Type I Photoreaction and Photoinduced Ferroptosis by a Ru(II) Complex to Overcome Tumor Hypoxia in Photodynamic Therapy

{"full_text": " RESEARCH ARTICLE\n\n Received: Apr. 24, 2022 | Accepted: July 15, 2022 | Published: Aug. 8, 2022\n\n\n\n\nType I Photoreaction and\nPhotoinduced Ferroptosis by a Ru(II)\nComplex to Overcome Tumor\nHypoxia in Photodynamic Therapy\nFen Qi1, Hao Yuan1,2, Yuncong Chen1,2*, Xin-Xin Peng1, Yanping Wu1, Weijiang He1* & Zijian Guo1,2*\n1\n State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and\nBiomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing 210023, Jiangsu, 2Nanchuang (Jiangsu)\nInstitute of Chemistry and Health, Nanjing 210000, Jiangsu\n\n*Corresponding authors: chenyc@nju.edu.cn; heweij69@nju.edu.cn; zguo@nju.edu.cn\n\nCite this: CCS Chem. 2023, 5, 1583\u20131591\nDOI: 10.31635/ccschem.022.202202074\n\n\nPhotodynamic therapy (PDT) usually shows limited develops a novel ferroptosis-inducing Ru(II) complex\nef\ufb01cacy in solid tumors since traditional PDT is O2- with the type I PDT process but also offers an effective\ndependent while solid tumors are inherently hypoxic. strategy to solve tumor hypoxia in PDT.\nIn addition, hypoxic tumor cells possess antiapoptotic\npathways that resist PDT-induced apoptosis. There-\nfore, developing photosensitizers (PSs) that show low\nO2-dependency and can induce nonapoptotic cell\ndeath pathways is critically needed. Herein, a series of\nRu(II) polypyridine complex-based PSs, RuNMe, RuH,\nand RuCN, were synthesized, and their applications\nagainst hypoxic tumor cells through PDT were investi-\ngated. All three complexes show the ability to generate\nthe superoxide anion radical (\u00b7O2\u2212), which is the type I\nphotoreaction and less O2-dependent. RuNMe shows\nthe best PDT performance against MCF-7 cells and\nthree-dimensional multicellular spheroids, due to its\nhigher cellular uptake and more reactive oxygen spe-\ncies generation. More importantly, RuNMe-incubated\nMCF-7 cells show photoinduced ferroptosis as evi- Keywords: ferroptosis, photodynamic therapy, O2-\ndenced by glutathione peroxidase 4 downregulation independent, type I photoreaction, superoxide an-\nand lipid peroxide accumulation. This work not only ion radical (\u00b7O2\u2212)\n\n\n\n\n systemic toxicity and minimal invasiveness.1,2 Generally,\nIntroduction photosensitizers (PSs) are injected and accumulated in\nIn the past few decades, photodynamic therapy (PDT) tumor tissues. Light irradiation on the tumor site then\nhas become a promising method for the treatment of produces cytotoxic reactive oxygen species (ROS), lead-\nvarious diseases, especially tumors, due to its low ing to suppression of the malignant tumors while\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1583\n\f RESEARCH ARTICLE\n\n\n\n\npreserving the surrounding normal tissues.3\u20135 PDT is main-\nly divided into type I (production of superoxide anion\nradicals, hydroxyl radicals, and hydrogen peroxide) and\ntype II (production of 1O2) photoreactions.6\u20138 Most of the\ncurrent reports on the PDT process are based on the type\nII photoreaction, which is highly dependent on the O2\nconcentration.7\u20139 However, hypoxia is a typical feature of\nthe solid tumor microenvironment.10\u201312 PDT-mediated O2\nconsumption further exacerbates tumor hypoxia and ul-\ntimately leads to unsatisfactory therapeutic effects.13,14 In\nthis regard, type I PSs are more favorable since they show\nless O2 dependence.15,16 On the other hand, PDT triggers\nvarious cellular responses and induces cell death through\nnecrosis and/or apoptosis.17,18 However, hypoxia will in-\nduce cellular adaptations,19 and one of these adaptations\nis the expression of multidrug resistance proteins (ABC\ntransporters) that make cells resistant to apoptosis.20\nTherefore, there is an urgent need to develop PSs that\nshow minimum O2-dependence and induce nonapoptotic\ncell death pathways to improve the therapeutic effect\nagainst hypoxic tumor cells.\n Ferroptosis is an iron-dependent, nonapoptotic form\nof cell death characterized by cytological changes, such\n Figure 1 | (a) Chemical structures of Ru(II) complexes.\nas decreased or vanished mitochondria cristae,21\u201323 and it\n (b) The schematic illustration of the photoinduced ferrop-\nhas been recognized as a novel strategy for anticancer\n tosis mechanism by RuNMe upon irradiation.\ntherapy.24,25 ROS generated by PSs will cause rapid glu-\ntathione (GSH) consumption, resulting in suppression of\nglutathione peroxidase 4 (GPX4) activity and accumula- RuNMe showed distinct mitochondrial atrophy, GSH con-\ntion of lipid peroxides (LPOs), \ufb01nally leading to ferrop- sumption, GPX4 downregulation, and LPO accumulation\ntosis.26 Metal complex-based PSs have shown their that can be reversed by cotreatment of ferrostatin-1\npotential to induce ferroptosis to effectively deplete (Fer-1, a ferroptosis inhibitor), suggesting typical ferrop-\nhypoxic tumor cells. Guo et al. reported a series of Ir(III) tosis in a GPX4-dependent manner (Figure 1b). This work\ncompounds as PSs that can generate superoxide anion demonstrates that Ru(II) complex-based PSs can induce\nradicals (\u00b7O2\u2212) and hydroxyl radicals (\u00b7OH) to overcome ferroptosis, providing a new and reliable method for\ntumor hypoxia and induce ferroptosis in cells.27 Ru(II) effective PDT to overcome tumor hypoxia.\npolypyridine complexes have also been widely used in\nPDT due to their excellent chemical and photophysical Experimental Methods\nproperties such as high-water solubility, high ROS gen-\neration, chemical stability, and photostability.28\u201339 The Details of the materials and instruments utilized are pro-\nruthenium polypyridine complex TLD-1433 has entered vided in the Supporting Information.\na phase II clinical trial for the treatment of nonmuscle\ninvasive bladder cancer.40,41 However, most of the Ru(II) Preparation of Ru complexes\ncomplexes cause apoptosis via photochemical process- The synthetic routes of Ru(II) compounds are shown in\nes. Ru(II)-based PSs with ferroptosis-inducing ability Supporting Information Scheme S1. Intermediate com-\nhave not been reported. pounds were synthesized according to the reported pro-\n In this study, three Ru(II) polypyridine complexes cedures,28 and the Ru(II) compounds RuNMe, RuH, and\n(RuNMe, RuH, and RuCN) were successfully synthesized RuCN (Figure 1a) have been successfully characterized\nas novel PSs for PDT against hypoxic tumor cells by nuclear magnetic resonance (NMR) spectroscopy and\n(Figure 1a). All the Ru(II) complexes showed a high quan- high-resolution mass spectrometry (HRMS) (Supporting\ntum yield of singlet oxygen and superoxide anion radical Information Figures S1\u2013S10).\n(\u00b7O2\u2212) upon light irradiation, suggesting the coexistence\nof both type I and type II PDT processes. Among them,\nRuNMe showed the best tumor cell inhibition effect due\n Cell culture\nto its higher cell uptake compared to that of RuH and MCF-7 cells were cultured in RPMI-1640 media. 1640\nRuCN. Upon light irradiation, MCF-7 cells cultured with media were supplemented with fetal bovine serum\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1584\n\f RESEARCH ARTICLE\n\n\n\n\n(10%, v/v), penicillin (100 units/mL), and streptomycin absorption peak of the o-phenanthroline ligand. The\n(50 units/mL) at 37 \u00b0C in a CO2 incubator (5% CO2). The broad emission band centered at 602 nm was MLCT\nexperiment was divided into the following four groups: emission. As can be seen from the emission spectra of\n three complexes in different solvents, the \ufb02uorescence\n 1. Dark + Normoxia group. The cells were incubated intensity increased with increasing solvent polarity\n with Ru(II) complex in the dark. (Supporting Information Figure S12). Additionally, due\n 2. Dark + Hypoxia group. The cells were sealed in an to the strong electron-donating properties of N,N-di-\n anaerobic airbag for 1 h after 3 h of incubation with methyl, the complex RuNMe exhibited the photoinduced\n Ru(II) complex in the dark. Cells were taken out of electron transfer effect, which caused its \ufb02uorescence\n the anaerobic airbag and put back in the normal to be signi\ufb01cantly quenched (Supporting Information\n incubator after 1 h. Oxygen concentration was below Figure S12).\n 0.1% during irradiation. The singlet oxygen (1O2) quantum yield is one of the\n 3. Light + Normoxia group. The cells were exposed to decisive factors for the PDT effect. Consequently, the\n photoirradiation (white light 30 min) after 4 h of dark 1\n O2 quantum yields of the Ru(II) compounds were in-\n incubation with Ru(II) complex and then taken back vestigated with Ru(bpy)3 as the standard sample. As\n into the incubator. Oxygen concentration was 21%. shown in Supporting Information Figure S13 and Table\n 4. Light + Hypoxia group. The cells were sealed in an S1, compounds RuH and RuNMe had high singlet oxygen\n anaerobic airbag 1 h before irradiation (white light\n quantum yields (0.55 for RuNMe, 0.65 for RuH, and 0.32\n 30 min) to create a hypoxic condition after 3 h\n for RuCN, respectively), indicating good potential for\n of incubation with Ru(II) complex in the dark, and\n effective PDT.\n the irradiation was carried out in the anaerobic\n airbag. Cells were taken out of the anaerobic airbag\n and put back into a normal incubator after irradia- Photoinduced anticancer activity of Ru(II)\n tion. Oxygen concentration was below 0.1% during complexes\n irradiation.\n Then the photocytotoxicities of RuNMe, RuH, and RuCN\n against MCF-7 cells were determined (Table 1). The IC50\nResults and Discussion values of RuH determined under normoxia and hypoxia\n upon light irradiation were 2.3 \u00b1 0.5 and 11.1 \u00b1 2.3 \u03bcM\nPhotophysical properties while the IC50 values for incubation under dark condi-\nThe photophysical properties of RuNMe, RuH, and RuCN tions were 168.0 \u00b1 5.6 and 146.2 \u00b1 10.3 \u03bcM, respectively.\nwere investigated as shown in Supporting Information The photocytotoxicity index (PI) values of RuH were\nFigures S11 and S12 and Table S1. There is no signi\ufb01cant determined to be 73 and 13 under normoxia and hypoxia\ndifference in the maximum absorption wavelength with by calculating the ratio of (IC50)Dark/(IC50)Light. For\ndifferent solvents (Supporting Information Figure S11). RuCN, the (IC50)Light values were 4.4 \u00b1 0.6 and\nThe absorption at 400\u2013500 nm in the visible light region 14.9 \u00b1 0.9 \u03bcM under normoxia and hypoxia, respectively.\nis attributed to the characteristic absorption of metal- The PI values were 42 and 12 under normoxia and hyp-\nto-ligand charge transfer (MLCT) of the Ru(II) complex, oxia, respectively (Table 1). The (IC50)Light values of\nwhich is the transition of d\u03c0-\u03c0* (Ru(II) to N, N ligand). The RuNMe under normoxia and hypoxia (0.4 \u00b1 0.1 and\nbroad and strong absorption at 450 nm and the shoulder 1.5 \u00b1 0.3 \u03bcM, respectively) were much lower than that\npeak that appeared at 420 nm can be attributed to of RuH and RuCN, concomitant with PIs of 244 and 58\nthe superposition of the absorption of the charge under normoxia and hypoxia. The above data show that\ntransfer state (MLCT) between metal Ru and different the PDT effect of RuNMe was much more ef\ufb01cient than\nligands, namely the d\u03c0 (Ru)-\u03c0*(bpy) (2, 2\u2032-bipyridine) that of RuH and RuCN. Meanwhile, the PIs of RuNMe\nand d\u03c0 (Ru)-\u03c0* (phenanthroline ligand) superposition. under normoxia and hypoxia were about 3\u20135 times\nThe absorption band of the complex at 330 nm was the higher than those of RuH and RuCN.\n\n\n Table 1 | IC50 (\u03bcM) of Ru(II) Complexes against MCF-7 Cell Line (24 h)\n\n Dark Light\n Complexes Normoxia Hypoxia Normoxia (PI) Hypoxia (PI)\n\n RuNMe 97.4 \u00b1 9.6 87.7 \u00b1 7.9 0.4 \u00b1 0.1 (244) 1.5 \u00b1 0.3 (58)\n RuH 168.0 \u00b1 5.6 146.2 \u00b1 10.3 2.3 \u00b1 0.5 (73) 11.1 \u00b1 2.3 (13)\n RuCN 185.4 \u00b1 19.4 183.9 \u00b1 11.7 4.4 \u00b1 0.6 (42) 14.9 \u00b1 0.9 (12)\n Note: PI: photocytotoxicity index, the ratio of (IC50)Dark/(IC50)Light.\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1585\n\f RESEARCH ARTICLE\n\n\n\n\n The cellular uptake of RuNMe, RuH, and RuCN was contribute to the differences in cellular uptake and cyto-\ndetermined by quanti\ufb01cation of Ru concentration in toxic activity.\nMCF-7 cells via inductively coupled plasma mass spec-\ntrometry (ICP-MS; Supporting Information Figure S14). Photoinduced reactive oxygen species\nThe results showed that the content of Ru in the cells generation in MCF-7 cells\nincubated with RuNMe was higher than that in the cells\n Photoinduced intracellular ROS generation by Ru(II)\nincubated with RuH and RuCN, both under normoxia and\n complexes was investigated in MCF-7 cells. First, the\nhypoxia. Therefore, the different cytotoxicities of differ- 1\n O2 generation was carefully examined with singlet oxy-\nent complexes may be related to the different uptake of\n gen sensor green (SOSG) as the probe. A signi\ufb01cant\nthe three compounds by the cells. The octanol/water\n increase in \ufb02uorescence under normoxia was observed\npartition coef\ufb01cients (logP) of RuNMe, RuH, and RuCN\n after photoirradiation of RuNMe, RuH, and RuCN-\nare \u22120.24, \u22121.30, and \u22121.50 (Supporting Information Table\n incubated MCF-7 cells by confocal imaging, indicating\nS1), respectively. This data demonstrate that the lipophi-\n the formation of 1O2 (Figure 2a). The \ufb02uorescence inside\nlicity of RuNMe was higher than that of RuH and RuCN,\n the cell was negligible under hypoxic conditions, suggest-\nresulting in higher cellular uptake. The \ufb02ow cytometric ing a typical O2-dependent manner of 1O2 generation.\nannexin V-FITC assay is a typical method to monitor the Then, the probe DHR123 was used to investigate the\ndestruction of cell membrane integrity.42,43 RuNMe, RuH, intracellular \u00b7O2\u2212 generation of the three Ru(II) complexes\nand RuCN were incubated for 4 h in the dark, then after irradiation. The distinct intracellular \u00b7O2\u2212 generation\nirradiated (white light, 30 min) and further incubated for ability of RuNMe was observed, which was signi\ufb01cantly\ndifferent times (1, 2, and 4 h). The FITC \ufb02uorescence of the stronger than that of RuH and RuCN under both normoxia\nRuH- and RuCN-incubated cells remained at a low level and hypoxia (Figure 2b). A similar phenomenon was\nduring the incubation while that of RuNMe-incubated observed when DHR123 was used as a \u00b7O2\u2212 indicator in\ncells increased gradually with time at a higher level phosphate-buffered saline (PBS) buffer (Supporting\n(Supporting Information Figure S15). The above results Information Figure S16). The above data reveal that\nindicated that the production of ROS after irradiation RuNMe enabled the type I photochemical process to form\ndestroyed the cell membrane43\u201345 and increased the cell \u00b7O2\u2212 in MCF-7 cells under hypoxia. Consequently, RuNMe\nuptake of RuNMe while the membrane of RuH- and RuCN- can function well as a PDT agent against hypoxic tumor\nincubated cells had no obvious changes. This may also cells.\n\n\n\n\nFigure 2 | Confocal imaging of photoinduced ROS in MCF-7 cells incubated with RuNMe, RuH, and RuCN (3 \u03bcM, 4 h,\n37 \u00b0C in the dark) under normoxia (O2 = 21%) and hypoxia (O2 < 0.1%) using (a) SOSG as \ufb02uorescence indicators for 1O2,\n(b) DHR123 as \ufb02uorescence indicators for \u00b7O2\u2212. Photoirradiation was imposed with white light (8.0 J/cm2, 30 min) after\n4 h of Ru complex incubation. Scale bar: 20 \u03bcm.\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1586\n\f RESEARCH ARTICLE\n\n\n\n\nCell death mechanism study decreased. Moreover, the photoinduced mitochondrial\n morphology change by RuNMe in MCF-7 cells was exam-\nRuNMe was chosen for the following assay since it\n ined using transmission electron microscopy (TEM). After\nshowed the highest phototoxicity among the three com-\nplexes. The photoinduced cell death mechanism of irradiation, the mitochondria of control cells remained\nRuNMe-incubated MCF-7 cells was investigated via coin- almost unchanged, while the mitochondria of RuNMe-\ncubation of RuNMe with different cell death inhibitors treated cells showed multilamellar globules, and the cris-\n(Figure 3a). The cell viabilities showed no distinct differ- tae in the inner membrane exhibited onion-like circles in\nence when MCF-7 cells were treated with inhibitors of both normoxia and hypoxia, indicating that mitochondria\napoptosis (z-VAD-fmk), autophagy (3-methyladenine, 3- were severely damaged (Figure 3b). The structural aber-\nMA), and necrosis (necrostatin-1, Nec-1), compared with rations of the mitochondria, including disrupted cristae\nthe control cells without cell death inhibitor treatment. and compromised membrane integrity, are typical hall-\nOnly the ferroptosis inhibitor (Fer-1) signi\ufb01cantly inhib- marks of ferroptosis.46 The above results demonstrate\nited cell death, and the cell survival rate was about 88% that ferroptosis was the main cell death mode upon PDT\nunder normoxia. A similar phenomenon was also ob- treatment using RuNMe as the PS.\nserved under hypoxia. The ferroptosis inhibitor effective- To verify the ferroptosis mechanism, other character-\nly increased the cell survival rate to 78%, suggesting that istics of ferroptosis were investigated (Figure 4). Tumor\nferroptosis is the main cell death pathway by RuNMe in cells exhibited relatively high GSH levels up to 10 mM to\nnormoxia and hypoxia upon being photoinduced. The maintain redox balance.47\u201349 GSH is a cofactor of GPX4,\nmortality of photoinduced cells is detected by coincubat- and rapid GSH consumption can inhibit the activity of\ning MCF-7 cells with deferoxamine (DFO, an iron chela- GPX4 as a cofactor.50,51 The cell viability of MCF-7 cells\ntor) or holo-transferrin (an iron transporter) and RuNMe with GSH and RuNMe coincubation was signi\ufb01cantly in-\n(Supporting Information Figure S17). The results show creased under both normoxia and hypoxia (Figure 4a),\nthat the death rate of MCF-7 cells coincubated with DFO indicating that GSH played an important role in the cell\nand RuNMe was reduced. Similar results were observed death pathway during RuNMe-mediated PDT. Then, the\nunder hypoxia, indicating that ferroptosis was sup- changes in intracellular GSH levels were investigated with\npressed when intracellular iron concentration was RuNMe-incubate MCF-7 cells after irradiation. The GSH\n\n\n\n\nFigure 3 | (a) Cell viabilities of MCF-7 cells upon coincubation (24 h) with RuNMe (3 \u03bcM) and different cell-death\ninhibitors: z-VAD-fmk (50 \u03bcM), 3-MA (100 \u03bcM), Fer-1 (50 \u03bcM), and Nec-1 (50 \u03bcM). (b) TEM images of the mitochondrial\nmorphology changes in MCF-7 cells with RuNMe incubation upon light irradiation.\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1587\n\f RESEARCH ARTICLE\n\n\n\n\nFigure 4 | (a) The viability of MCF-7 cells treated with RuNMe (3 \u03bcM) and GSH (5 mM) with light irradiation. (b) GSH\nand oxidized glutathione disul\ufb01de (GSSG) levels in MCF-7 cells incubated with RuNMe (3 \u03bcM) with light irradiation.\n(c) Western blotting results of GPX4 expression in MCF-7 cells after treatment with RuNMe (3 \u03bcM). (d) The activity\nof GPX4 in MCF-7 cells upon incubation with RuNMe (3 \u03bcM) after irradiation. (e) LPOs assay of MCF-7 cells using\nC11-BODIPY581/591 after coincubation with RuNMe (3 \u03bcM).\n\nlevel in RuNMe-incubated MCF-7 cells after irradiation PDT activity in three-dimensional\nwas signi\ufb01cantly lower than that in untreated cells under multicellular spheroids\nnormoxia and hypoxia (Figure 4b). These results suggest\n The above results have demonstrated that RuNMe can\nthat RuNMe ef\ufb01ciently reduces GSH levels and disturbs\n undergo the type I PDT process and induce ferroptosis,\nthe redox balance in tumor cells.\n which can be very attractive as a potent PS for combating\n GSH consumption of cells can inhibit the expression of\n hypoxic tumor cells. Three-dimensional (3D) multicellular\nGPX4 protein, thereby reducing the activity of GPX4.\n spheroids (MCSs) are often used to simulate the micro-\nUpon light irradiation, distinctly decreased expression\n environment of tumor tissues.11,54 3D MCSs possess a\nand activity of GPX4 protein in RuNMe-incubated MCF-\n diffusion limit of approximately 150\u2013200 \u03bcm for many\n7 cells were observed under both normoxia and hypoxia\n(Figure 4c,d). Generally, to reduce cytotoxicity, LPOs will molecules, which makes them an excellent model for\nbe converted into nontoxic lipid alcohols (LOHs). How- hypoxia. Therefore, 3D MCSs with diameters of about\never, the deactivation of GPX4 will inhibit the conversion 500 \u03bcm were used to simulate the hypoxic environment\nof LPOs so the accumulation of LPOs can be regarded as of tumor tissue (Figure 5). In the control group without\nan important hallmark of ferroptosis.23,52,53 LPOs levels Ru(II) complexes, the volume of the 3D MCSs increased\nwere monitored using the lipid peroxidation probe C11- distinctly after 4 days of incubation with and without\nBODIPY581/591. Confocal imaging disclosed that RuNMe irradiation. The growth of Ru(II) complexes incubated\nenhanced the probe \ufb02uorescence remarkably upon light 3D MCSs was inhibited slightly under dark incubation\nirradiation, implying the obvious LPO accumulation under while the growth of Ru(II) complexes treated MCSs were\nboth normoxia and hypoxia (Figure 4e). These results distinctly inhibited after irradiation. The above results\ncon\ufb01rm that RuNMe can induce ferroptosis via a GPX4- show that the three Ru(II) complexes can be used as\ndependent pathway upon PDT. excellent type I PDT agents to overcome tumor hypoxia\n\nDOI: 10.31635/ccschem.022.202202074\nCitation: CCS Chem. 2023, 5, 1583\u20131591\nLink to VoR: https://doi.org/10.31635/ccschem.022.202202074\n\n\n 1588\n\f RESEARCH ARTICLE\n\n\n\n\nFigure 5 | Microscopic images of MCF-7 MCSs. MCSs were incubated in the culture medium with or without 10 \u03bcM Ru\ncomplexes. Photoirradiation was imposed by white light 30 min. Scale bar: 500 \u03bcm.\n\n\nand have the potential to effectively inhibit the growth of\nsolid tumors. Irradiation of RuNMe-treated 3D MCSs Con\ufb02ict of Interest\ncaused the almost complete collapse of the 3D structures There is no con\ufb02ict of interest to report.\nof the MCSs, showing a superior PDT effect to that of RuH\nand RuCN.\n Funding Information\nConclusion This work was \ufb01nancially supported by the National\n Natural Science Foundation of China (grant nos.\nThree Ru(II) complexes, RuNMe, RuH, and RuCN, were\n 22122701, 21731004, 91953201, 92153303, 21977044, and\nsuccessfully synthesized and used as PSs to overcome\n 21907050), the Natural Science Foundation of Jiangsu\ntumor hypoxia during the PDT process. RuNMe displayed\n Province (grant nos. BK20202004 and BK20190282),\nenhanced cellular uptake, higher \u00b7O2\u2212 generation and ex-\n and the Excellent Research Program of Nanjing Univer-\ncellent PDT ef\ufb01ciency toward MCF-7 cells compared with\n sity (grant no. ZYJH004).\nRuH and RuCN. Meanwhile, RuNMe-incubated MCF-7 cells\nshowed evident GPX4-dependent ferroptosis upon light\nirradiation under both normoxia and hypoxia. The identi-\n\ufb01cation of ferroptosis and its signal transduction mecha-\n Acknowledgments\nnism may help to develop new therapeutic approaches The authors wish to acknowledge technicians Mingyi Xie,\nthat ef\ufb01ciently suppress the growth of hypoxic tumor Jun Cai, Dejun Liu, and Yufei Jiang for their helpful\ncells. suggestions on the experimental tests.\n\n\nSupporting Information References\nSupporting Information is available and includes (1) syn- 1. Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic\nthesis routes and methods, (2) experimental methods, Therapy and Anti-Tumour Immunity. Nat. Rev. Cancer 2006,\n(3) 1H and 13C NMR spectra, (4) HRMS spectra, (5) UV 6, 535\u2013545.\nand FL spectra, (6) ICP-MS, (7) \ufb02ow cytometric Annexin 2. Lovel, J. F.; Tracy, W.; Liu, B.; Chen, J.; Zheng, G. 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