Fig.1 Synthetic characterization, photophysical properties, and ROS generation capability. (A) Synthetic scheme of mitoTPS. (B) Chemical stability assessment of mitoTPS and PS with corresponding DFT-calculated HOMO and LUMO energy levels. (C) UV–vis absorption spectra of mitoTPS and PS in aqueous (H2O) and organic environments (DMSO). (D) Time-dependent decay of DPBF absorbance upon irradiation in aqueous mitoTPS solution. Inset: Normalized absorbance of DPBF at 415 nm in the solutions of mitoTPS and MB under LED irradiation. (E) ESR spectra of mitoTPS in aqueous solution using TEMP and DMPO as spin traps under irradiation. (Irradiation conditions: 660 nm, 50.0 mW cm−2).#

Fig.2 In vitro mitochondrial targeting, phototoxicity, and induction of GSDME-mediated pyroptosis. (A) Representative images showing the distribution of mitoTPS (red) in the organelles (green) of B16F10 cells (Scale bar = 15.0 µm). (B) Evaluation of intracellular ROS generation via DCF fluorescence in B16F10 cells treated with mitoTPS (5.0 µM) with or without irradiation (Scale bar = 40.0 µm). (C) Mitochondrial membrane potential assessment using JC-1 staining (Scale bar = 100.0 µm). (D) Cell viability assays of B16F10 cells under normoxic and hypoxic conditions. (E) Representative flow cytometric plots evaluating apoptotic cell profiles (n = 3 per group). (F) Confocal images of DiO-labeled cell membranes showing characteristic pyroptotic morphological changes (Scale bar = 15.0 µm). (G) Confocal visualization and quantitative analysis of cytochrome c released from mitochondria into the cytosol (Scale bar = 25.0 µm; n = 3 per group). (H,I) Western blot analysis and corresponding quantification of cleaved caspase-3 and GSDME expression (n = 3 per group). (J,K) Immunofluorescence imaging of DAMP expression, including CRT surface exposure and HMGB1 nuclear-to-cytoplasmic translocation (Scale bar = 10.0 µm). (L) Quantification of extracellular ATP secretion (n = 3 per group). (M) Schematic illustration of the mitoTPS-induced signaling cascade leading from mitochondrial oxidative stress to GSDME-mediated pyroptosis. (Irradiation conditions: 660 nm, 50.0 mW cm−2, 10 min).#

在放疗协同机制方面，研究证实mitoTPS光动力预处理能将肿瘤细胞同步阻滞于高辐射敏感的G2/M期，与后续X射线协同使胞内活性氧激增12.5倍，同时抑制了DNA同源重组与非同源末端连接修复通路，导致不可逆的DNA双链断裂积累与克隆存活率骤降（如图3）。基因敲低及抑制剂实验进一步揭示，GSDME不仅负责执行焦亡和释放炎性因子，还与活性氧构成了相互放大的正反馈循环，是维持高效放疗增敏的必要中枢（如图4）。为实现体内高效递送，团队将mitoTPS载入快速溶解的透明质酸微针阵列，微针展现出优异的穿刺力学性能，可在入肤后迅速溶解并将药物深度递送至肿瘤纵深400至500微米处，原位药物蓄积率高达75.1% ID/g（如图5）。

Fig.3 Mitochondrial ROS amplification and cell cycle synchronization for potentiated radiotherapy. (A) Representative flow cytometric histograms of DCF fluorescence of B16F10 cells with mitoTPS upon LED irradiation or x-ray exposure (n = 3 per group). (B,C) Representative flow cytometry plots and quantitative analysis of cell cycle distributions (n = 3 per group). (D,E) Immunofluorescence imaging and corresponding quantification of γ-H2AX foci (Scale bar = 10.0 µm; n = 3 per group). (F) Clonogenic survival curves of B16F10 cells subjected to increasing radiation doses (0.0–8.0 Gy; n = 3 per group). (G) Colony-forming unit assay was carried out, and (H) Representative images and quantitative analysis of the colony-forming unit assay (n = 3 per group). (I) Schematic illustration of the ROS amplification for enhanced radiotherapy. (Irradiation: 660 nm, 50.0 mW cm−2, 10 min; x-ray exposure: 6.0 Gy).#

Fig.4 Mechanistic validation of GSDME-dependent pyroptosis in dictating the ROS amplification loop, immunogenic cell death, and the ensuing radiosensitization cascade. (A) Representative immunoblots of GSDME cleavage in B16F10 cells pretreated with a pharmacological inhibitor (DMF) or subjected to siRNA-mediated GSDME knockdown (KD), followed by the indicated treatments. (B) The expression of FL-GSDME and N-GSDME was measured by Western blotting (n = 3 per group). (C) Effects of mitoTPS on LDH release in B16F10 cells as determined by the LDH release assay (n = 3 per group). (D-G) The cell supernatant of IL-6, CXCL1, CCL2, and CCL5 measured by ELISA (n = 3 per group). (H) Representative flow cytometric histograms of DCF fluorescence of B16F10 cells pretreated with inhibitors and GSDME knockdown as indicated, followed by the indicated treatments (n = 3 per group). (I) Clonogenic survival assay of B16F10 cells treated with different formulations under a series of radiation doses. (J) The corresponding surviving fraction of mitoTPS/hv treated B16F10 cells after exposure to x-ray of varied doses (n = 3 per group). (K) γ-H2AX immunofluorescence staining of B16F10 cells treated with different treatments (Scale bar = 30.0 µm). (L) The corresponding quantification of γ-H2AX foci (n = 3 per group).#

Fig.5 Fabrication and physicochemical characterization of dissolving microneedles. (A) Schematic illustration of the fabrication process for the fast-dissolving HA microneedle patches. (B,C) Brightfield and SEM images of the mitoTPS-MNs array (Scale bar = 200.0 µm in B and 500.0 µm in C). (D) Representative fluorescence microscopy image of mitoTPS-MNs patches (Scale bar = 200.0 µm). (E) Mechanical compression behavior of mitoTPS-MNs (Scale bar = 200.0 µm). (F,G) Quantitative cumulative release profiles and real-time optical microscopic images showing the rapid dissolution of the HA matrix and subsequent release of mitoTPS (Scale bar = 500.0 µm). (H) Representative photograph of the mouse tumor surface 60 min post-insertion, showing the formation of microchannels and the successful delivery of the therapeutic payload into the tumor tissue (Scale bar = 200.0 µm). (I) Depth-dependent quantification of mitoTPS release percentages, demonstrating efficient cargo distribution across the longitudinal axis of the tumor tissue.#

在黑色素瘤荷瘤小鼠模型中，微针光动力联合放疗实现了98.1%的原发肿瘤生长抑制，使60%的小鼠维持长期无瘤存活，且未见任何器官损伤或血液生化异常（如图6、图7）。在双侧肿瘤转移模型中，仅对单侧原发灶进行局部微针免疫放疗，便显著促进了引流淋巴结内树突状细胞的成熟（达71.6%），大幅提升了原发及未受照远端肿瘤内CD8+细胞毒性T细胞的浸润与炎性趋化因子的表达，成功抑制了未照射远端转移瘤的生长（如图8）。小鼠体内CD8+ T细胞清除实验最终确证，该远端抑瘤效应完全由系统性适应性抗肿瘤免疫介导，证实了该系统作为原位肿瘤疫苗的强大全身免疫保护能力。

Fig.6 In vivo antitumor efficacy, survival analysis, and biosafety evaluation. (A) Schematic illustration of the experimental timeline for the treatment of B16F10 melanoma-bearing C57BL/6 mice. (B) Average tumor volume growth curves across various treatment groups (n = 5 per group). (C–G) Individual tumor growth kinetics for each treatment group. (H) Relative survival curves from the mice treated with various groups (n = 5 per group). (I) Changes in body weight profile (n = 5 per group). (J) Serum biochemical analysis 24 h posttreatment, including markers for ALT, AST, ALP, and urea (n = 3 per group). (K) Representative H&E staining and immunofluorescence images of tumor sections, indicating intratumoral ROS generation (DCFH-DA; Sclale bar = 200.0 µm) and DNA fragmentation (TUNEL; Scale bar = 100.0 µm).#

Fig.7 In vivo synergistic radiosensitization and mechanistic validation. (A) Schematic illustration of the sequential treatment protocol for B16F10 melanoma-bearing mice (n = 5 per group). (B) Average tumor growth curves of the B16F10 tumor-bearing C57BL/6 mice after various treatments (n = 5 per group). (C–H) Individual tumor growth kinetics for each treatment group. (I) Relative survival curves from the mice treated with various groups. (J) Changes in body weight profile (n = 5 per group). (K) ROS analysis of tumor tissues by DCFH-DA in the different treatments and quantitative analysis (Scale bar = 200.0 µm; n = 3 per group). (L) Flow cytometric analysis of intracellular ROS levels by detecting DCF fluorescence in tumor tissues treated with mitoTPS-MNs upon LED irradiation (50.0 mW cm−2, 35 min) or x-ray exposure (6.0 Gy; n = 5 per group).#

Fig.8 Induction of systemic antitumor immunity and evaluation of the abscopal effect. (A) Proportion of infiltrated DC maturation in lymph nodes after various treatments analyzed by flow cytometry. (B) Proportion of infiltrated CD8+ T cells and CD4+ T cells in tumors after various treatments analyzed by flow cytometry. (C) Quantitative analysis of DC maturation (n = 5 per group). (D) Quantitative analysis of CD8+/CD4+ T cells (n = 5 per group). (E) Quantitative analysis of CD3+CD8+ killer T cells (n = 5 per group). (F) Regulation of immune cells by mitoTPS-MNs-induced pyroptosis through ROS. (G) Schematic illustration of the construction of bilateral in C57BL/6 mice with subcutaneously implanted B16F10 cells and in vivo therapy process (n = 5 per group). (H, I) Average growth kinetics of treated primary tumors and untreated distant tumors. (J,K) Quantitative assessment of primary and distant tumor weights at the study endpoint. (L) Changes in body weight profile (n = 5 per group).#