【Angew.Chem.】北师大杨清正、滕坤旭|破译细胞死亡新密码：首例双靶向光动力制剂，同步激活Caspase-3/GSDME轴与心磷脂外翻实现16倍高效细胞焦亡#

(a) The molecular structure of photosensitizers. (b) Schematic illustration of assembly induced ROS generation. (c) Schematic diagram of the mechanism of pyroptosis triggered by photogenerated ROS through dual activation of the Caspase-3/GSDME pathway and cardiolipin externalization.#

(a) Absorption spectra of 1 in DMF/water mixtures with different water fractions (f water, vol %) from 0% to 100%. (b) Normalized absorption and emission of 1 in the DMF and 1a in water, respectively. (c) Crystal-packing pattern of compound 1. H atoms have been omitted for clarity. Color code: C, pale gray; N, blue; B, yellow; O, red; F, green. Absorption spectra of ABDA (12 µM) after irradiation (white light, 30 mW cm−2) for increasing irradiation times in the presence of (d) 1 in DMF and (e) 1a in water. (f) Time-dependent plots of ΔAbs (A0−A) of ABDA at 379 nm under light irradiation (white light, 30 mW cm−2) in the presence of compound 1 in DMF/water mixtures with different water fractions (_f_w, 0–100 vol%). (g) ESR spectra to detect 1O2 generated by 1a (10 µM) under illumination (white light, 100 mW cm−2), using TEMP (25 mM) as a spin trap agent.#

在微观形貌与生化水平的表征中，研究人员捕捉到了细胞焦亡的特征性变化。接受光照处理后的肿瘤细胞表现出明显的细胞溶胀、泡沫状外翻以及质膜破裂，并伴随着大量乳酸脱氢酶的释放，这证实了非凋亡性的裂解死亡形式。印迹杂交定量分析结果显示，光照激活后细胞内的剪切态Caspase-3水平较对照组骤增了接近八倍，同时胞质内的细胞色素c水平显著升高。更重要的是，在利用特定药物阻断下游剪切体生成的前提下，流动式细胞术和特异性抗体染色证实，光敏剂产生的活性氧能够直接驱动线粒体表面的心磷脂发生高达16倍的外翻，使表面心磷脂阳性线粒体的比例飙升至百分之七十四。通过钙黄绿素钴离子淬灭实验可以观察到，外翻的心磷脂与转位至线粒体膜上的气孔蛋白片段结合，迅速破坏了线粒体纵深结构的完整性，形成了跨膜孔道并加速了促焦亡因子的释放，构建起一条不可逆的细胞死亡正反馈信号链。

(a) Co-localization imaging of HepG 2 cells stained with MitoTracker Green (500 nm) and 1a@DSPE-PEG (10 µM) at 37°C. Scale bar: 20 µm. (b) The linear analysis of the selected regions in (a). (c) Time-dependent change in the fluorescence of mitochondrial-localized PS and Hoechst 3342 before and after the application of FCCP. Images were acquired at 0-, 3-, and 10-min post-addition. Scale bar: 20 µm. (d) The linear analysis of selected regions in (c). (e) ROS generation of the PS in HepG 2 with DCFH-DA measured under light irradiation (660 nm, 30 mW cm−2). Scale bar, 50 µm. (f) Colocalization imaging of the PS-stained cells with BODIPY 581/591 C11 after 10 min light irradiation (660 nm, 30 mW cm−2) or dark incubation. Enhanced green fluorescence indicates lipid peroxidation. Scale bar, 50 µm.#

(a) Cell viability of HepG 2 cells incubated with various concentrations of 1a@DSPE-PEG (mean ± SD, n = 5). (b) Real-time confocal imaging of HepG 2 Cells under continuous irradiation stained with 1a@DSPE-PEG. The cells marked with red arrows show possible pyroptotic morphology with bubbling. Scale bar: 20 µm. (c) The LDH release of cells co-incubated with 1a@DSPE-PEG before and after irradiation. ns > 0.05, **** p < 0.0001. (d) Western blot analysis of protein expression in the pyroptosis in HepG 2 cells under different treatments. Uncropped blots are shown in Figure S33. (e) Cells were untreated (UNT) or treated with ethidium bromide (EB) for 20 days. Scale bar: 20 µm. (f) Western blot analysis of protein expression in the pyroptosis in HepG 2 cells under different treatments. Uncropped blots are shown in Supplementary Figure S34.#

为了验证该体系在生物体内的抗肿瘤实际转化价值，研究团队在小鼠皮下乳腺癌肿瘤模型中开展了体内光动力治疗评价。全身荧光成像结果表明，经静脉注射后的纳米制剂能够在肿瘤部位实现长达数小时的高效富集与持久留存。在连续二十天的疗效追踪中，接受低剂量制剂注射并配合红光照射的治疗组小鼠，其肿瘤生长受到了极大的抑制，肿瘤抑制率达到了百分之七十三点二。与此同时，单纯注射制剂而不给予光照的对照组小鼠，其肿瘤生长轨迹以及动物整体体重变化与空白组基本一致，这充分证明了该超分子纳米材料具有极低的体内暗毒性以及优异的系统耐受性，通过同步双靶向机制在体内成功驱动了强效的光动力抗肿瘤焦亡效应。

(a) Confocal fluorescence images and (b) TEM images of 1a@DSPE-PEG treated cells before and after light irradiation. The higher-magnification images (top row) correspond to the boxed regions shown in the lower-magnification panels directly below. Black arrows indicate normal mitochondria; yellow arrows, mitochondria with loss of cristae or membrane damage; red arrow, damaged mitochondria within an autophagosome. Scale bar: 1 µm. (c) Flow cytometry gating strategy for isolated mitochondria and (d) Flow cytometry histograms of APC-anti-cardiolipin stained mitochondria. (e) Schematic illustration of principle of mitochondrial permeability detection: Calcein AM readily enters live cells and is hydrolyzed by intracellular esterases into Calcein, which emits intense green fluorescence throughout the cytoplasm and mitochondria. Treatment with cobalt chloride (CoCl2) provides Co2+ ions that quench the fluorescence of Calcein in the cytoplasm. Co2+ cannot cross the intact mitochondrial membrane, so the fluorescence within mitochondria remains visible. Upon pore formation by GSDME-NT on the mitochondrial membrane, its integrity is compromised, allowing Co2+ to enter the mitochondrial matrix and quench the intra-mitochondrial Calcein fluorescence, thereby reporting increased permeability. (f) Calcein green fluorescence in 1a@DSPE-PEG treated cells without or with DEVD, before and at indicated time points after irradiation. Scale bar: 10 µm.#

(a) Treatment schedule for evaluation of the antitumor efficacy induced by 1a@DSPE-PEG mediated PDT in 4T1 tumor bearing mice. (b) In vivo fluorescence imaging of the 4T1 tumor-bearing mice at 1, 3, 5, 7, 10, and 15 h after intravenous injection of 1a@DSPE-PEG. Red circle indicates the tumor sites. (c) Tumor growth profiles during the observation. (d) Body weight changes in different groups. (e) Representative photographs of tumor tissues obtained on day 20. (f) Average tumor weights of mice at the end of different treatments. light source: 660 nm, 0.1 W cm−2, 15 min irradiation with three replicates. Statistical significance was assessed via unpaired two-sided Student t-test, ***p < 0.001.#