【Biomaterials】大连理工彭孝军院士团队|一种通过杂环硫代化和D-A增强作用制备的、稳定性导向的无重原子尼罗红光敏剂，用于铁细胞焦亡光动力免疫疗法#

Scheme 1. Design strategy of NRS-N-Me and its Ferro-Pyroptosis synergy mechanism.#

Fig. 1. Characterization of photophysical and photochemical properties of NRS-N-Me, NRS-N and NRS. a) Chemical structures of NRS-N-Me, NRS-N and NRS. b) Normalized absorption spectra of NRS-N-ME, NRS-N and NRS in dichloromethane. c) Normalized fluorescence emission spectra of NRS-N-ME, NRS-N and NRS in dichloromethane. d) The absorbance spectra of DPBF with NRS-N-Me following light irradiation (660 nm, 1 mW cm−2). e) The fluorescence emission spectra of DCFH with NRS-N-Me following light irradiation (660 nm, 10 mW cm−2). f) DPBF absorbance activation rates by NRS-N-Me, NRS-N and NRS under light irradiation (660 nm, 550 nm, 1 mW cm−2). g) DCFH (5 μM) fluorescence activation rates by NRS-N-Me (5 μM), NRS-N (5 μM) and NRS (5 μM) under light irradiation (660 nm, 10 mW cm−2). (h-j) Triplet excited-state lifetimes of NRS-N-Me, NRS-N and NRS in degassed DCM after different treatments.#

Fig. 2. The results of three molecular quantum chemical calculations. a) The optimized ground-state molecular structures of NRS-N-Me, NRS-N and NRS, as calculated by DFT (B3LYP/def2svp, in DCM), along with HOMO, LUMO, energy levels and energy differences. b) The Jablonski diagram of the excitation processes of NRS-N-Me, NRS-N and NRS-N (calculated by DFT, B3LYP/def2svp, with DCM as the solvent) is obtained.#

在细胞水平的实验中，研究者深入探讨了NRS-N-Me诱导细胞死亡的深层机制。如图2所示，通过特定的荧光探针监测发现，光照后的肿瘤细胞内出现了明显的脂质过氧化现象，这是铁死亡的典型标志。与此同时，透射电镜观察到细胞膜出现了大量气泡样突起，这一现象与焦死亡的特征完全吻合。进一步的分子生物学检测（如图3所示）显示，NRS-N-Me诱导了谷胱甘肽过氧化物酶4（GPX4）的下调以及焦死亡关键蛋白Gasdermin E（GSDME）的剪切。这意味着，该光敏剂并非简单地诱导细胞凋亡，而是通过触发铁死亡与焦死亡的“双管齐下”，最大程度地释放肿瘤细胞内的损伤相关分子模式（DAMPs）。

Fig. 3. Evaluation of NRS-N-Me-mediated PDT efficacy in cells. a-b) Confocal images demonstrate the intracellular ROS and •O2− levels in MCF-7 cells as detected by DCFH-DA and DHE probes. Scale bar: 30 μm. c) CLSM images of MCF-7 cells stained with Calcein-AM/PI after various treatments Scale bars: 150 μm. d) CLSM images of MMP damage in JC-1 stained MCF-7 cells after different treatments. Scale bar: 30 μm. e) Cell viability of 4T1, MCF-7 and mc38 cells treated with NRS-N-Me, NRS-N and NRS, with and without light irradiation (660 nm, 20 mW cm−2, 20 min, n = 6). f) Analysis of apoptosis and necrosis in 4T1 cells stained with Annexin V-FITC/PI after different treatments via flow cytometry.#

Fig. 4. Detection of multiple cell death pathways mediated by NRS-N-Me-mediated PDT in 4T1 cells. a) CLSM images of LPO in BDPY 581/591 C11 stained 4T1 cells after different treatments. Scale bar: 50 μm. b) CLSM images of Fe2+ stained 4T1 cells after different treatments. Scale bar: 30 μm. Immunofluorescence staining CLSM images of c) HMGB1 and d) CRT in 4T1 cells after coincubation with NRS-N-Me under light. Scale bar: 30 μm. e) The release of LDH from 4T1 cells after different treatments (n = 3). f) The release of ATP from 4T1 cells after different treatments (n = 3). g) The relative expression of NADH in 4T1 cells after different treatments (n = 3). The release of h) IL-1β and i) IL-18 from 4T1 cells after different treatments (n = 3). j) The relative expression of LPO in 4T1 cells after different treatments. k) The relative expression of FE2+ in 4T1 cells after different treatments. l) The WB analysis of FSP1, xCT, GPX4, Pro-caspase 1, N-GSDMD and GSDMD in 4T1 cells following various treatments (660 nm, 20 mW cm−2, 10 min). m) Schematic illustration of NRS-N-Me-mediated PDT targeting mitochondria, inducing pyroptosis, ferroptosis, and immunogenic cell death. Statistical significance was calculated using two-tailed Student’s t-test (∗p < 0.05, ∗∗p ≤ 0.01, or ∗∗∗p ≤ 0.001).#

为了验证该策略在复杂生物体内的实际应用价值，研究团队开展了荷瘤小鼠实验。如图4所示，在接种肿瘤后，仅通过静脉注射低剂量的NRS-N-Me并配合近红外光照射，肿瘤生长便受到了极大的抑制，肿瘤体积相较于对照组缩小了显著比例。更令人兴奋的是，在肿瘤组织切片中观察到了大量浸润的CD8+ T细胞。流式细胞术分析结果（如图5所示）进一步证实，治疗后的小鼠不仅原位肿瘤得到了清除，其体内的树突状细胞成熟度也显著提高。这一系列发现表明，NRS-N-Me驱动的光动力过程成功点燃了机体的抗肿瘤免疫反应，将“冷”肿瘤转化为了对免疫治疗敏感的“热”肿瘤。

Fig. 5. In vivo antitumor PDT evaluation. a) Schematic overview of the 4T1 syngeneic breast cancer model: tumor inoculation, growth monitoring, and therapeutic intervention protocol. b) Time-course in vivo fluorescence imaging of NRS-N-Me distribution in mice post-injection. c) Quantitative analysis of tumor-associated fluorescence signal intensity (mean ± SD, n = 3). d) Photographs of surgically resected breast tumor specimens following distinct therapeutic regimens. e) Tumor volume growth curves of mice after different therapeutic regimens over 14 days (n = 5). f) The weight of tumor after different therapeutic regimens over 14 days (n = 5). g) Relative expression of TUNEL after different therapeutic regiments. h) Immunofluorescence staining images of TUNEL and Ki67 staining from tumor tissues in different therapeutic regimens. Scale bars: 50 μm. i) Relative expression of Ki67 after different therapeutic regiments. Statistical significance was calculated using two-tailed Student’s t-test (∗p < 0.05, ∗∗p ≤ 0.01, or ∗∗∗p ≤ 0.001).#

Fig. 6. Evaluation of NRS-N-Me-mediated PDT performance in lung metastasis model in vivo a) Orthotopic breast cancer-lung metastasis model establishment & therapy regimen diagram. b) Tumor volume growth curves of mice after different therapeutic regimens over 14 days (n = 5). c) The weight changes of mice after different treatment regimens within 14 days (n = 5). d) H&E staining of tumor tissues from different therapeutic regiments. Scale bars: 50 μm. e) Typical images of excised lung tissues across various treatment groups. Yellow arrows denote metastatic foci. f–i) Quantification of HGF, MTA2, PECAM-1 and VCAM-1 expression levels in Immunofluorescence staining of tumor slices (n = 3). j) H&E staining of lung tissues after various therapeutic regiments. Scale bars: 2000 μm (Conventional graph) 100 μm (enlarged graph). Statistical significance was calculated using two-tailed Student’s t-test (∗p < 0.05, ∗∗p ≤ 0.01, or ∗∗∗p ≤ 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)#

Fig. 7. Evaluation of the in vivo immune effect of NRS-N-Me-mediated photodynamic therapy. Flow cytometry assay of a) matured DCs (CD11c+CD80+CD86+), c) helper T cells (CD45+CD3+CD4+), and e) cytotoxic T cells (CD45+CD3+CD8+) in the tumors after various therapeutic regiments. Quantitative analysis of b) matured DCs (CD11c+CD80+CD86+), d) helper T cells (CD45+CD3+CD4+), and f) cytotoxic T cells (CD45+CD3+CD8+) after various therapeutic regiments (n = 3). g) Immunofluorescence staining images of CRT and HHMGB1 staining from tumor tissues in different therapeutic regimens. Scale bars: 50 μm. Quantitative analysis of h) CRT and i) HMGB1 after various therapeutic regiments (n = 3). Statistical significance was calculated using two-tailed Student’s t-test (∗p < 0.05, ∗∗p ≤ 0.01, or ∗∗∗p ≤ 0.001).#