In situ oxygen generation by a low-toxicity ruthenium electrocatalyst for multimodal radiotherapy sensitization

Abstract

The efficacy of radiotherapy is often significantly compromised due to tumor hypoxia. We developed a novel strategy to overcome tumor hypoxia and enhance radiotherapy using a low-toxicity catalyst with high-Z atoms. We employed in situ electrocatalytic oxygen generation in the tumor to improve the hypoxic state and sensitize radiotherapy. By employing multi-dentate chelating ligands in conjunction with a high-Z Ru metal center, we constructed a low-toxicity electrocatalyst for water oxidation: Ru(bbp)(Py)₂Cl. On the one hand, Ru(bbp)(Py)₂Cl served as a low-toxicity catalyst for electrocatalytic oxygen production, improving the hypoxic condition in the tumor. On the other hand, Ru enhanced the sensitivity of radiotherapy in response to X-ray, significantly boosting the therapeutic effect. In vitro and vivo experimental results revealed that our in situ electrocatalytic oxygen-production strategy could directly generate oxygen within the body, effectively alleviating tumor hypoxia. Furthermore, this strategy employed a multi-faceted sensitization mechanism by producing excess reactive oxygen species, which disrupted mitochondrial function and induced activation of the apoptosis-regulating proteins caspase-3 and caspase-9, ultimately triggering apoptosis and achieving significant anti-cancer effects. This research provides a novel approach to improving the hypoxic environment in tumors, but also opens new avenues for sensitizing radiotherapy, potentially leading to breakthrough advancements in cancer treatment.

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Introduction

Radiotherapy is one of the most important means of tumor treatment,^1–6 but the existence of tumor hypoxia severely limits the efficacy of radiotherapy.^7–12 Currently, oxygen microbubbles,¹³in situ generation of O₂ by metal ions or a catalase reaction with intratumoral H₂O₂,¹⁴ and oxygen-carrying perfluorocarbons¹⁵ have been studied to improve tumor hypoxia. However, low intratumor H₂O₂ concentration and insufficient O₂ supply limit their applications.^16–21 Therefore, it is necessary to explore more efficient strategies for O₂ generation to address tumor hypoxia.

Electrotherapy and electrodynamic therapy are used to kill tumor cells by generating large amounts of reactive oxygen species (ROS) through electrochemical reactions at electrode sites using direct and alternating current. This action induces changes in pH, and alters the transmembrane transport of ions.^22–27 The large amount of water in tumor tissues and various inorganic salts contained in body fluids are ideal electrolyte systems in the electrochemical system. Under electro/electrodynamic therapy, the OER reaction is carried out to electrocatalyze the decomposition of water to produce oxygen, which can achieve the precise release of oxygen.^28–32 However, conventional electrochemical therapy for the OER reaction is inefficient and occurs under harsh conditions.^33–36 Therefore, we are interested in designing a low-toxicity metal drug with water oxidation catalytic activity to achieve in situ electrocatalytic oxygen production in tumors to improve tumor hypoxia while using metal atoms of high atomic number in response to X-ray sensitizing radiotherapy.

Ruthenium is a metal with a high atomic number. It is rich in inner electrons, which become excited to jump under the applied energy to produce photoelectrons to realize the sensitization of radiotherapy.^37–42 Meanwhile, bipyridine ruthenium complexes can electrocatalyze water oxidation.^43–50 Based on these premises, in the present work, low-toxicity ruthenium complexes with electrocatalytic water oxidation activity were synthesized by coordinating with pyridine using benzimidazole ligands as co-ligands. On the one hand, the synthesized ruthenium complexes efficiently produce oxygen by lowering the in situ electrocatalytic oxygen production voltage, which improves the tumor hypoxia and, thus, enhances the radiotherapy effect. On the other hand, high-atomic-number ruthenium responds to radiophysical sensitization radiotherapy. Under multiple sensitization effects, the sensitization ratio of radiotherapy at the cellular level reached 5.3. Meanwhile, by constructing electrode arrays, the sensitization effect of radiotherapy in vivo was demonstrated to be increased 13.7-times compared with that in the control group in nude mice subcutaneous tumors and C57 mouse melanoma models. In summary, this study provides a new method for improvement in tumor hypoxia for the clinic and a new idea for subsequent tumor treatment (Scheme 1).

Scheme 1 Ru(bbp)(Py)₂Cl in situ electrocatalytic oxygen production to improve tumor hypoxia and enhance the radiotherapy effect (schematic).

Results and discussion

Design, synthesis and characterization of low-toxicity ruthenium complexes as multiple radiotherapy sensitizers

Tumor hypoxia is a key factor affecting tumor treatment.^51–54 In order to overcome the tumor hypoxia caused by radiotherapy tolerance, we used water (which exists in large quantities in cells) as a raw material. Also, we utilized electrocatalytic water decomposition to generate oxygen controllably in the tumor in situ, which generates oxygen to alleviate the tumor hypoxia and simultaneously sensitizes the radiotherapy. In order to achieve controlled in situ oxygen release at the tumor site and sensitization for radiotherapy, we selected ruthenium as the metal center which, on the one hand, utilizes the excellent electrocatalytic ability of ruthenium metal to achieve in situ water oxidation of tumors to improve tumor hypoxia and sensitize radiotherapy. On the other hand, it utilizes the compton effect and photoelectric effect under the rays of the elemental ruthenium with a high atomic number to sensitize radiotherapy (Fig. 1a). In order to synthesize stable and low-toxic ruthenium complexes, we chose the tridentate ligand 2,6-bis (2-benzimidazolyl)pyridine (bbp) and the auxiliary ligand pyridine (Py) to synthesize Ru(bbp)(Py)₂Cl. Electrospray ionization mass spectrometry (ESI-MS) (Fig. S1a†), UV/vis spectroscopy (Fig. S1b†), ¹H NMR spectroscopy (Fig. S1c†) and single-crystal X-ray diffraction (Fig. S1d and Table S1†) revealed the synthesis of the complexes. The purity of the complexes was verified by PXRD (Fig. S1e†). The stability of the complexes was also confirmed by UV/vis in PBS (Fig. S2a†) and DMEM (Fig. S2b†). Meanwhile, in order to verify the biosafety of Ru(bbp)(Py)₂Cl, the MTT assay showed that the IC₅₀ of Ru(bbp)(Py)₂Cl was 9.9 μM for normal cervical (Ect1/E6E7) cells, 9.2 μM for human renal tubular epithelial cells, and 4.1 μM for cervical cancer (HeLa) cells (Fig. 1b). These experimental results demonstrated that our synthesized Ru(bbp)(Py)₂Cl possessed good catalytic activity, but also had enhanced biosafety because it significantly reduced the toxicity to normal cells while maintaining efficient antitumor activity.

Fig. 1 In situ oxygen production by Ru(bbp)(Py)₂Cl electrocatalysis. (a) Synthesis of Ru(bbp)(Py)₂Cl and electrocatalytic oxygen production (schematic). (b) Toxicity test of Ru(bbp)(Py)₂Cl on normal cervical (Ect1/E6E7) cells and in cervical cancer (HeLa) cells. (c) Cyclic voltammograms of the complex (0.5 mM) in tetrabutylammonium chloride acetonitrile solution as well as in acetonitrile/water (100 μL) mixed solution. Scan rate = 100 mV s⁻¹. (d) An oxygen probe was used to detect the amount of oxygen produced by the blank group, electrocatalytic alone (3 V) group and the electrocatalytic combined Ru(bbp)(Py)₂Cl group (60 μM). (e) Detection of oxygen content in HeLa cells after treatment with control, electrocatalysis alone (3 V) and electrocatalysis with Ru(bbp)(Py)₂Cl (5 μM) groups using an oxygen probe. (f) Detection of the amelioration of oxygen deprivation in mouse slices after treatments in control, electrocatalytic alone (3 V) and electrocatalytic with Ru(bbp)(Py)₂Cl (2.5 mg kg⁻¹) groups using an oxygen-deprivation probe. (g) Oxygen production after in situ electrocatalytic oxygen-production treatment of mouse tumors was monitored using B-ultrasound.

In situ electrocatalytic oxygen generation in vitro and in vivo

As a cutting-edge and highly efficient energy conversion technology, electrocatalytic water oxidation has shown great potential in biomedical fields (especially in tumor therapy) in recent years. In tumor therapy, the in situ generation of oxygen by electrocatalysis can effectively alleviate the anoxic state of tumor tissues and, thus, improve the therapeutic effect. In view of whether our synthesized Ru(bbp)(Py)₂Cl could undertake electrocatalytic water oxidation as a key indicator for tumor treatment, we first validated it using an electrochemical workstation, and examined its redox properties and electrocatalytic water decomposition ability using cyclic voltammetry (CV). Ru(bbp)(Py)₂Cl alone presented a quasi-reversible peak at a potential of 0.6 V, which was identified as the redox peak of Ru^II/III. When a trace amount of water was added to the system, a water oxidation catalytic peak was observed at a potential of 1.2 V (Fig. 1c), which indicated that the complex, as a catalyst, could effectively catalyze the decomposition of water at 1.2 V. It was a suitable catalyst for in situ oxygen production under electrochemical conditions, and exhibited a great potential for further application for the in situ electrocatalytic production of oxygen in tumors.

In order to further verify whether the ruthenium complex Ru(bbp)(Py)₂Cl could generate oxygen under electrocatalytic conditions, we conducted comprehensive in vitro, cellular, animal-tissue section and in vivo animal assays using the oxygen probe RDPP, the hypoxia-probe pimozole and in situ ultrasound techniques, respectively. First, in in vitro experiments, we evaluated the oxygen-generation capacity of the complexes under electrocatalysis using the oxygen probe RDPP. The oxygen content of electrocatalytic group 3 alone increased by 9.6% compared with that in the control group 1, whereas the oxygen content of co-treated group 4 upon the addition of Ru(bbp)(Py)₂Cl increased significantly by 80.13% (Fig. 1d). This result was validated in cell and animal experiments. We incubated cells and animal-tissue sections with the oxygen probe RDPP (oxygen causes its fluorescence to burst, so it is used as an oxygen-detection tool) and the lack-of-oxygen probe pimozole. In the combined treatment group of the complex and electrocatalysis, the fluorescence of RDPP in HeLa cells (1: 100%; 3: 66.08%; 4: 10.92%) and sections burst, while the lack-of-oxygen region was reduced (Fig. 1e and f). These data further confirmed that the complexes could generate oxygen under electrocatalytic conditions, thus alleviating tumor hypoxia. Meanwhile, in order to observe the oxygen production more intuitively, we examined the treated mice using in situ ultrasound technology. A signal of oxygen production was not detected in control mice, whereas obvious oxygen signals were observed in the electrocatalytic group 3 as well as in the complex-combined electrocatalytic group 4. Also, the oxygen signals in the combined group 4 were more pronounced compared with those in the electrocatalytic group 3 alone (Fig. 1g). These results demonstrated that the complexes could produce oxygen in situ under electrocatalytic conditions, effectively overcoming the problem of tumor hypoxia.

In situ oxygen production regulates redox homeostasis

We validated the ability of our in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy to generate oxygen. This strategy effectively improved the tumor hypoxic environment, but also absorbs X-ray energy and converts it into highly active ROS which, in turn, damage tumor cells (Fig. 2a). In this process, the stability of the drug under physiological conditions is a key factor before its entry into clinical applications. Therefore, we first verified the stability of ruthenium complexes under different treatment conditions. The structure of the complex was not affected after X-ray (dose of 2 Gy) and electrocatalytic (voltage of 3 V for 3 min) treatments (Fig. S2c†). This structural stability in physiological solution and after treatment further validated the feasibility of our proposed in situ electrocatalytic oxygen-producing sensitizing radiotherapy strategy.

Fig. 2 The electrocatalytic oxygen-production strategy promotes ROS production in HeLa cells. (a) Effect of the in situ electrocatalytic oxygen-production strategy on intracellular reactive oxygen species (schematic). (b) Detection of the superoxide anion produced by different treatment groups at different times of treatment in vitro using the DHE probe. (c) Detection of the hydroxyl radical produced by different treatment groups at different times of treatment using methylene blue in vitro. (d) Effect of different treatment groups of treatment on the total reactive oxygen species in HeLa cells. (e) Effect of different treatment groups of treatment on single-linear state oxygen species in HeLa cells. (f) Effect of different treatment groups of treatments on the superoxide anion in HeLa cells. (g) Principle of detection of reactive oxygen species by DCFH-DA, DPBF and DHE probes (schematic).

Notably, although the characteristic UV absorption peaks of the complexes did not change after treatment, we found that their aqueous solutions showed increasing absorption peaks at 225 nm after different durations of treatment (Fig. S2d†). Through literature research, we hypothesized that this could be due to the changes in UV absorption caused by production of the superoxide anion after treatment. To further validate the production of superoxide anion as well as other ROS, we used ESR to detect in situ the free-radical changes of the DMPO radical trap after different treatments. Several control and experimental groups were set up (1: control group; 2: Ru(bbp)(Py)₂Cl group; 3: electrocatalytic group; 4: Ru(bbp)(Py)₂Cl combined electrocatalytic group; 5: X-ray group; 6: Ru(bbp)(Py)₂Cl combined X-ray group; 7: electrocatalytic combined with X-ray group; 8: Ru(bbp)(Py)₂Cl combined with electrocatalytic group combined with X-ray group). The groups without combined radiotherapy treatment (1–4) did not produce significant hydroxyl radicals, whereas the radiotherapy-treated groups (5–7) showed absorption peaks of hydroxyl radicals. In the final combined group (8), we observed the characteristic peak of the superoxide anion (Fig. S3a†). In addition, we captured the free-radical production in the different treatment groups in situ using a free radical-capture reagent (TEMP). The groups without combined radiotherapy (1, 2, 5, 6) did not produce single-linear state oxygen, while the groups with combined electrocatalysis or radiotherapy (3, 4, 7, 8) showed absorption peaks of single-linear state oxygen (Fig. S3b†). Meanwhile, we further confirmed, by in situ DHE probe, that the final combined superoxide anion radical (O₂˙⁻) was generated to produce different types of radicals by disproportionation or reaction with other substances. In order to further verify the effect of the in situ electrocatalytic oxygen-production strategy on other kinds of ROS, we examined each of them in vitro by DCFH-DA, MB (hydroxyl radicals cause the degradation of methylene blue), and TEMP probes. Relative to the other treatment groups, total ROS (Fig. S3c†) (1: 100%; 2: 103.0%; 3: 897.7%; 4: 1029.5%; 5: 209.5%; 6: 235.2%; 7: 3820.8%; 8: 5228.1%), and hydroxyl radicals (Fig. 2c and S3d†) and, ultimately, the combined group (8), could induce stronger production of ROS, which further suggested that our in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy induced ROS overproduction after ameliorating hypoxia.

To further validate the feasibility of our in situ electrocatalytic oxygen-production strategy, we detected ROS at the cellular level using DCFH-DA, DPBF, and DHE probes, respectively (Fig. 2g). The final combined group (8) could elicit stronger production of total ROS (Fig. 2d) relative to the other treatment groups (1: 100%; 2: 146.8%; 3: 135.3%; 4: 205.7%; 5: 162.0%; 6: 229.3%; 7: 225.8%; 8: 421.6%), singlet oxygen (Fig. 2e) (1: 100%; 2: 0.2%; 3: 10.5%; 4: 18.0%; 5: 29.6%; 6: 22.7%; 7: 39.5%; 8: 51.0%) and the superoxide anion (Fig. 2f) (1: 100%; 2: 112.3%; 3: 98.1%; 4: 105.3%; 5: 103.0%; 6: 126.2%; 7: 126.6%; 8: 144.3%). These data further illustrated that we utilized electrocatalytic oxygen production by Ru(bbp)(Py)₂Cl as a strategy to alleviate tumors. And sensitizing radiotherapy strategy to alleviate the anoxic state of tumor tissues for tumor treatment.

In situ oxygen generation achieves efficient anticancer activity in vitro

We verified in vitro experiments that the in situ electrocatalytic oxygen generation sensitizing radiotherapy strategy could trigger the generation of large amounts of ROS after improving the oxygen-deprived environment of tumors by generating oxygen, and that ROS overproduction was effective in inducing cell death (Fig. 3a). In order to deeply investigate the anti-tumor activity of Ru(bbp)(Py)₂Cl, we chose cervical cancer as a model to investigate its antitumor effect during in situ electrocatalytic oxygen production at the cellular level. Prior to cellular experiments, we first conducted comprehensive assessment of the physicochemical properties and biosafety of Ru(bbp)(Py)₂Cl. Our synthesized Ru(bbp)(Py)₂Cl had good biocompatibility and did not trigger the change of erythrocyte morphology or hemolysis after entering into the organism (Fig. S4a and S4b†). We employed the MTT assay to verify the antitumor activity of the in situ electrocatalytic oxygen generation and sensitization radiotherapy strategy. This strategy exhibited significant anti-tumor effects under oxygen-producing conditions (Fig. 3b) and under anoxic conditions (Fig. 3c). Especially in the anoxic environment, this strategy improved the anoxic state of tumor tissues by generating oxygen, which further enhanced its anti-tumor effect. In addition, this strategy significantly inhibited the population growth of tumor cells (Fig. 3d) (1: 100%; 2: 93.71%; 3: 24.87%; 4: 20.57%; 5: 65.84%; 6: 44.02%; 7: 22.18%; 8: 8.46%). These results further demonstrated that our strategy could generate oxygen to improve the lack of oxygen and, under X-ray irradiation, to generate more ROS which, in turn, sensitized radiotherapy.

Fig. 3 Electrocatalytic oxygen-production strategy causes mitochondrial breakage, leading to the death of cancer cells. (a) Cellular electrocatalytic oxygen generation and sensitization of death mechanism in radiotherapy (schematic). (b) Effects of different treatment groups on the survival of HeLa cells under normoxic conditions at Ru(bbp)(Py)₂Cl concentrations of 0, 2.5 and 5.0 μM, respectively. (c) Effects of different treatment groups on the survival of HeLa cells under hypoxia at Ru(bbp)(Py)₂Cl of 0, 2.5 and 5.0 μM, respectively. (d) Effect of different treatment groups on the formation of HeLa cells communities and quantitative analyses. (e) Effects of different treatments on caspase 3 and caspase 9 in HeLa cells. (f) Effect of different treatment groups on the mitochondrial morphology of HeLa cells. (g) Effects of different treatment groups on protein expression in HeLa cells. (h) Effect of different treatment groups on the cell-cycle distribution of HeLa cells.

In situ oxygen-generation system through a mitochondrial apoptotic pathway to kill cancer cells

We fully validated the feasibility of the in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy and the remarkable antitumor activity it exhibited. In order to gain deeper understanding of the specific mechanisms by which this strategy leads to cell death, we performed further investigations. It has been reported that tumor cells can be killed by changing the pH after electrotherapy. We examined the pH of the culture medium before and after treatment using our strategy. The in situ electrocatalytic oxygenation-sensitizing radiotherapy strategy did not lead to a change in pH while killing tumors (Fig. S4c†), which excluded the possibility that the strategy was activated by altering the pH of the environment. Our study provides further insight into the mechanism by which the in situ electrocatalytic oxygen-sensitizing radiotherapy strategy triggers the death of cancer cells. Previous studies have shown that this strategy improves the tumor hypoxic environment and generates large amounts of ROS, and that ROS overproduction can activate caspase-family proteins which, in turn, triggers the apoptosis of cancer cells.

To verify this hypothesis, we used specific fluorescent probes (Ac-DEVD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC) to detect the activating effect of this strategy on caspase activity in HeLa cells. The in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy significantly activated caspase-9 in HeLa cells, whereas it had no significant effect on the activity of caspase-8. The activation of caspase-9 further led to the activation of caspase-3 (Fig. 3e, S4d, S4e and S4f†), which is a key step in the mitochondrial apoptotic pathway. To further validate the activation of the mitochondrial apoptotic pathway, we observed the mitochondrial morphological changes in HeLa cells after different treatments and measured the expression of related proteins. The in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy could disrupt the mitochondrial morphology (Fig. 3f) and activate the phosphorylation of Bax, Bad, and p-p38, as well as inhibit the expression of Mcl-1, Bcl-2, Bcl-xl, p-AKT and p-ERK (Fig. 3g). All these changes are typical signs of activation of the mitochondrial apoptotic pathway. In addition, we evaluated the cell-cycle distribution after different treatments using flow cytometry. The in situ electrocatalytic oxygen-production strategy resulted in blockade of the G2/M step of the cell cycle, as well as an increase in the proportion of Sub-G1 apoptotic peaks (Fig. 3h). These changes further confirmed the ability of our strategy to trigger apoptosis in cancer cells. Our results suggest that the in situ electrocatalytic oxygen-generation sensitizing radiotherapy strategy killed tumor cells by activating the mitochondrial apoptotic pathway.

In situ oxygen-generation strategy achieves antitumor activity in vivo

Based on the remarkable results of in vitro cellular experiments, we further explored the active manifestation of the drug in vivo. Specifically, we employed a subcutaneous tumor transplantation tumor model in nude mice to evaluate the antitumor activity of the compounds in situ in our electrocatalytic oxygen-producing and sensitizing radiotherapy strategy (Fig. 4a). To ensure the validity and accuracy of animal experiments, we first optimized the strategy using in situ ultrasound technology. By adjusting the distance between electrodes and energization time, we observed the diffusion of oxygen in solid tumors. We could detect more intense oxygen signal generation when the electrode spacing was 1 cm compared with a setting of 0.5 cm (Fig. S5†). Moreover, this oxygen could diffuse rapidly and efficiently within tumor tissue after generation. Based on this finding, we used a 1 cm electrode spacing for subsequent animal experiments.

Fig. 4 In situ electrocatalytic oxygen-production strategy affects mouse immunity. (a) Assessment of in vivo antitumor activity of in situ electrocatalytic oxygen generation and sensitization radiotherapy (schematic). (b) Tumor-growth curves of different treatment groups within 15 days of treatment. (c) Effects of different treatment groups on the maturation of DC cells in lymph nodes after 15 days of treatment. (d) Effects of different treatment groups on macrophage polarization in tumor cells after 15 days of treatment. (e) Measurement of the hypoxia index in tumor tissues by different treatment groups after 15 days of treatment.

Tumor growth was more significantly inhibited in mice with the in situ electrocatalytic oxygen-generation strategy compared with that in the control group according to tumor-growth curves (Fig. 4b), and the strategy did not cause toxicity to mice (Fig. S6†). To more deeply evaluate the effect of our strategy on the immune response in mice in vivo, we further examined the maturation status of dendritic cells (DC cells) in the lymph nodes of mice as well as the macrophage phenotype in tumor cells. We documented a significant increase in the number of CD80 and CD86 double-positive mature DCs within the lymph nodes of mice after treatment with our strategy (Fig. 4c) (1: 7.71%; 2: 9.56%; 3: 7.60%; 4: 11.51%; 5: 16.19%; 6: 22.68%; 7: 26.04%; 8: 26.96%). Meanwhile, the number of CD206- and F4/80-double-positive M2-type macrophages in tumor cells was significantly reduced (Fig. 4d) (1: 43.75%; 2: 25.69%; 3: 32.49%; 4: 33.91%; 5: 20.85%; 6: 17.41%; 7: 5.18%; 8: 3.57%). These results demonstrated that our strategy was effective in inducing the maturation of DC cells in mice, but also in regulating the conversion of M2-type macrophages to M1-type macrophages after improving tumor hypoxia and enhancing the sensitivity to radiotherapy. The reason for generation of this immune response may be the increase in ROS in tumor tissues. We detected the hypoxia index in tumor tissues using pimonidazole (Fig. 4e). As a result, we found that the hypoxic status of tumor tissues in mice treated with our strategy was significantly improved compared with that in the control group, which was consistent with our speculation.

In situ oxygen-generation strategy inhibits highly malignant melanoma growth in vivo

Based on the remarkable results achieved in vitro using cellular experiments and the HeLa subcutaneous tumor model, the in vivo activity of the in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy was further explored in a more malignant melanoma model. We employed the C57 mouse melanoma model to investigate the anti-tumor effects of this strategy in depth (Fig. 5a). The strategy significantly inhibited melanoma growth in mice according to the tumor-growth curve (Fig. 5b). In order to evaluate the effectiveness of the strategy more comprehensively, we sectioned animal tissues. After treatment with the in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy, the cell density in tumor tissues was significantly reduced (Fig. S7a†), which indicated that tumor growth was effectively inhibited. Further analysis revealed that the strategy could activate the p53 pathway in tumor cells (Fig. S7b†), and its activation helped to slow down or stop the growth of tumor cells. Meanwhile, our strategy also inhibited the expression of the hypoxia-inducible factor HIF-α and induced apoptosis in tumor cells (Fig. 5c). In addition, compared with the control group, our strategy had no significant effect on important organs such as the heart, liver, spleen, lungs or kidneys in mice (Fig. 5d), which further confirmed its good safety profile. In summary, through in vivo experiments, we further validated the antitumor activity and safety of the in situ electrocatalytic oxygen-producing and sensitizing radiotherapy strategy in a melanoma model, which provides strong support for its future clinical application.

Fig. 5 Electrocatalytic oxygen-production strategy against melanoma. (a) Assessment of in vivo antitumor activity of in situ electrocatalytic oxygen generation and sensitization radiotherapy (schematic). (b) Tumor-growth curves within 15 days of treatment in different treatment groups. (c) Assays for TUNEL and HIF-α in tumor tissues after 15 days of treatment with in situ electrocatalytic oxygen generation and sensitization radiotherapy. (d) H&E staining of the heart, liver, spleen, lungs, and kidneys of mice after 15 days of treatment in different treatment groups.

Conclusions

The challenge of tumor hypoxia, which often severely weakens the efficacy of radiotherapy, prompted us to explore innovative strategies to overcome tumor hypoxia and enhance the efficacy of radiotherapy. We pioneered a novel strategy to effectively improve the hypoxic microenvironment and significantly enhance the sensitivity of radiotherapy by in situ electrocatalytic generation of oxygen at the tumor site using a low-toxicity catalyst containing high-Z atoms. Specifically, we combined a polydentate chelating ligand with a high Z-value Ru metal center to develop a low-toxicity water-oxidation electrocatalyst, Ru(bbp)(Py)₂Cl. The latter served as a low-toxicity electrocatalytic oxygen-generating tool to dramatically improve the hypoxic state of tumors, but also contained Ru element to enhance the sensitivity of radiotherapy to X-rays, thus dramatically improving the therapeutic efficacy. As validated by in vitro and in vivo experiments, our strategy achieved in situ electrocatalytic oxygen production at the tumor site, effectively alleviating tumor hypoxia. In addition, the strategy employed multiple sensitization mechanisms to achieve significant anticancer effects by generating excessive ROS, disrupting the normal function of mitochondria, and inducing activation of the apoptosis-regulating proteins caspase-3 and caspase-9 which, ultimately, triggered apoptotic cell death. Our study provides a new idea and method to improve the hypoxic environment of tumors, but also opens up a new path for sensitized radiotherapy, which is expected to achieve breakthrough progress in cancer treatment.

Author contributions

Conceptualization, L.M. and T.C. Data analyses, M.C., H.H., and J.D. Investigation, M.C., H.H., J.D., P.X. and J.C. Writing (original draft), M.C., H.H., and J.D. Writing (review and editing), M.C., H.H., J.D., L.M. and T.C. Resources, L.M. and T.C. Visualization, L.M. and T.C. Supervision, L.M. and T.C. Project administration, L.M. and T.C. Funding acquisition, L.M. and T.C.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by the National Science Fund for Distinguished Young Scholars (82225025), the National Natural Science Foundation of China (22177038, 21877049, 32171296, 82372103), China National Postdoctoral Program for Innovative Talent (BX20240141) and K.C. Wong Education Foundation.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qi03065f

‡ These authors contributed equally to this work.

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