An osmium-peroxo complex for photoactive therapy of hypoxic tumors

An osmium-peroxo complex for photoactive therapy of hypoxic tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An osmium-peroxo complex for photoactive therapy of hypoxic tumors Nong Lu, Zhihong Deng, Jing Gao, Chao Liang, Haiping Xia, Pingyu Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1088102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Its limited therapeutic effect on hypoxic and refractory solid tumors has hindered the practical application of photodynamic therapy (PDT). Herein, we report our investigation of an osmium-peroxo complex ( Os2 ), which is inactive in the dark, but upon light irradiation, can release a peroxo ligand O 2 •− , and is transformed into a cytotoxic osmium complex ( Os1 ). The osmium-peroxo complex Os2 produces O 2 •− under light irradiation even in the absence of oxygen, and retains its phototoxicity in hypoxic tumors. Os1 is cytotoxic in the presence or absence of irradiation, behaves as a chemotherapeutic drug. The light-activated Os2 induces distinct ferroptosis, which is mediated by GSH degradation, lipid peroxide accumulation and down-regulation of glutathione peroxidase 4 (GPX4). In addition, Os2 causes photocatalytic oxidation of endogenous 1,4-dihydronicotinamide adenine dinucleotide (NADH) in living cancer cells, leading to ferroptosis. In vivo studies have confirmed that the Os2 can effectively inhibit the growth of solid hypoxic tumors in mice. A new strategy is proposed for the treatment of hypoxic tumors with metal-based drugs. Bioinorganic chemistry Applied Biochemistry Medicinal Chemistry Drug Discovery, Design, & Development osmium-peroxo complex photoactive therapy hypoxia metals in medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The hypoxic microenvironment of solid tumors caused by abnormal proliferation and vascularization of cells can greatly compromise the therapeutic effect of traditional photodynamic therapy (PDT), [1] which usually requires O 2 for the production under light activation of reactive oxygen species (ROS) by a photosensitizer (PS). [2] Many innovative methods have recently been introduced to solve the problem of tumor hypoxia. Some additives such as perfluorocarbons, artificial red blood cells and covalent organic frameworks (COF), [3] carry O 2 directly to the tumor site, and others utilize unique in situ features of the tumor microenvironment to generate O 2 . These include CaO 2 and graphite phase-carbon nitride (gC 3 N 4 ) carbonization [4] or high H 2 O 2 concentrations in tumor cells resulting from MnO 2 or H 2 O 2 decomposition [5] , or even photocatalytic oxygen production by photosynthetic bacteria. [6] The results however are still not ideal and there are numerous possible adverse side effects. [7] In addition, in vivo studies have shown that enrichment of O 2 promotes proliferation and inhibits apoptosis of malignant cells, results which are not conducive to PDT. [8] Therefore, improvement of the efficacy of phototherapy is necessary. Photoactive chemotherapy (PACT) has the potential to overcome the limitations imposed by hypoxia. In PACT, metal complexes can be photoactivated in a controlled manner to produce cytotoxic substances. [2a] For example, Chao et al. designed an iridium (III) complex that upon irradiation, produces free carbon radicals. [9] Toxic gases such as NO [10] and CO [11] , or ligand-centered cytotoxic substances can also be released by photoactivation. [12] The superoxide radical (O 2 •− ) is one of the most toxic ROS and has been identified as a most useful oxidant for cancer treatment and an adjuvant in synergistic chemotherapy. [13] Under the action of intracellular superoxide dismutase, O 2 •− can form H 2 O 2 and O 2. The accumulated H 2 O 2 is further transformed into a hydroxyl radical (OH•) with enhanced toxicity and reactivity, exacerbating oxidative damage of cancer cells and improving the anticancer effect. [14] The design of a photoactive compound that does not rely on O 2 but is photo-controlled to release ROS has significant application prospects in hypoxic tumor treatment. We have investigated an osmium-peroxo complex ( Os2 ), whose structure significantly improves the thermal stability of the osmium-peroxo moiety. [15, 16] Upon photoactivation with 465 nm light, Os2 releases O 2 •− even in severely hypoxic conditions (1% O 2 ), and is transformed into the cytotoxic Os1 in a Cl - containing PBS solvent (Figure 1a) , thus maintaining useful photoactivation efficacy in hypoxia. Os1 displays toxicity in both light and dark, contributing to a synergistic effect of chemotherapy and photoactive chemotherapy of Os2 under light irradiation. We also found that irradiation of Os2 induces distinct ferroptosis mediated by GSH degradation, lipid peroxide accumulation and GPX4 down-regulation. Furthermore, Os2 can photocatalytically oxidize endogenous NADH in living cancer cells, triggering ferroptosis. In vivo studies confirm that Os2 effectively inhibits the growth of solid hypoxic tumors in mice. Thus this work is developing a new process to release O 2 •− for the treatment of hypoxic tumor cells through the mechanism of ferroptosis. Results Photoactivation property The synthesis of the osmium-peroxo complex Os2 was based on our previously reported method, [16] and the characterization data can be found in Figures S1-S3 in the Supporting Information (SI). Os2 is highly stable in phosphate-buffered saline (PBS) at pH = 7.4 or in Dulbecco's Modified Eagle Medium (DMEM) cell culture media in the dark at room temperature (rt) ( Figures S4 and S5 ), and is also stable in the presence of reducing agents such as NADH, GSH, or Cys ( Figure S5 ). However, with 465 nm light irradiation, the solution of Os2 undergoes a gradual color change from brown to light pink ( Figure 1b , insets), which led us to investigate this transformation in detail. High-resolution mass spectrometry (HRMS) measurements were performed to identify the photolytic products, and a prominent peak was observed at m/z = 1175.2351 ( Figure S6 ). The mass and isotope distribution in this ion correspond well to Os1 . The formation of Os1 was further evidenced by HPLC analysis. As shown in Figure 1c (blue line), the retention time of the new peak is consistent with that of the independently synthesized Os1 ( Figures S7-S11 , Tables S1-S2 . See also SI for the synthesis and characterization of Os1 ). These results show that Os1 is one of the main photoproducts of Os2 . ROS detection Due to the dissociation of O 2 unit from the metal center of Os2 under light irradiation, we sought to verify that the O 2 unit is released in the form of a superoxide anion (O 2 •− ). We first tried to detect O 2 •− by using the non-fluorescent dihydrorhodamine 123 (DHR123) probe, [17] which can react with O 2 •− and emit strong green fluorescence around 526 nm. As shown in Figure 2a and Figure S12 , when irradiated at 465 nm (13 mW/cm 2 ), the solution of Os2 showed an increasing emission at 526 nm, suggesting the generation of O 2 •− . We also simulated the Fenton reaction process. [18a] When Os2 (15 μM) and a small amount of superoxide dismutase (SOD) were added into a PBS solution containing Fe 2+ , the absorbance of methylene blue probe (MB, 5 μg/mL) at 665 nm decreased with a degradation rate of 70.26 % ( Figure S13 ). On the contrary, in control experiments using only Os2 and Os2 +SOD, respectively, no changes were observed in the characteristic UV−Vis absorption band of MB. On the basis of these findings and published data, we propose the mechanism in Figure 2f . Under the action of SOD enzyme, O 2 •− disproportionately generates H 2 O 2 and O 2 . The OH• is generated from H 2 O 2 under the action of Fe 2+ , and then reacts with MB, thus decreasing its absorption. These findings confirmed that Os2 can produce O 2 •− under light irradiation. To gain more insight into the nature of ROS, we made electron spin resonance (ESR) measurements using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and (2,2,6,6-tetramethylpiperidine)oxyl (TEMPO) as spin traps. Specifically, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) [18] was used to trap O 2 •− generated by Os2 under 465 nm light irradiation. After the mixture containing DMPO and Os2 was exposed to light irradiation, several signal peaks of DMPO-OOH appeared in the 3440 – 3480 G ( Figure 2b ), which explained the generation of O 2 •− . In order to study whether Os2 produces 1 O 2 under light irradiation, we used 2,2,6,6-tetramethylpiperidine (TEMP) to measure 1 O 2 generation. [19] As shown in Figure 2c , a three-line signal with the intensity of 1:1:1 between 3480 and 3530 G was observed in the ESR spectrum of Os2 and TEMP mixing solution under light irradiation. A similar signal was observed in the presence of Os1 (the light product of Os2 ). These ESR results suggest that Os2 can generate both O 2 •− and 1 O 2 under light irradiation. In addition, 9,10-anthracenediyl-bis-(methylene) dimalonic acid (ABDA) was further used to detect the 1 O 2 quantum yields of Os1 and Os2 under light irradiation. As shown in Figures 2d and S14 , the characteristic absorption peak of ABDA at about 378 nm decreased gradually as the irradiation time increased. The 1 O 2 quantum yields of Os1 and Os2 were calculated as 0.044 and 0.045 by comparison with Ru(bpy) 3 2+ ( Figure S14 and Table S3 ). The above experiments were all conducted under aerobic condition. Next, we studied the production of ROS in the absence of oxygen (hypoxia, deaeration with nitrogen). As shown in Figure S15 , the emission spectra showed enhancing green fluorescence in the presence of Os2 and DHR123 probe. This result proves that Os2 also produces O 2 •− under hypoxia. We further measured whether 1 O 2 generation under hypoxia. As shown in Figure S16 , the absorption of ABDA in the presence of both Os1 and Os2 failed to change obviously under hypoxia, confirming that no 1 O 2 generation occurred under hypoxia. However, the absorption of ABDA was reduced in the presence of Os2 + SOD. This is due to Os2 produces O 2 •− under hypoxia, and that O 2 •− disproportionately generates H 2 O 2 and O 2 under the action of the SOD enzyme, the O 2 being available for 1 O 2 generation by the photoproduct Os1 ( Figure 2f ). However, Os1 is unable to produce any ROSs under hypoxia ( Figure S16 ). Generation of cellular ROS We studied the photo-induced ROS generation ability of Os2 in living HeLa cells. Dihydroethidium (DHE), hydroxyphenyl fluorescein (HPF) and singlet oxygen sensor green (SOSG) were used as probes to monitor the production of O 2 •− , OH• and 1 O 2 , respectively. [18a,b] The detection mechanisms of these probes for various free radicals are shown in Figure 3a . Confocal microscopy showed that when Os2 -incubated HeLa cells were exposed to light under normoxia (20% O 2 , 465 nm, 13 mW/cm 2 , 1 h), the fluorescence signal in DHE-stained cells is significantly enhanced ( Figure 3b,c ). This confirms the ability of Os2 to release O 2 •− in cells under light irradiation. Under hypoxia (1% O 2 ), the fluorescence intensity after light irradiation was similar to that obtained under normoxia, indicating that the process of O 2 •− generation from Os2 is independent of O 2 . This is different from the mechanism of O 2 -dependent type I photodynamic mechanism. Because it is known that O 2 •− can produce OH• in the presence of intracellular SOD enzyme and Fe 2+ ions, [18a] we further studied the generation of OH• in the cells by HPF staining. Confocal imaging showed that Os2 can produce OH• in HeLa cells under normoxia or hypoxia ( Figures 3b, 3c ). In addition, we also compared the ability of 1 O 2 generation of Os2 and the photoproduct Os1 by using a SOSG probe, and found that under hypoxia, Os2 has more advantages in producing 1 O 2 than Os1 . The result showed that the green fluorescence in the Os2 treated cells is strong under both normoxia and hypoxia. However, the fluorescence in the Os1 treated cells was much weaker under hypoxia than that observed under normoxia ( Figure S17 ). These results are consistent with those in Figure 2f , showing that Os2 firstly induces the release of O 2 •− and production of Os1 , accompanied by the formation of O 2 (as an oxygen source for Os1 ) and OH• by a Fenton reaction. Finally 1 O 2 is generated by the photoproduct, Os1 . These data all show that Os2 may be useful as a new effective drug for photoactive treatment of hypoxic tumor cells. Phototoxicity in vitro The dark- and photo-cytotoxicities of Os2 and Os1 against HeLa cells were determined with an MTT method. [19] As showed in Figure 4 and Table 1 , Os2 had low dark-cytotoxicity and high photo-cytotoxicity under both normoxia and hypoxia ( Figures 4a, 4c ). The (IC 50 ) dark values, determined with only dark incubation, were 89.2 and >100 μM, respectively ( Table 1 ). The (IC 50 ) Light values of Os2 , determined by photoirradiation under normoxia or hypoxia were 1.23 and 5.86 μM, respectively. The photo-cytotoxicity under hypoxia can be explained in terms of the release of O 2 •− by Os2 and light irradiation, followed by the production of O 2 , which may be used by the light product Os1 to generate 1 O 2 , and OH• with stronger toxicity via the Fenton reaction. In contrast, the light product ( Os1 ) shows high dark-cytotoxicity and photo-cytotoxicity under both normoxia and hypoxia ( Figures 4b, 4d ). The (IC 50 ) dark values of Os1 under normoxia and hypoxia are 8.12 and 9.95 μM, and the (IC 50 ) Light values under normoxia and hypoxia are 1.31 and 7.51 μM, respectively ( Table 1 ). This result suggests that Os1 is high photo- and dark- cytotoxic, and can be seen as a chemotherapeutic drug. However, with hypoxia, due to the lack of oxygen, Os1 shows no obvious photodynamic effect. The PI value of Os2 (>18) under hypoxia is much higher than that of Os1 (1.3). Staining with alcein acetoxymethyl ester (Calcein-AM) or propidium iodide (PI) were also used to distinguish living cells (green) from dead cells (red). As shown in Figure S18 , the dark group of Os2 in normoxia and hypoxia shows strong green fluorescence (living cells) but no red fluorescence (dead cells), and the control group in the dark or under light irradiation also shows no dead cells. In contrast, the light group of Os2 shows weak green fluorescence and strong red fluorescence, indicating that Os2 leads to a large number of dead cells under light irradiation. Table 1. The dark- and photo-IC 50 values of Os2 and Os1 against HeLa cells under normoxia and hypoxia. Compounds Dark [μM] Light [c] [μM] PI [d] Os2 [a] 89.2±2.6 1.23±0.09 72.5 Os2 [b] ＞ 100 5.86±0.19 ＞ 18 Os1 [a] 8.12±0.26 1.31±0.05 6.2 Os1 [b] 9.95±0.14 7.51±0.35 1.3 [a] under normoxia (20% O 2 ); [b] under hypoxia (1% O 2 ); [c] photoirradiation was imposed (465 nm, 13 mW/cm 2 , 1 h) after 8 h of the complexes incubation; [d] photocytotoxicity index, the ratio of (IC 50 ) dark /(IC 50 ) Light . Ferroptosis mechanism It has been reported that the photo-chemical process of ROS generation could cause ferroptosis in cancer cells. [2e, 20] This led us to consider if Os2 can induce ferroptosis. GSH is closely related to ferroptosis, [21] and we first detected the ability of Os2 to consume GSH. As shown in Figures 5a-5b , with an increase of light irradiation time, the absorption at 412 nm decreased, indicating that Os2 when irradiated, could consume GSH. We determined the GSH levels in the cells after treatment with Os2 , and found that the GSH level in the irradiated group was significantly lower than that in the non-irradiated group ( Figure 5c ). Therefore, we concluded that Os2 can consume cellular GSH under irradiation conditions. Ferroptosis is a type of iron-dependent cell death caused by excessive lipid peroxidation. The main feature of ferroptosis is that after the inactivation of cell antioxidant capacity, phospholipids containing polyunsaturated fatty acids are peroxidated on the cell membrane, destroying the cell membrane and leading to ferroptosis. [22] The antioxidant glutathione peroxidase 4 (GPX4) specifically catalyzes loss of oxidative activity in the lipid peroxides in a glutathione-dependent manner, [23] and subsequent inhibition of GPX4 induces ferroptosis. GSH consumption can indirectly inhibit the expression of GPX4. We speculated that Os2 could further inhibit the expression of GPX4 and we verified this hypothesis by western blot analysis of GPX4. As shown in Figures 5f-5g , Os2 fails to reduce the expression of GPX4 in the dark. Upon light irradiation, Os2 significantly reduces the expression of GPX4, and the GSH consumption caused by ROS and the inhibition of GPX4 will further lead to the accumulation of lipid peroxides and induce ferroptosis. We used C11-BODIPY as a lipid peroxide probe with which to monitor intracellular accumulation of lipid peroxides ( Figures 5d, 5e ). Confocal microscopy showed that the fluorescence of HeLa cells treated with Os2 was significantly enhanced after exposure to light, indicating a significant accumulation of lipid peroxides, which could be effectively inhibited by Ferrostatin-1 (Fer-1, a ferroptosis inhibitor). All the above results confirm that Os2 induces ferroptosis as shown in Figure 5h . NADH photocatalytic oxidation As a cofactor, 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) regulates the redox balance of cellular mitochondria, and plays an important role in regulating energy production. If NADH is oxidized to NAD + , it can destroy the whole respiratory chain and kill cells. [24] We studied whether NADH can be oxidized by Os2 under light irradiation, which could provide a photocatalytic oxidation pathway to kill cancer cells. The photocatalytic efficiency of Os2 (20 μM) towards NADH (175 μM) was first determined by UV-Vis absorption spectroscopy. As shown in Figure 6a , the absorbance at 339 nm decreases and the absorbance at 259 nm increases gradually with increase of the irradiation time. In contrast, the absorption of the non-illuminated Os2 group exhibits no obvious changes ( Figure S19 ). This indicates that Os2 could reduce the enzyme activity of NADH under light irradiation. We also calculated the NADH oxidation turnover number (TON) of Os2 at 339 nm to evaluate its photocatalytic efficiency. The TON value of NADH oxidation by Os2 under light irradiation is 3.808, which is 30-times higher than that of the non-illuminated group ( Figure 6b ). Similarly, Os1 also shows a similar photocatalytic oxidation effect on NADH ( Figure S20 ). The photocatalytic oxidation of NADH was also monitored by 1 H NMR in D 2 O/CD 3 OD (1/3, v/v) at 298 K. As shown in Figure 6c , In the Os2 and NADH illuminated group, new peaks from hydrogens on the nicotinamide ring of NAD + are observed at 6.13, 8.31, 8.55, 8.99, 9.36 and 9.58, but no new NAD + peak is observed in either the non-illuminated group or the non- Os2 group ( Figure 6c ). Intuitively, this shows that Os2 could oxidize NADH under light irradiation, transforming it into NAD + . Subsequently, we measured the NADH photocatalytic oxidation ratio at the cellular level using a NAD/NADH-Glo TM method, which is a bioluminescence method for the detection of NAD + and NADH. As shown in Figure 6d , the chemical luminescence intensity of Os2 -light group proved to be lower than that of other groups. These results show that Os2 can effectively oxidize NADH at the cellular level by light irradiation, thereby killing cancer cells. As can be seen in Figure 6e , NADH can not only be converted to NAD + by interacting with Os1 * (the excited state of Os1 ), but can also be oxidized to NAD + by O 2 •− released from Os2 . The down-regulation of NADH indirectly aids the reduction of oxidized glutathione (GSSG) to GSH by glutathione reductase (GR), [25] resulting in the accumulation of lipid peroxides, and ultimately achieving the synergistic induction of ferroptosis. Photoactive antitumor therapy in vivo We studied the feasibility of photoactive therapy in vivo by Os2. Since Os2 is a small molecule and there is no specific targeted group, it can be administered intratumorally. As shown in Figure 7b, compared with the other three groups, the tumor growth in mice treated with Os2-light group was inhibited. The tumor size of the Os2-light group was the smallest in the four groups (Figure 7d), and the average tumor weight of the Os2-light group was significantly lower than that of the other groups (Figure 7c). The tumor tissues after final treatment were collected for histological assessment. The hematoxylin and eosin (H&E) staining showed obvious destruction of tumor tissues in the Os2-light group, while tumor tissues in the other three groups were not affected (Figure 7e). In order to evaluate the biological safety of Os2, we first analyzed the H & E staining slices of the main organs of healthy mice i.v. injected with three times of the therapeutic dose (2.69 mg kg −1 ). The results showed no obvious tissue damage in these slices (Figure S21). We also used Singapore wild-type zebrafish to test the biological safety of Os2 (Figure S22) with the green fluorescent protein (GFP) commonly used as a biomarker to visualize the physiological processes. After 5 days of incubation with Os2, the blood vessels of the zebrafish were apparently not damaged, showing good biocompatibility in vivo . Conclusions In summary, we have demonstrated that an osmium-peroxo complex ( Os2 ) can release O 2 •− under light irradiation in the absence of O 2 , and at the same time is transformed into another active osmium complex ( Os1 ), which exhibits both chemotherapeutic and photodynamic properties, thus maintaining good phototoxicity in hypoxic tumors. The osmium-peroxo complex Os2 can induce ferroptosis, which is characterized by GSH degradation, GPX4 down-regulation and lipid peroxide accumulation. In addition, under light irradiation, the same osmium-peroxo complex oxidizes NADH into NAD + , further helping induction of ferroptosis. At the in vivo level, the osmium-peroxo complex achieves highly effective photoactive therapy of solid hypoxic tumors. This study reports the first example of a metal-peroxo complex for O 2 -independent photoactive therapy and provides a promising strategy for combating hypoxic tumors. Declarations Acknowledgements We appreciate the financial support of the National Natural Science Foundation of China (NSFC, 22077085, 22007104 and 21931002), and the Science and Technology Foundation of Shenzhen (JCYJ20190808153209537 and JCYJ20200109140812302). We appreciate the Instrumental Analysis Center of Shenzhen University. Author contributions N. L., J. G., H. X., and P. Z. designed the study. Z. D. synthesized and characterized the complexes. N. L. and C. L. performed the experiments in vitro and in vivo. N. L., Z. D., J. G., H. X. and P. Z. analyzed the data and wrote the paper. All authors contributed to the general discussion. 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Additional Declarations There is NO Competing Interest. Supplementary Files SI.docx SI floatimage8.png Graphical Abstract. We develop a metal-peroxo complex for O2-independent photoactive therapy that can overcome the hypoxia problem of traditional PDT. This provides a promising strategy for combating hypoxic tumors. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1088102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":66208162,"identity":"091a0b85-ed56-470b-94d9-ba5623b63946","order_by":0,"name":"Nong Lu","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nong","middleName":"","lastName":"Lu","suffix":""},{"id":66208163,"identity":"c6c68fad-6a1e-42d7-8ff1-c4ac660dda47","order_by":1,"name":"Zhihong Deng","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Deng","suffix":""},{"id":66208164,"identity":"4e34b44c-e7b8-461d-95bd-29b7846b0cc5","order_by":2,"name":"Jing Gao","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Gao","suffix":""},{"id":66208165,"identity":"7288dd41-844c-44fa-a9c7-3df0664fd44b","order_by":3,"name":"Chao Liang","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Liang","suffix":""},{"id":66208166,"identity":"81955ef0-cded-47cb-9215-1cbb01fad5a3","order_by":4,"name":"Haiping Xia","email":"","orcid":"https://orcid.org/0000-0002-2688-6634","institution":"Southern University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haiping","middleName":"","lastName":"Xia","suffix":""},{"id":66208167,"identity":"6ea22371-9c4a-439e-9771-99a6242d8de2","order_by":5,"name":"Pingyu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYHACNhAhxw/hMBOvxViygVQtiQYHiNVizsD+7MHPHbUJxuePP5NgqLBObGA/ewCvFssGhnTD3jPH88xuJKRJMJxJT2zgyUvAqwXonmMSvG3His1uABmMbYcTGyR4DAhoYWyT/Nt2LHFz/8E2CcZ/RGlhZpPmbatJ3MCQzCbB2ECEFstmNjZp2bYDxhI30pgtEo6lG7fx5ODXYs7e/kzybVudHH//8Yc3PtRYy/aznyHgMEhEHIbwEhig0YRXC4SqI6RuFIyCUTAKRjIAAJhAQO4CVt+0AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2921-9490","institution":"Shenzhen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pingyu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2021-11-17 07:15:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1088102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1088102/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16047043,"identity":"4d77201c-f453-41b0-82ec-5c004621abb1","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132494,"visible":true,"origin":"","legend":"(a) Schematic diagram of light-mediated Os2 release of O2•− and Os1. (b) The UV-Visible spectral changes of Os2 (20 μM) in PBS solution (pH = 7.4, 1% DMSO) under light illumination (465 nm, 13 mW/cm2) at rt. (c) HPLC analysis of Os2 in PBS solution (1% DMSO) in the dark or after irradiating for 60 min. Mobile phase was CH3CN and water (v/v 1:1). ★: Individual control of Os1.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/f7798a8ba5cb1673c466c261.png"},{"id":16047295,"identity":"86cad945-80ee-41b1-801e-d2ecaa4bf6cd","added_by":"auto","created_at":"2021-11-30 22:30:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":260363,"visible":true,"origin":"","legend":"(a) The emission spectra for monitoring O2•− generation by Os2 (10 μM) using DHR 123 probe (λex = 500 nm) in normoxia at 298 K. (b) The ESR signal of O2•− trapped by DMPO after light irradiation in normoxia. (c) The ESR signal of 1O2 trapped by TEMP after light irradiation in normoxia. (d) The UV-Vis absorption spectra of ABDA for monitoring of 1O2 generated by Os2 under light irradiation in normoxia. (e) The plot of A0-A of ABDA at 378 nm versus irradiation time in the presence of Os1 or Os2 in hypoxia or normoxia. (f) Schematic diagram of generating O2•−, 1O2 and OH• by light-activating Os2. Light: 465 nm, 13 mW/cm2.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/cc8b1eaf82ab49e522930bb7.png"},{"id":16047296,"identity":"e7cd6de3-3d1f-4849-8739-8c4b89b113da","added_by":"auto","created_at":"2021-11-30 22:30:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318955,"visible":true,"origin":"","legend":"(a) Schematic diagram of detection of O2•−, OH• and 1O2 with DHE, HPF and SOSG probes, respectively. (b) Confocal microscope images of O2•−, OH• and 1O2 in HeLa cells under normoxia (20% O2) or hypoxia (1% O2) probed by DHE, HPF and SOSG, respectively. (c) The average fluorescence intensities calculated from the images in b. (**P ≤ 0.05, ***P ≤ 0.001). HeLa cells incubated with Os2 (20 μM) for 8 h, and then treated with 10 μM of DHE, HPF or SOSG for 30 min. DHE: λex = 488 nm, λem = 600 ± 30 nm; HPF: λex = 488 nm, λem = 530 ± 30 nm; SOSG: λex = 488 nm, λem =525 ± 30 nm. Scale bar: 20 μm.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/6c59c44f95dfeb9babeca69a.png"},{"id":16047047,"identity":"eddce816-4057-47de-86f1-c5053f23e689","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96275,"visible":true,"origin":"","legend":"The viability of HeLa cells treated with different concentrations of Os2 or Os1 in the dark or upon light irradiation under normoxia (20% O2) or hypoxia (1% O2).","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/9f168b4a973b5ff2c1d08e9b.png"},{"id":16047051,"identity":"1e4c18ee-36cf-4ffa-b902-4b2ca66d6c7a","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":406961,"visible":true,"origin":"","legend":"(a) Irradiation time-dependent GSH depletion by Os2 upon blue light irradiation and (b) the absorption at 412 nm was decreased by increasing the irradiation time. (c) The GSH levels in cells after different treatments. (d) The fluorescence images of lipid peroxides in the treated cells detected by C11-BODIPY probe. C11-BODIPY: λex = 488 nm, λem = 570 ± 80 nm. (e) The average fluorescence intensities calculated from the images in (d). (f) Western blot analysis of GPX4 in HeLa cells after treatment with Os2 with or without light treatment. RSL3 is the positive control group. Light: 465 nm, 13 mW/cm2. (g) The relative expression levels of GPX4 calculated from (f). (h) The process of ferroptosis in this photoactive antitumor therapy.","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/df3efa16b4304e282a8c5feb.png"},{"id":16047046,"identity":"325c3db0-ed5a-4439-b5f8-cdefe81a3cf3","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":195975,"visible":true,"origin":"","legend":"(a) Reaction of Os2 (20 μM) and NADH (175 μM) in PBS solution under blue light irradiation monitored by UV-Vis spectra at 298 K. (b) TON of Os2 under dark or irradiation conditions. (c) Photocatalytic oxidation of NADH (3.5 mM) by Os2 (0.25 mM) under dark or irradiation conditions monitored by 1H NMR spectroscopy. Peaks associated with blue triangles represent NADH, those with red squares represent NAD+. (d) NADH concentrations in the treated HeLa cells. (e) Schematic diagram of the photocatalytic oxidation of NADH by Os2 and followed by induced ferroptosis. Light: 465 nm, 13 mW/cm2.","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/4dc1d6144002bdfdf24d4577.png"},{"id":16047297,"identity":"66d6a489-bce3-4462-938e-9dc1803796f4","added_by":"auto","created_at":"2021-11-30 22:30:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":451447,"visible":true,"origin":"","legend":"(a) Schematic of the in vivo (HeLa tumor-bearing Balb/c mice) therapeutic protocol. Mice were irradiated by light (465 nm, 13 mW/cm2) for 60 min after i.t. injection with 25 μL of PBS containing 500 μM Os2. (b) Tumor growth curves after treatment. Error bars are standard errors calculated based on five mice/group. (c) Tumor weights of mice at day 16 after various treatments. (d) The digital photos of representative mice after various treatments. (e) H\u0026E staining images of HeLa tumor tissues in mice after various treatments. ***P ≤ 0.001.","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/6a1582e3e442656d7d549cad.png"},{"id":16047299,"identity":"731f702c-69b4-47ab-acca-4dc3b428f7c3","added_by":"auto","created_at":"2021-11-30 22:30:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":759831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/e31d452e-354e-45ab-80ed-384a8ff4d447.pdf"},{"id":16047049,"identity":"86b4e18c-e2cc-4cdd-a816-2d77b578cf59","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4485200,"visible":true,"origin":"","legend":"SI","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/da1de0960d8e470df3bc0444.docx"},{"id":16047045,"identity":"9e1e60b3-8028-4e27-9072-b53f64aebc13","added_by":"auto","created_at":"2021-11-30 22:27:03","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":392041,"visible":true,"origin":"","legend":"Graphical Abstract. We develop a metal-peroxo complex for O2-independent photoactive therapy that can overcome the hypoxia problem of traditional PDT. This provides a promising strategy for combating hypoxic tumors.","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-1088102/v1/4fdc16d2bc12352bcbc9ae16.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"An osmium-peroxo complex for photoactive therapy of hypoxic tumors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hypoxic microenvironment of solid tumors caused by abnormal proliferation and vascularization of cells\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecan greatly compromise the therapeutic effect of traditional photodynamic therapy (PDT),\u003csup\u003e[1]\u003c/sup\u003e which usually requires O\u003csub\u003e2\u003c/sub\u003e for the production under light activation of reactive oxygen species (ROS) by a photosensitizer (PS).\u003csup\u003e[2]\u003c/sup\u003e Many innovative methods have recently been introduced to solve the problem of tumor hypoxia. Some additives such as perfluorocarbons, artificial red blood cells and covalent organic frameworks (COF),\u003csup\u003e[3]\u003c/sup\u003e carry O\u003csub\u003e2\u003c/sub\u003e directly\u003csub\u003e\u0026nbsp;\u003c/sub\u003eto the tumor site, and others utilize unique \u003cem\u003ein situ\u0026nbsp;\u003c/em\u003efeatures of the tumor microenvironment to generate O\u003csub\u003e2\u003c/sub\u003e. These include CaO\u003csub\u003e2\u003c/sub\u003e and graphite phase-carbon nitride (gC\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) carbonization\u003csup\u003e[4]\u003c/sup\u003e or high H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations in tumor cells resulting from MnO\u003csub\u003e2\u003c/sub\u003e or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition\u003csup\u003e[5]\u003c/sup\u003e, or even photocatalytic oxygen production by photosynthetic bacteria.\u003csup\u003e\u0026nbsp;[6]\u003c/sup\u003e The results however are still not ideal and there are numerous possible adverse side effects.\u003csup\u003e[7]\u003c/sup\u003e In addition, \u003cem\u003ein vivo\u003c/em\u003e studies have shown that enrichment of O\u003csub\u003e2\u003c/sub\u003e promotes proliferation and inhibits apoptosis of malignant cells, results which are not conducive to PDT.\u003csup\u003e[8]\u003c/sup\u003e Therefore, improvement of the efficacy of phototherapy is necessary.\u003c/p\u003e\n\u003cp\u003ePhotoactive chemotherapy (PACT) has the potential to overcome the limitations imposed by hypoxia. In PACT, metal complexes can be photoactivated in a controlled manner to produce \u0026nbsp;cytotoxic substances.\u003csup\u003e[2a]\u003c/sup\u003e For example, Chao et al. designed an iridium (III) complex that upon irradiation, produces free carbon radicals.\u003csup\u003e[9]\u003c/sup\u003e Toxic gases such as NO\u003csup\u003e[10]\u003c/sup\u003e and CO\u003csup\u003e[11]\u003c/sup\u003e, or ligand-centered cytotoxic substances can also be released by photoactivation.\u003csup\u003e[12]\u003c/sup\u003e The superoxide radical (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) is one of the most toxic ROS and has been identified as a most useful oxidant for cancer treatment and an adjuvant in synergistic chemotherapy.\u003csup\u003e[13]\u003c/sup\u003e Under the action of intracellular superoxide dismutase, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e can form H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2.\u003c/sub\u003e The accumulated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is further transformed into a hydroxyl radical (OH\u0026bull;) with enhanced toxicity and reactivity, exacerbating oxidative damage of cancer cells and improving the anticancer effect.\u003csup\u003e[14]\u003c/sup\u003e The design of a photoactive compound that does not rely on O\u003csub\u003e2\u003c/sub\u003e but is photo-controlled to release ROS has significant application prospects in hypoxic tumor treatment.\u003c/p\u003e\n\u003cp\u003eWe have investigated an osmium-peroxo complex (\u003cstrong\u003eOs2\u003c/strong\u003e), whose structure significantly improves the thermal stability of the osmium-peroxo moiety.\u003csup\u003e[15, 16]\u003c/sup\u003e Upon photoactivation with 465 nm light,\u003cstrong\u003e\u0026nbsp;Os2\u003c/strong\u003e releases O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e even in \u0026nbsp;severely hypoxic conditions (1% O\u003csub\u003e2\u003c/sub\u003e), and is transformed into the cytotoxic \u003cstrong\u003eOs1\u0026nbsp;\u003c/strong\u003ein a Cl\u003csup\u003e-\u003c/sup\u003e containing PBS solvent \u003cstrong\u003e(Figure 1a)\u003c/strong\u003e, thus maintaining useful photoactivation efficacy in hypoxia. \u003cstrong\u003eOs1\u003c/strong\u003e displays toxicity in both light and dark, contributing to a synergistic effect of chemotherapy and photoactive chemotherapy of \u003cstrong\u003eOs2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eunder light irradiation. We also found that irradiation of \u003cstrong\u003eOs2\u003c/strong\u003e induces distinct ferroptosis mediated by GSH degradation, lipid peroxide accumulation and GPX4 down-regulation. Furthermore, \u003cstrong\u003eOs2\u003c/strong\u003e can photocatalytically oxidize endogenous NADH in living cancer cells, triggering ferroptosis. \u003cem\u003eIn vivo\u003c/em\u003e studies confirm that \u003cstrong\u003eOs2\u003c/strong\u003e effectively inhibits the growth of solid hypoxic tumors in mice. Thus this work is developing a new process to release O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e for the treatment of hypoxic tumor cells through the mechanism of ferroptosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhotoactivation property\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis of the osmium-peroxo complex \u003cstrong\u003eOs2\u003c/strong\u003e was based on our previously reported method,\u003csup\u003e[16]\u003c/sup\u003e and the characterization data can be found in \u003cstrong\u003eFigures S1-S3\u0026nbsp;\u003c/strong\u003ein the Supporting Information (SI). \u003cstrong\u003eOs2\u003c/strong\u003e is highly stable in phosphate-buffered saline (PBS) at pH = 7.4 or in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) cell culture media in the dark at room temperature (rt) (\u003cstrong\u003eFigures S4\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;S5\u003c/strong\u003e), and is also stable in the presence of reducing agents\u0026nbsp;such as NADH, GSH, or Cys (\u003cstrong\u003eFigure S5\u003c/strong\u003e). However, with 465 nm light irradiation, the solution of \u003cstrong\u003eOs2\u003c/strong\u003e undergoes a gradual color change from brown to light pink (\u003cstrong\u003eFigure 1b\u003c/strong\u003e, insets), which led us to investigate this transformation in detail. High-resolution mass spectrometry (HRMS) measurements were performed to identify the photolytic products, and a prominent peak was observed at \u003cem\u003em/z\u003c/em\u003e = 1175.2351 (\u003cstrong\u003eFigure S6\u003c/strong\u003e). The mass and isotope distribution in this ion correspond well to \u003cstrong\u003eOs1\u003c/strong\u003e. The formation of \u003cstrong\u003eOs1\u0026nbsp;\u003c/strong\u003ewas further evidenced by HPLC analysis. As shown in \u003cstrong\u003eFigure 1c\u003c/strong\u003e (blue line), the retention time of the new peak is consistent with that of the independently synthesized \u003cstrong\u003eOs1\u003c/strong\u003e (\u003cstrong\u003eFigures S7-S11\u003c/strong\u003e, \u003cstrong\u003eTables S1-S2\u003c/strong\u003e. See also SI for the synthesis and characterization of \u003cstrong\u003eOs1\u003c/strong\u003e). These results show that \u003cstrong\u003eOs1\u0026nbsp;\u003c/strong\u003eis one of the main photoproducts of \u003cstrong\u003eOs2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eROS detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the dissociation of O\u003csub\u003e2\u003c/sub\u003e unit from the metal center of \u003cstrong\u003eOs2\u003c/strong\u003e under light irradiation, we sought to verify that the O\u003csub\u003e2\u003c/sub\u003e unit is released in the form of a superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e).\u0026nbsp;We first tried to detect O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by using the non-fluorescent dihydrorhodamine 123 (DHR123) probe,\u003csup\u003e[17]\u003c/sup\u003e which can react with O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and emit strong green \u0026nbsp;fluorescence around 526 nm. As shown in \u003cstrong\u003eFigure 2a\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eFigure S12\u003c/strong\u003e, when irradiated at 465 nm (13 mW/cm\u003csup\u003e2\u003c/sup\u003e), the solution of \u003cstrong\u003eOs2\u003c/strong\u003e showed an increasing emission at 526 nm, suggesting the generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. We also simulated the Fenton reaction process.\u003csup\u003e[18a]\u003c/sup\u003e When \u003cstrong\u003eOs2\u003c/strong\u003e (15 \u0026mu;M) and a small amount of superoxide dismutase (SOD) were added into a PBS solution containing Fe\u003csup\u003e2+\u003c/sup\u003e, the absorbance of methylene blue probe (MB, 5 \u0026mu;g/mL) at 665 nm decreased with a degradation rate of 70.26 % (\u003cstrong\u003eFigure S13\u003c/strong\u003e). On the contrary, in control experiments using only \u003cstrong\u003eOs2\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eOs2\u003c/strong\u003e+SOD, respectively, no changes were observed in the characteristic UV\u0026minus;Vis absorption band of MB. On the basis of these findings and published data, we propose the mechanism in \u003cstrong\u003eFigure 2f\u003c/strong\u003e. Under the action of SOD enzyme, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003edisproportionately generates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e. The OH\u0026bull; is generated from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eunder the action of Fe\u003csup\u003e2+\u003c/sup\u003e, and then reacts with MB, thus decreasing its absorption. These findings confirmed that \u003cstrong\u003eOs2\u003c/strong\u003e can produce O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under light irradiation.\u003c/p\u003e\n\u003cp\u003eTo gain more insight into the nature of ROS, we made electron spin resonance (ESR) measurements using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and (2,2,6,6-tetramethylpiperidine)oxyl (TEMPO) as spin traps. Specifically, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)\u003csup\u003e[18]\u003c/sup\u003e was used to trap\u003csup\u003e\u0026nbsp;\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generated by \u003cstrong\u003eOs2\u003c/strong\u003e under 465 nm light irradiation. After the mixture containing DMPO and\u003cstrong\u003e\u0026nbsp;Os2\u003c/strong\u003e was exposed to light irradiation, several signal peaks of DMPO-OOH appeared in the 3440 \u0026ndash; 3480 G (\u003cstrong\u003eFigure 2b\u003c/strong\u003e), which explained the generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. In order to study whether \u003cstrong\u003eOs2\u003c/strong\u003e produces \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under light irradiation, we used 2,2,6,6-tetramethylpiperidine\u0026nbsp;(TEMP) to measure \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egeneration.\u003csup\u003e[19]\u003c/sup\u003e As shown in \u003cstrong\u003eFigure 2c\u003c/strong\u003e, a three-line signal with the intensity of 1:1:1 between 3480 and 3530 G was observed in the ESR spectrum of \u003cstrong\u003eOs2\u003c/strong\u003e and TEMP mixing solution under light irradiation. A similar signal was observed in the presence of \u003cstrong\u003eOs1\u003c/strong\u003e (the light product of \u003cstrong\u003eOs2\u003c/strong\u003e). These ESR results suggest that \u003cstrong\u003eOs2\u003c/strong\u003e can generate both O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u0026nbsp;\u003c/sup\u003eand\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under light irradiation.\u003c/p\u003e\n\u003cp\u003eIn addition, 9,10-anthracenediyl-bis-(methylene) dimalonic acid (ABDA) was further used to detect the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e quantum yields of \u003cstrong\u003eOs1\u003c/strong\u003e and \u003cstrong\u003eOs2\u003c/strong\u003e under light irradiation. As shown in \u003cstrong\u003eFigures 2d\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;S14\u003c/strong\u003e, the characteristic absorption peak of ABDA at about 378 nm decreased gradually as the irradiation time increased. The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e quantum yields of \u003cstrong\u003eOs1\u003c/strong\u003e and \u003cstrong\u003eOs2\u003c/strong\u003e were calculated as 0.044 and 0.045 by comparison with Ru(bpy)\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e (\u003cstrong\u003eFigure S14\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Table S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe above experiments were all conducted under aerobic condition. Next, we studied the production of ROS in the absence of oxygen (hypoxia, deaeration with nitrogen). As shown in \u003cstrong\u003eFigure S15\u003c/strong\u003e, the emission spectra showed enhancing green fluorescence in the presence of \u003cstrong\u003eOs2\u003c/strong\u003e and DHR123 probe. This result proves that \u003cstrong\u003eOs2\u003c/strong\u003e also produces O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under hypoxia. We further measured whether \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation under hypoxia. As shown in \u003cstrong\u003eFigure S16\u003c/strong\u003e, the absorption of ABDA in the presence of both \u003cstrong\u003eOs1\u003c/strong\u003e and \u003cstrong\u003eOs2\u003c/strong\u003e failed to change obviously under hypoxia, confirming that no \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation occurred under hypoxia. However, the absorption of ABDA was reduced in the presence of \u003cstrong\u003eOs2\u003c/strong\u003e + SOD. This is due to \u003cstrong\u003eOs2\u003c/strong\u003e produces O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under hypoxia, and that O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003edisproportionately generates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e under the action of the SOD enzyme, the O\u003csub\u003e2\u003c/sub\u003e being available for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation by the photoproduct \u003cstrong\u003eOs1\u003c/strong\u003e (\u003cstrong\u003eFigure 2f\u003c/strong\u003e). However, \u003cstrong\u003eOs1\u003c/strong\u003e is unable to produce any ROSs under hypoxia (\u003cstrong\u003eFigure S16\u003c/strong\u003e).\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eGeneration of cellular ROS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe studied the photo-induced ROS generation ability of \u003cstrong\u003eOs2\u003c/strong\u003e in living HeLa cells. Dihydroethidium (DHE), hydroxyphenyl fluorescein (HPF) and singlet oxygen sensor green (SOSG) were used as probes to monitor the production of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, OH\u0026bull; and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, respectively.\u003csup\u003e[18a,b]\u0026nbsp;\u003c/sup\u003eThe detection mechanisms of these probes for various free radicals are shown in \u003cstrong\u003eFigure 3a\u003c/strong\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eConfocal microscopy showed that when \u003cstrong\u003eOs2\u003c/strong\u003e-incubated HeLa cells were exposed to light under\u0026nbsp;normoxia\u0026nbsp;(20% O\u003csub\u003e2\u003c/sub\u003e, 465 nm, 13 mW/cm\u003csup\u003e2\u003c/sup\u003e, 1 h), the fluorescence signal in DHE-stained cells is significantly enhanced (\u003cstrong\u003eFigure 3b,c\u003c/strong\u003e). This confirms the ability of \u003cstrong\u003eOs2\u003c/strong\u003e to release O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e in cells under light irradiation. Under hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e), the fluorescence intensity after light irradiation was similar to that obtained under normoxia, indicating that the process of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e generation from \u003cstrong\u003eOs2\u003c/strong\u003e is independent of O\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;This is different from the mechanism of O\u003csub\u003e2\u003c/sub\u003e-dependent type I photodynamic mechanism.\u003c/p\u003e\n\u003cp\u003eBecause it is known that O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e can produce OH\u0026bull; in the presence of intracellular SOD enzyme and Fe\u003csup\u003e2+\u003c/sup\u003e ions,\u003csup\u003e[18a]\u003c/sup\u003e we further studied the generation of OH\u0026bull; in the cells by HPF staining. Confocal imaging showed that \u003cstrong\u003eOs2\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003c/strong\u003ecan produce OH\u0026bull; in HeLa cells under normoxia or hypoxia (\u003cstrong\u003eFigures 3b, 3c\u003c/strong\u003e). In addition, we also compared the ability of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egeneration of \u003cstrong\u003eOs2\u003c/strong\u003e and the photoproduct\u003cstrong\u003e\u0026nbsp;Os1\u0026nbsp;\u003c/strong\u003eby using a SOSG probe, and found that under hypoxia,\u003cstrong\u003e\u0026nbsp;Os2\u003c/strong\u003e has more advantages in producing \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e than \u003cstrong\u003eOs1\u003c/strong\u003e. The result showed that the green fluorescence in the \u003cstrong\u003eOs2\u003c/strong\u003e treated cells is strong under both normoxia and hypoxia. However, the fluorescence in the \u003cstrong\u003eOs1\u003c/strong\u003e treated cells was much weaker under hypoxia than that observed under normoxia (\u003cstrong\u003eFigure S17\u003c/strong\u003e). These results are consistent with those in \u003cstrong\u003eFigure 2f\u003c/strong\u003e, showing that \u003cstrong\u003eOs2\u003c/strong\u003e firstly induces the release of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and production of \u003cstrong\u003eOs1\u003c/strong\u003e, accompanied by the formation of O\u003csub\u003e2\u003c/sub\u003e (as an oxygen source for \u003cstrong\u003eOs1\u003c/strong\u003e) and OH\u0026bull; by a Fenton reaction. Finally \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eis generated by the photoproduct, \u003cstrong\u003eOs1\u003c/strong\u003e. These data all show that \u003cstrong\u003eOs2\u003c/strong\u003e may be useful as a new effective drug for photoactive treatment of hypoxic tumor cells.\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003ePhototoxicity \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dark- and photo-cytotoxicities of \u003cstrong\u003eOs2\u003c/strong\u003e and \u003cstrong\u003eOs1\u003c/strong\u003e against HeLa cells were determined with an MTT method.\u003csup\u003e[19]\u003c/sup\u003e As showed in \u003cstrong\u003eFigure 4\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eTable 1\u003c/strong\u003e, \u003cstrong\u003eOs2\u003c/strong\u003e had low dark-cytotoxicity and high photo-cytotoxicity under both normoxia and hypoxia (\u003cstrong\u003eFigures 4a, 4c\u003c/strong\u003e). The (IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003edark\u0026nbsp;\u003c/sub\u003evalues, determined with only dark incubation, were 89.2 and \u0026gt;100 \u0026mu;M, respectively (\u003cstrong\u003eTable 1\u003c/strong\u003e). The (IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003eLight\u003c/sub\u003e values of \u003cstrong\u003eOs2\u003c/strong\u003e, determined by photoirradiation under normoxia or hypoxia were 1.23 and 5.86 \u0026mu;M, respectively. The photo-cytotoxicity under hypoxia can be explained in terms of the release of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by \u003cstrong\u003eOs2\u003c/strong\u003e and light irradiation, followed by the production of O\u003csub\u003e2\u003c/sub\u003e, which may be used by the light product \u003cstrong\u003eOs1\u003c/strong\u003e to generate \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, and OH\u0026bull; with stronger toxicity \u003cem\u003evia\u003c/em\u003e the Fenton reaction. In contrast, the light product (\u003cstrong\u003eOs1\u003c/strong\u003e) shows high dark-cytotoxicity and photo-cytotoxicity under both normoxia and hypoxia (\u003cstrong\u003eFigures 4b, 4d\u003c/strong\u003e). The (IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003edark\u0026nbsp;\u003c/sub\u003evalues of \u003cstrong\u003eOs1\u003c/strong\u003e under normoxia and hypoxia are 8.12 and 9.95 \u0026mu;M, and the (IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003eLight\u003c/sub\u003e values under normoxia and hypoxia are 1.31 and 7.51 \u0026mu;M, respectively (\u003cstrong\u003eTable 1\u003c/strong\u003e). This result suggests that \u003cstrong\u003eOs1\u003c/strong\u003e is high photo- and dark- cytotoxic, and can be seen as a chemotherapeutic drug. However, with hypoxia, due to the lack of oxygen, \u003cstrong\u003eOs1\u003c/strong\u003e shows no obvious photodynamic effect. The PI value of \u003cstrong\u003eOs2\u003c/strong\u003e (\u0026gt;18) under hypoxia is much higher than that of \u003cstrong\u003eOs1\u0026nbsp;\u003c/strong\u003e(1.3).\u003c/p\u003e\n\u003cp\u003eStaining with alcein acetoxymethyl ester (Calcein-AM) or propidium iodide (PI) were also used to distinguish living cells (green) from dead cells (red). As shown in \u003cstrong\u003eFigure S18\u003c/strong\u003e, the dark group of \u003cstrong\u003eOs2\u003c/strong\u003e in normoxia and hypoxia shows strong green fluorescence (living cells) but no red fluorescence (dead cells), and the control group in the dark or under light irradiation also shows no dead cells. In contrast, the light group of \u003cstrong\u003eOs2\u003c/strong\u003e shows weak green fluorescence and strong red fluorescence, indicating that \u003cstrong\u003eOs2\u003c/strong\u003e leads to a large number of dead cells under light irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp id=\"isPasted\" style='margin-top:11.5pt;margin-right:0in;margin-bottom:23.0pt;margin-left:0in;text-align:justify;line-height:150%;font-size:9px;font-family:\"Arial\",sans-serif;margin:0in;'\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable 1.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:14px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eThe dark- and photo-IC\u003csub\u003e50\u003c/sub\u003e values of \u003cstrong\u003eOs2\u003c/strong\u003e and \u003cstrong\u003eOs1\u003c/strong\u003e against HeLa cells under normoxia and hypoxia.\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"border: none;width:100.0%;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28.56%;border-color: black currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003eCompounds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.14%;border-color: black currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003eDark [\u0026mu;M]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.56%;border-color: black currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003eLight\u003csup\u003e[c]\u003c/sup\u003e [\u0026mu;M]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.74%;border-color: black currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003ePI\u003csup\u003e[d]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28.56%;border: medium none;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003eOs2\u003c/strong\u003e\u003csup\u003e[a]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.14%;border: medium none;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e89.2\u0026plusmn;2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.56%;border: medium none;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e1.23\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.74%;border: medium none;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e72.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28.56%;padding: 0in 5.4pt;height: 17.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003eOs2\u003c/strong\u003e\u003csup\u003e[b]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.14%;padding: 0in 5.4pt;height: 17.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cspan style=\"font-family: DengXian;\"\u003e＞\u003c/span\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.56%;padding: 0in 5.4pt;height: 17.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e5.86\u0026plusmn;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.74%;padding: 0in 5.4pt;height: 17.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cspan style=\"font-family: DengXian;\"\u003e＞\u003c/span\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28.56%;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003eOs1\u003c/strong\u003e\u003csup\u003e[a]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.14%;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e8.12\u0026plusmn;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.56%;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e1.31\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.74%;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 28.56%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003eOs1\u003c/strong\u003e\u003csup\u003e[b]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.14%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e9.95\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.56%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e7.51\u0026plusmn;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.74%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 16.8pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;line-height:150%;'\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:11.5pt;margin-right:0in;margin-bottom:23.0pt;margin-left:0in;text-align:justify;line-height:150%;font-size:9px;font-family:\"Arial\",sans-serif;margin:0in;'\u003e\u003csup\u003e\u003cspan style='font-size: 14px; line-height: 150%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003e[a]\u003c/span\u003e\u003c/sup\u003e\u003cspan style='font-size: 14px; line-height: 150%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003e\u0026nbsp;under normoxia (20% O\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e[b]\u003c/sup\u003e under hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e[c]\u003c/sup\u003e photoirradiation was imposed (465 nm, 13 mW/cm\u003csup\u003e2\u003c/sup\u003e, 1 h) after 8 h of the complexes incubation; [d] photocytotoxicity index, the ratio of (IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003edark\u003c/sub\u003e/(IC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003eLight\u003c/sub\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eFerroptosis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been reported that the photo-chemical process of ROS generation could cause ferroptosis in cancer cells.\u003csup\u003e[2e, 20]\u003c/sup\u003e This led us to consider if \u003cstrong\u003eOs2\u003c/strong\u003e can induce ferroptosis. GSH is closely related to ferroptosis,\u003csup\u003e[21]\u003c/sup\u003e and we first detected the ability of \u003cstrong\u003eOs2\u003c/strong\u003e to consume GSH. As shown in \u003cstrong\u003eFigures 5a-5b\u003c/strong\u003e, with an increase of light irradiation time, the absorption at 412 nm decreased, indicating that \u003cstrong\u003eOs2\u003c/strong\u003e when irradiated, could consume GSH. We determined the GSH levels in the cells after treatment\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewith\u003cstrong\u003e\u0026nbsp;Os2\u003c/strong\u003e, and found that the GSH level in the irradiated group was significantly lower than that in the non-irradiated group (\u003cstrong\u003eFigure 5c\u003c/strong\u003e). Therefore, we concluded that \u003cstrong\u003eOs2\u003c/strong\u003e can consume cellular GSH under irradiation conditions.\u003c/p\u003e\n\u003cp\u003eFerroptosis is a type of iron-dependent cell death caused by excessive lipid peroxidation. The main feature of ferroptosis is that after the inactivation of cell antioxidant capacity, phospholipids containing polyunsaturated fatty acids are peroxidated on the cell membrane, destroying the cell membrane and leading to ferroptosis.\u003csup\u003e[22]\u003c/sup\u003e The antioxidant glutathione peroxidase 4 (GPX4) specifically catalyzes loss of oxidative activity in the lipid peroxides in a glutathione-dependent manner,\u003csup\u003e[23]\u003c/sup\u003e and subsequent inhibition of GPX4 induces ferroptosis. GSH consumption can indirectly inhibit the expression of GPX4. We speculated that \u003cstrong\u003eOs2\u003c/strong\u003e could further inhibit the expression of GPX4 and we verified this hypothesis by western blot analysis of GPX4. As shown in \u003cstrong\u003eFigures 5f-5g\u003c/strong\u003e, \u003cstrong\u003eOs2\u003c/strong\u003e fails to reduce the expression of GPX4 in the dark. Upon light irradiation, \u003cstrong\u003eOs2\u003c/strong\u003e significantly reduces the expression of GPX4, and the GSH consumption caused by ROS and the inhibition of GPX4 will further lead to the accumulation of lipid peroxides and induce ferroptosis. We used C11-BODIPY as a lipid peroxide probe with which to monitor intracellular accumulation of lipid peroxides (\u003cstrong\u003eFigures 5d, 5e\u003c/strong\u003e). Confocal microscopy showed that the fluorescence of HeLa cells treated with \u003cstrong\u003eOs2\u003c/strong\u003e was significantly enhanced after exposure to light, indicating a significant accumulation of lipid peroxides, which could be effectively inhibited by Ferrostatin-1 (Fer-1, a ferroptosis inhibitor). All the above results confirm that \u003cstrong\u003eOs2\u003c/strong\u003e induces ferroptosis as shown in \u003cstrong\u003eFigure 5h\u003c/strong\u003e.\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eNADH photocatalytic oxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a cofactor, 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) regulates the redox balance of cellular mitochondria, and plays an important role in regulating energy production. If NADH is oxidized to NAD\u003csup\u003e+\u003c/sup\u003e, it can destroy the whole respiratory chain and kill cells.\u003csup\u003e[24]\u003c/sup\u003e We studied whether NADH can be oxidized by\u003cstrong\u003e\u0026nbsp;Os2\u003c/strong\u003e under light irradiation, which could provide a photocatalytic oxidation pathway to kill cancer cells. The photocatalytic efficiency of \u003cstrong\u003eOs2\u003c/strong\u003e (20 \u0026mu;M) towards NADH (175 \u0026mu;M) was first determined by UV-Vis absorption spectroscopy. As shown in \u003cstrong\u003eFigure 6a\u003c/strong\u003e, the absorbance at 339 nm decreases and the absorbance at 259 nm increases gradually with increase of the irradiation time. In contrast, the absorption of the non-illuminated \u003cstrong\u003eOs2\u003c/strong\u003e group exhibits no obvious changes (\u003cstrong\u003eFigure S19\u003c/strong\u003e). This indicates that \u003cstrong\u003eOs2\u003c/strong\u003e could reduce the enzyme activity of NADH under light irradiation. We also calculated the NADH oxidation turnover number (TON) of \u003cstrong\u003eOs2\u003c/strong\u003e at 339 nm to evaluate its photocatalytic efficiency. The TON value of NADH oxidation by \u003cstrong\u003eOs2\u003c/strong\u003e under light irradiation is 3.808, which is 30-times higher than that of the non-illuminated group (\u003cstrong\u003eFigure 6b\u003c/strong\u003e). Similarly, \u003cstrong\u003eOs1\u003c/strong\u003e also shows a similar photocatalytic oxidation effect on NADH (\u003cstrong\u003eFigure S20\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe photocatalytic oxidation of NADH was also monitored by \u003csup\u003e1\u003c/sup\u003eH NMR in D\u003csub\u003e2\u003c/sub\u003eO/CD\u003csub\u003e3\u003c/sub\u003eOD (1/3, v/v) at 298 K. As shown in\u003cstrong\u003e\u0026nbsp;Figure 6c\u003c/strong\u003e, In the \u003cstrong\u003eOs2\u0026nbsp;\u003c/strong\u003eand NADH illuminated group, new peaks from hydrogens on the nicotinamide ring of NAD\u003csup\u003e+\u003c/sup\u003e are observed at 6.13, 8.31, 8.55, 8.99, 9.36 and 9.58, but no new NAD\u003csup\u003e+\u003c/sup\u003e peak is observed in either the non-illuminated group or the non-\u003cstrong\u003eOs2\u003c/strong\u003e group (\u003cstrong\u003eFigure 6c\u003c/strong\u003e). Intuitively, this shows that \u003cstrong\u003eOs2\u003c/strong\u003e could oxidize NADH under light irradiation, transforming it into NAD\u003csup\u003e+\u003c/sup\u003e. Subsequently, we measured the NADH photocatalytic oxidation ratio at the cellular level using a NAD/NADH-Glo\u003csup\u003eTM\u003c/sup\u003e method, which is a bioluminescence method for the detection of NAD\u003csup\u003e+\u003c/sup\u003e and NADH. As shown in \u003cstrong\u003eFigure 6d\u003c/strong\u003e, the chemical luminescence intensity of \u003cstrong\u003eOs2\u003c/strong\u003e-light group proved to be lower than that of other groups. These results show that \u003cstrong\u003eOs2\u003c/strong\u003e can effectively oxidize NADH at the cellular level by light irradiation, thereby killing cancer cells. As can be seen in \u003cstrong\u003eFigure 6e\u003c/strong\u003e, NADH can not only be converted to NAD\u003csup\u003e+\u003c/sup\u003e by interacting with Os1\u003csup\u003e*\u0026nbsp;\u003c/sup\u003e(the excited state of \u003cstrong\u003eOs1\u003c/strong\u003e), but can also be oxidized to NAD\u003csup\u003e+\u003c/sup\u003e by O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e released from \u003cstrong\u003eOs2\u003c/strong\u003e. The down-regulation of NADH indirectly\u0026nbsp;aids the reduction of oxidized glutathione (GSSG) to GSH by glutathione reductase (GR),\u003csup\u003e[25]\u003c/sup\u003e resulting in the accumulation of lipid peroxides, and ultimately achieving the synergistic induction of ferroptosis.\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003ePhotoactive antitumor therapy \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe studied the feasibility of photoactive therapy \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eby\u0026nbsp;Os2. Since\u0026nbsp;Os2\u0026nbsp;is a small molecule and there is no specific targeted group, it can be administered intratumorally. As shown in\u0026nbsp;Figure 7b, compared with the other three groups, the tumor growth in mice treated with\u0026nbsp;Os2-light group was inhibited. The tumor size of the\u0026nbsp;Os2-light group was the smallest in the four groups (Figure 7d), and the average tumor weight of the\u0026nbsp;Os2-light group was significantly lower than that of the other groups (Figure 7c). The tumor tissues after final treatment were collected for histological assessment. The hematoxylin and eosin (H\u0026amp;E) staining showed obvious destruction of tumor tissues in the\u0026nbsp;Os2-light group, while tumor tissues in the other three groups were not affected (Figure 7e).\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the biological safety of\u0026nbsp;Os2, we first\u0026nbsp;analyzed\u0026nbsp;the H \u0026amp; E staining slices of the main organs of healthy mice i.v. injected with three times of the therapeutic dose (2.69 mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e). The results showed no obvious tissue damage in these slices\u0026nbsp;(Figure S21).\u0026nbsp;We also used Singapore wild-type zebrafish to test the biological safety of\u0026nbsp;Os2\u0026nbsp;(Figure S22) with the green fluorescent protein (GFP) commonly used as a biomarker to visualize the physiological processes. After 5 days of incubation with\u0026nbsp;Os2, the blood vessels of the zebrafish were apparently not damaged, showing good biocompatibility \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we\u0026nbsp;have\u0026nbsp;demonstrated that an osmium-peroxo complex\u0026nbsp;(\u003cstrong\u003eOs2\u003c/strong\u003e)\u0026nbsp;can release O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under light irradiation in the absence of O\u003csub\u003e2\u003c/sub\u003e, and at the same time is transformed into another active osmium complex (\u003cstrong\u003eOs1\u003c/strong\u003e), which exhibits both chemotherapeutic and photodynamic properties, thus maintaining good phototoxicity in hypoxic tumors. The\u0026nbsp;osmium-peroxo\u0026nbsp;complex \u003cstrong\u003eOs2\u003c/strong\u003e can induce ferroptosis, which is characterized by GSH degradation, GPX4 down-regulation and lipid peroxide accumulation. In addition, under light irradiation, the same\u0026nbsp;osmium-peroxo\u0026nbsp;complex oxidizes NADH into NAD\u003csup\u003e+\u003c/sup\u003e, further helping induction of ferroptosis. At the \u003cem\u003ein vivo\u003c/em\u003e level, the\u0026nbsp;osmium-peroxo\u0026nbsp;complex\u0026nbsp;achieves\u0026nbsp;highly effective photoactive therapy of solid hypoxic tumors.\u0026nbsp;This study reports the first example of a\u0026nbsp;metal-peroxo\u0026nbsp;complex for O\u003csub\u003e2\u003c/sub\u003e-independent photoactive therapy and provides a promising strategy for combating hypoxic tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the financial support of the National Natural Science Foundation of China (NSFC, 22077085, 22007104 and 21931002), and the Science and Technology Foundation of Shenzhen (JCYJ20190808153209537 and JCYJ20200109140812302). We appreciate the Instrumental Analysis Center of Shenzhen University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN. L., J. G., H. X., and P. Z. designed the study. Z. D. synthesized and characterized the complexes. N. L. and C. L. performed the experiments in vitro and in vivo. N. L., Z. D., J. G.,\u0026nbsp;H. X. and P. Z. analyzed the data and wrote the paper. All authors contributed to the general discussion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e[1]\u0026nbsp;X. Zhao, J. Liu, J. Fan, H. Chao, X. Peng, \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 50 (6), 4185-4219.\u003c/p\u003e\n\u003cp\u003e[2]\u0026nbsp;a) C. Imberti, P. Zhang, H. Huang, P. J. Sadler, \u003cem\u003eAngew. Chem. Int. Ed.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, 59 (1), 61-73; b) S. Monro, K. L. Colon, H. Yin, J. 3\u003csup\u003erd\u003c/sup\u003e. Roque, P. Konda, S. Gujar, R. P. 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Herein, we report our investigation of an osmium-peroxo complex (\u003cb\u003eOs2\u003c/b\u003e), which is inactive in the dark, but upon light irradiation, can release a peroxo ligand O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, and is transformed into a cytotoxic osmium complex (\u003cb\u003eOs1\u003c/b\u003e). The osmium-peroxo complex \u003cb\u003eOs2\u003c/b\u003e produces O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e under light irradiation even in the absence of oxygen, and retains its phototoxicity in hypoxic tumors. \u003cb\u003eOs1\u003c/b\u003e is cytotoxic in the presence or absence of irradiation, behaves as a chemotherapeutic drug. The light-activated \u003cb\u003eOs2\u003c/b\u003e induces distinct ferroptosis, which is mediated by GSH degradation, lipid peroxide accumulation and down-regulation of glutathione peroxidase 4 (GPX4). In addition, \u003cb\u003eOs2\u003c/b\u003e causes photocatalytic oxidation of endogenous 1,4-dihydronicotinamide adenine dinucleotide (NADH) in living cancer cells, leading to ferroptosis. \u003cem\u003eIn vivo\u003c/em\u003e studies have confirmed that the \u003cb\u003eOs2\u003c/b\u003e can effectively inhibit the growth of solid hypoxic tumors in mice. A new strategy is proposed for the treatment of hypoxic tumors with metal-based drugs.\u003c/p\u003e","manuscriptTitle":"An osmium-peroxo complex for photoactive therapy of hypoxic tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-30 22:27:01","doi":"10.21203/rs.3.rs-1088102/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e453dcc-c7e3-4e22-9386-e241856dbab9","owner":[],"postedDate":"November 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":8853368,"name":"Bioinorganic chemistry"},{"id":8853370,"name":"Applied Biochemistry"},{"id":8853371,"name":"Medicinal Chemistry"},{"id":8853372,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2022-04-11T09:51:00+00:00","versionOfRecord":[],"versionCreatedAt":"2021-11-30 22:27:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1088102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1088102","identity":"rs-1088102","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}