞出现了线粒体体积缩小、膜密度增加等典型的铁死亡形态学特征（如图4所示）。生化分析表明，TBC介导的光动力作用导致细胞内GPX4和xCT蛋白表达下调，还原型谷胱甘肽显著耗竭，丙二醛积累量大幅上升，证明细胞内积累了致命的脂质过氧化物。亚细胞定位追踪发现，TBC展现出独特的时空级联靶向特性，在孵育30分钟时优先定位于细胞膜，而在6至12小时内逐渐转移并富集于高尔基体与内质网（如图5所示）。光照引发的局部氧化应激导致了高尔基体碎裂以及内质网网状结构破坏，进而诱发了严重的内质网应激与钙离子释放，极大放大了脂质过氧化效应。转录组测序分析进一步证实，受TBC光动力处理影响的基因显著富集于内质网蛋白加工、谷胱甘肽代谢、钙信号通路及铁死亡等关键代谢路径（如图6所示）。

Fig.4 (A) Characterization of specific ROS produced in MDA-MB-231 cells upon TBC-mediated PDT. Total ROS levels after TBC + Light treatment were measured by CLSM in cells pre-incubated with the indicated ROS inhibitors. (B) Effects of various inhibitors on TBC-mediated photocytotoxicity. Cell viability was measured by MTT assay in MDA-MB-231 cells that were pre-incubated for 1 h with the indicated inhibitors and then treated with TBC + Light (2 µm, 20 mW cm−2, 10 min) (n = 3). (C) TEM images of TBC + Light-treated MDA-MB-231 cells under light irradiation. (D) Western blot analysis of GPX4, xCT, and GAPDH expression in MDA-MB-231 cells after treatment with TBC + Light. Impact of TBC on the ratios of GSSG/GSH (E) and MDA (F). All data are presented as mean ± SD (n = 3). Statistical significance was determined using one‑way analysis of variance (ANOVA). (G) Schematic illustration of TBC-mediated CAIX photodegradation inducing ferroptosis.#

Fig.5 (A) CLSM images of MDA-MB-231 cells collected at different times after TBC or TBN treatment (2 µm). TBC/TBN: _λ_ex = 450 nm, _λ_em = 660 ± 30 nm. Scale bar: 10 µm. (B) Colocalization of TBC with Golgi (left) and ER (right)#

Fig.6 RNA sequencing. (A–C) Gene ontology categorization of cellular component, molecular function and biological process for assembled unigenes of the transcriptome induced by TBC + Light treatment (2 µm, 20 mW cm−2, 10 min). (D) KEGG enrichment analysis of differentially expressed genes after TBC + Light treatment. (E) GSEA demonstrated significant enrichment of pathways involved in biological, metabolic, and immune functions in the TBC + Light-treated groups.probes in MDA‑MB‑231 cells (2 µm, 12 h). BODIPY TR ceramide/ER-Tracker Red: _λ_ex = 560 nm, _λ_em = 615 ± 20 nm. Scale bar: 5 µm. (C) TEM images of MDA-MB-231 cells after treatment with TBC + Light (2 µm, 20 mW cm−2, 10 min). (D) Western blot of GRASP55, GM130, p-IRE1, CHOP, and GAPDH expression in MDA-MB-231 cells after treatment with TBC + Light. (E) CLSM images of intracellular Ca2+ assays with Fluo-4 AM in MDA-MB-231 cells after treatment with TBC + Light. Scale bar: 20 µm. (F) Spatiotemporal cascade targeting of TBC: from cell membrane to Golgi/ER stress and ferroptosis.#

TBC诱导的细胞铁死亡同时展现出强烈的免疫原性。免疫荧光与生化检测显示，光照处理后的肿瘤细胞表面钙网织蛋白表达量剧增，高核黄素蛋白HMGB1从细胞核向细胞质迁移，并且细胞外ATP分泌量呈剂量依赖性增加（如图7所示）。在同种移植4T1乳腺癌小鼠模型中，TBC结合光照治疗展现出卓越的体内抑瘤效果，小鼠肿瘤生长抑制率高达84%，且未观察到明显的全身毒副作用或主要器官损伤。流式细胞术分析表明，该治疗策略显著促进了肿瘤组织中树突状细胞的成熟，成熟度从9.7%提升至42.4%，同时使辅助性CD4+ T细胞和杀伤性CD8+ T细胞的浸润比例分别大幅提升至69.2%与22.2%，成功激活了强效的抗肿瘤适应性免疫应答。

Fig.7 In vivo anti-tumor efficacy and anti-tumor immune response. (A) CLSM analysis of ecto-CRT and HMGB1 in MDA-MB-231 cells after treatment with TBC + Light (2 µm, 20 mW cm−2, 10 min). Scale bar: 20 µm. (B) ATP levels in cell culture supernatants after treatment with TBC + Light. Data are presented as mean ± SD (n = 3). Statistical significance was determined using one‑way analysis of variance (ANOVA). (C) The photographs of the tumor were collected at the end of the therapeutic period. (D) Volume changes of the tumors: (grey dot) Ctrl, (black dot) Ctrl + Light, (light red dot) TBC, (red dot) TBC + Light. Data are presented as mean ± SD (n = 5). Populations of CD80/CD86 (E), CD4+ (F) and CD8+ T cells (G) in tumors. Data are presented as mean ± SD (n = 3). Statistical significance was determined using an unpaired, two‑tailed Student’s t‑test. *p < 0.05, **p < 0.01, *p < 0.001.#