Dual Reactive Oxygen Species Generator Independent of Light and Oxygen for Tumor Imaging and Catalytic Therapy

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE14 Jul 2022Dual Reactive Oxygen Species Generator Independent of Light and Oxygen for Tumor Imaging and Catalytic Therapy Shu Sheng, Feng Liu, Meng Meng, Caina Xu, Huayu Tian and Xuesi Chen Shu Sheng Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 University of Chinese Academy of Sciences, Beijing 100049 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 Google Scholar More articles by this author , Feng Liu Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 University of Chinese Academy of Sciences, Beijing 100049 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 Google Scholar More articles by this author , Meng Meng Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 University of Science and Technology of China, Hefei 230026 Google Scholar More articles by this author , Caina Xu Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 Google Scholar More articles by this author , Huayu Tian *Corresponding author: E-mail Address: [email protected] Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 University of Chinese Academy of Sciences, Beijing 100049 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 University of Science and Technology of China, Hefei 230026 Google Scholar More articles by this author and Xuesi Chen Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 University of Chinese Academy of Sciences, Beijing 100049 Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022 University of Science and Technology of China, Hefei 230026 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101103 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Currently, reactive oxygen species (ROS) generation primarily depends upon light and O2, which hampers its further biomedical application. Here, we report that a manganese(III) salen-based complex (MnS) can continuously catalyze overexpressed hydrogen peroxide (H2O2) in the tumor microenvironment to 1O2, while the nanocarrier (MIL-100) as a Fenton reagent can convert H2O2 to hydroxyl radicals (•OH) through the Fenton reaction, inducing noticeable intracellular DNA strand scission and lipid peroxidation to provoke tumor cell apoptosis without the involvement of light and O2. Moreover, MIL-100 depleted the antioxidant glutathione, further amplifying intracellular oxidative pressure, which in turn led to the self-degradation of MIL-100, suggesting the long-term biosafety of the nanoplatform. Owing to the excellent magnetic resonance imaging performance of MnS, the diagnosis and specific treatment of tumors were eventually achieved. This work provides a novel approach for the realization of effective tumor catalytic therapy independent of light and O2 and a promising reference for the development of a wide range of catalytic therapeutic agents. Download figure Download PowerPoint Introduction The sustained development of nanocatalysis medicine has promoted the exploitation and utilization of multifunctional nanomaterials with catalytic activity for tumor therapy.1–3 Antitumor effects are realized by evoking intratumoral chemical reactions that convert nontoxic substances to highly toxic species with negligible effects on normal tissues.4,5 For example, photodynamic therapy (PDT) generally uses a photosensitizer that is irradiated by external light in the presence of O2 to produce highly cytotoxic singlet oxygen (1O2) for oncotherapy,6–9 but this inevitably depends upon light and O2.10 Besides, several approaches have been established to catalyze O2 in the tumor site to superoxide radicals (O2•−) to combat tumors,11 and O2 deficiency in the tumor critically diminishes the therapeutic effects on the tumor.12 Chemodynamic therapy usually converts hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to cytotoxic hydroxyl radicals (•OH) via Fenton/Fenton-like reaction for tumor therapy,13–16 but the types of reactive oxygen species (ROS) produced are single. Therefore, there is an urgent need to establish a paradigm that can simultaneously produce multiple ROS independent of light and O2 to combat tumors in the field of nanocatalysis. Herein, we synthesized a manganese complex (Mn(III)-salen) and leveraged a metal–organic framework (MOF) (MIL-100) to carry Mn(III)-salen (MnS) and then coated it with hyaluronic acid (HA) to prepare [email protected]/HA nanoparticles (MnSMH NPs) (Figure 1). MnSMH NPs were injected intravenously into 4T1 tumor-bearing mice to achieve effective tumor accumulation through passive and active targeting. The catalytic performance of the MnSMH NPs could be evoked by the participation of endogenous H2O2 in the TME and continued to produce ROS without the involvement of light and O2. The cargo MnS catalyzed the conversion of endogenous H2O2 to 1O2, while the nanocarrier MIL-100 could act as a Fenton reaction agent to generate •OH. Noteworthy DNA damage and lipid peroxidation were evoked by the two exceedingly cytotoxic ROS. Moreover, MIL-100 could consume overexpressed glutathione (GSH), further amplifying intracellular oxidative pressure. The synergy of these functions eventually achieved a highly effective antitumor effect. Due to the existence of manganese-containing complexes, the MnSMH NPs had excellent magnetic resonance imaging (MRI) capabilities for tumor diagnosis, while the degradation of nanocarrier MIL-100 under the action of GSH indicated the long-term biosafety of the nanoplatform. To the best of our knowledge, this was the first report to demonstrate the simultaneous conversion of endogenous H2O2 to 1O2 and •OH to combat tumors without the involvement of light and O2, opening a new avenue for the development of catalytic therapeutic agents. Figure 1 | Schematic illustration of dual ROS generator independent of light and O2 for MRI and catalytic therapy. Download figure Download PowerPoint Experimental Methods First, we synthesized the MnS complex, and the structure was verified by proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS). Subsequently, the 1O2 production and MRI capabilities were measured. Then, we employed MIL-100 to load MnS (MnSM NPs), and MnSMH NPs was obtained after HA shielded. The physicochemical properties were characterized in detail. Next, we comprehensively evaluated the performance of MnSMH NPs in vitro, including endocytosis, 1O2 and •OH production, GSH consumption, DNA damage, and lipid peroxidation. Finally, biodistribution, ROS production, and the antitumor effects of MnSMH NPs were investigated in vivo. All animal experiments were performed following the guidelines for laboratory animals established by the Animal Care and Use Committee of Jilin University. More experimental procedures are available in the Supporting Information. Results and Discussion Synthesis and characterization of MnS To realize the effective loading of trivalent manganese, we first synthesized the salen ligand by an aldimine condensation reaction between ethylenediamine (EDA) and salicylaldehyde.17–20 Subsequently, the structure of salen was characterized by 1H NMR ( Supporting Information Figure S1). In the 1H NMR spectrum, the hydrogen atom peaks at 13.39, 8.59, and 7.51–6.80 ppm were allocated to the exposure of salicylaldehyde. The hydrogen atoms peaks at 3.92 ppm were attributed to the methylene of EDA, which appeared after the bonding, verifying the successful synthesis of salen. The synthesis was further demonstrated through MS characterization ( Supporting Information Figure S2). Next, salen was employed to coordinate with manganese(III) acetate to obtain the MnS complex. Effective synthesis was verified by MS characterization ( Supporting Information Figure S3). We exploited singlet oxygen sensor green (SOSG) as a specific probe to evaluate 1O2 generated from H2O2 catalyzed by MnS.21 After MnS (10 μg/mL) was incubated with H2O2 (100 μM) in phosphate-buffered saline (PBS), the fluorescence intensity of SOSG was significantly enhanced and gradually boosted with increasing H2O2 concentration (Figure 2a and Supporting Information Figure S4). We used 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) as a probe to examine the 1O2 generating capability of MnS.22 As shown in Supporting Information Figure S5, compared with the other control groups, a sharp decrease in ABDA absorbance in the MnS/H2O2 group was attributed to the generation of 1O2. Similar results were attained with 1,3-diphenylisobenzofuran (DPBF) as a probe ( Supporting Information Figure S6).23 Furthermore, we explored the efficiency of 1O2 generation under hypoxic or normoxic conditions ( Supporting Information Figure S7 and Figure 2b). The absorbance of ABDA gradually lessened with the prolonging of incubation time regardless of hypoxic or normoxic conditions, and the trend and level of decrease remained almost unchanged. These findings suggested that MnS could continuously and efficiently convert H2O2 to 1O2 independent of O2. For that usually depends upon O2, hypoxia in the TME, and the consumption of O2 during PDT exacerbated the hypoxia, making the O2 levels insufficient to achieve the desired effects. However, MnS-generated 1O2 independent of O2, representing the potential to treat hypoxic tumors. Figure 2 | (a) The generation of 1O2 was verified by fluorescence spectra following SOSG incubation with various groups for 1 h. (b) Under hypoxic or normoxic conditions, ABDA was incubated with MnS (10 μg/mL) in the presence of H2O2 (100 μM), and the relative absorbance of ABDA at 379 nm was measured at different times to reflect 1O2 production. MR images and r1 relaxivity of MnS at different concentrations in the absence (c) or presence (d) of H2O2 (100 μM). Download figure Download PowerPoint Furthermore, manganese ions as an effective contrast agent for MRI motivated us to explore the MRI performance of MnS.24–26 As displayed in Figures 2c and 2d, the T1 relaxation rate (r1) of MnS was 2.38 mM−1s−1, and r1 was almost unchanged with the introduction of H2O2, demonstrating that the capability of MnS as a T1 contrast agent for MRI was not susceptible to H2O2. Therefore, we speculated that MnS displayed the potential for the diagnosis and therapy of tumors. Preparation and characterization of MnSMH NPs The MOF, a series of crystalline porous structural materials constructed by the coordination between metal ions/clusters and organic ligands, possesses several advantages such as high porosity, large surface area, and facile modification, attracting considerable attention in biomedical applications.27–29 We fabricated MIL-100 by a microwave reaction, which was formed by the coordination of Fe3+ ions with trimesic acid.5 Due to the presence of Fe3+ ions, MIL-100 could act as a Fenton reagent to catalyze the production of •OH from H2O2. Then, MIL-100 was performed as a nanocarrier to load MnS. The loading content of MnS displayed a tendency toward gradual growth until becoming roughly unchanged as MnS:MIL-100 weight ratios increased ( Supporting Information Table S1). The maximum loading content of MnS was 22.7 % (MnS/MIL-100 at 2.5/1). Multifunctional nanoparticles (MnSMH NPs) were established by shielding with HA. The prepared nanoparticles had spherical morphology and were uniform in size (Figure 3a). The hydrodynamic size of the MnSMH NPs gradually increased to 155 nm after carrying MnS and HA shielding (Figure 3b and Supporting Information Figure S8a). Furthermore, Supporting Information Figure S8b showed that the size of MnSMH NPs negligibly changed following incubation in PBS and the cell culture medium for 48 h, demonstrating the outstanding size stability of MnSMH NPs in physiological conditions. The zeta potential of MnSMH NPs was −25.9 mV (Figure 3c). We leveraged X-ray diffraction (XRD) to characterize the structural changes of the MIL-100 during the process of functionalization ( Supporting Information Figure S9). The crystal structures of MnSMH NPs and MIL-100 were consistent except that the reflection intensity was slightly attenuated, which confirmed that the structure of MIL-100 was maintained. The Brunauer–Emmett–Teller (BET) surface area of MIL-100 was lessened due to MnS loading and shielding with HA (Figure 3d). The Fourier transform infrared (FT-IR) spectroscopy spectra of the MnSM and MnSMH groups showed a band at 1290 cm−1, which was attributed to the C–O stretching vibration of phenol in MnS,30 indicating that MnS was successfully carried by the NPs (Figure 3e). Figure 3 | (a) SEM images of MIL-100 NPs, MnSM NPs, and MnSMH NPs. Scale bar: 200 nm. (b) Dynamic light scattering (DLS) measurement, (c) zeta potential, (d) nitrogen adsorption–desorption isotherms, and (e) FTIR spectra of MIL-100 NPs, MnSM NPs, and MnSMH NPs. Download figure Download PowerPoint MRI, ROS evaluation, GSH depletion, and degradation of MnSMH NPs The MRI contrast capacity of MnSMH NPs was investigated due to the presence of MnS (Figure 4a). The T1 relaxation rate of MnSMH NPs displayed superior image contrast (r1 = 4.58 mM−1s−1) compared with the commercial MRI T1 contrast agent (Gd-DTPA, r1 = 3.69 mM−1s−1),31 signifying that the MnSMH NPs could perform as a high-efficiency MRI contrast agent. Figure 4 | (a) MR images and r1 relaxivity of MnSMH at a series of concentrations. (b) UV–vis absorption spectra of TMB treated with various groups to evaluate the production of •OH. (c) Fluorescence spectra of SOSG treated with various groups to confirm the production of 1O2. (d) Depletion of GSH after incubation with different concentrations of MnSMH NPs. (e) UV–vis absorption spectra of Phe treatment with various groups to detect the Fe2+. (f) The release of iron following MnSMH NPs (1 mg/mL) incubated with GSH (10 mM) for 0, 12, 24, and 48 h. (g) SEM images of MnSMH NPs degradation following the incubation of MnSMH NPs and GSH for different times. Scale bar: 200 nm. *p<0.05 (significant), **p<0.01 (moderately significant), and ***p<0.001 (extremely significant). Download figure Download PowerPoint It is widely accepted that iron ions catalyze H2O2 to produce •OH via the Fenton reaction.32–36 We chose 3,3′,5,5′-tetramethylbenzidine (TMB) as an •OH probe, which could be oxidized to oxTMB with an absorbance at 652 nm.32 The following control experiment was implemented to eliminate the possibility that MnS catalyzed the conversion of H2O2 to 1O2 and affected the chromogenic reaction of TMB ( Supporting Information Figure S10). In the presence of MnS and H2O2, weak absorbance at 652 nm was measured, indicating that TMB could be employed to detect •OH without interference from 1O2. As shown in Figure 4b, neither MnSMH NPs nor H2O2 displayed evident absorbance when present alone, but a noteworthy absorbance at 652 nm appeared when MnSMH NPs and H2O2 were present together. After MnSMH NPs were incubated with H2O2, SOSG showed an obvious emission peak at 525 nm compared with the other control groups (Figure 4c), suggesting that MnS in MnSMH NPs were still capable of converting H2O2 to 1O2. Highly expressed GSH in tumor cells could damage the efficiency of ROS-mediated therapies.15 Thus, we examined whether the constructed MnSMH NPs could deplete GSH. As displayed in Supporting Information Figure S11, while various concentrations of MIL-100 and MNs were incubated with GSH, only MIL-100 showed the capability of GSH depletion. After loading MnS and shielding with HA, MnSMH NPs still could consume GSH (Figure 4d). These results indicated that MnSMH NPs could not only catalyze H2O2 to produce 1O2 and •OH, but also exhaust GSH, and eventually achieve an improved effect of catalytic therapy. Fe3+ could be reduced by GSH to Fe2+, which could coordinate with 1,10-phenanthroline (Phe) to form an orange complex with characteristic absorbance at 510 nm.37 As shown in Figure 4e, when Phe, MnSMH NPs, and GSH were present together, an evident absorbance at 510 nm appeared due to the reduction of Fe3+ in MnSMH NPs by GSH to produce Fe2+. A standard curve was established according to the Beer–Lambert law, which was conducted to quantitatively analyze Fe2+ ( Supporting Information Figure S12 and Figure 4f). The amount of iron content released from the MnSMH NPs gradually increased with the duration of incubation, and 30.5% was released at 48 h. Moreover, the morphological changes during degradation were observed by scanning electron microscopy (SEM) (Figure 4g). After incubation with GSH for 24 h, the size of MnSMH NPs decreased detectably compared with that at 0 h. The nanoparticles were further degraded into lumps at 48 h. These data indicated that GSH reduced Fe3+ to Fe2+ to promote the degradation of MnMH NPs, which could achieve biodegradation under the trigger of a high level of GSH in tumor cells and thus long-term biosafety in vivo. Cellular uptake, ROS evaluation, GSH depletion, and catalytic therapy in vitro Due to the existence of Fe and Mn in MnSMH NPs, their intracellular content could be exploited as an indicator of endocytosis. The amount of intracellular Fe and Mn was measured following the incubation of 4T1 cells with different concentrations of MnSMH NPs (Figure 5a and Supporting Information Figure S13). The intracellular Fe and Mn content was gradually improved with increasing MnSMH NPs concentrations, indicating that MnSMH NPs exhibited superior endocytosis efficacy in 4T1 cells. We leveraged cyanine 5 (Cy5) as a substituted cargo to analyze endocytosis through flow cytometry analysis ( Supporting Information Figure S14). The fluorescence intensity of the [email protected]/HA (CMH) group was stronger than that of the [email protected] (CM) group, suggesting that endocytosis efficacy was enhanced after shielding with HA, which was ascribed to the fact that HA could bind to the CD44 receptor overexpressed on 4T1 cell membranes and demonstrated active targeting. Figure 5 | (a) After incubating MnSMH NPs with 4T1 cells, the intracellular iron content was quantitatively detected by ICP-MS. (b) Fluorescence microscopy images of intracellular ROS levels in 4T1 cells pretreated with various groups for 12 h. Scale bar: 200 μm. (c) GSH depletion was measured in 4T1 cells following treatment with different concentrations of MnSMH NPs. (d) Viability of 4T1 cells following incubation with various formulations. (e) Fluorescence images of 4T1 cells incubated with various groups by costaining with calcein-AM (green) and propidium iodide (red). Scale bar: 200 μm. (f) Plasmid nicking assay and agarose gel electrophoresis were employed to assess ROS-evoked DNA damage. (g) γ-H2AX immunofluorescence staining of 4T1 cells following treatment with different formulations. Nuclei and γ-H2AX were stained blue and red, respectively. Scale bar: 100 μm. (h) Changes in MDA content in 4T1 cells after different treatments. *p<0.05 (significant), **p<0.01 (moderately significant), and ***p<0.001 (extremely significant). Download figure Download PowerPoint To study the intracellular ROS generating capability of MnSMH NPs, fluorescence microscopic observation was conducted by employing 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as a probe (Figure 5b).38 As a control group, the addition of extra H2O2 barely affected the intracellular oxidation pressure. Obvious fluorescence intensity was observed with the further introduction of MnSMH NPs, confirming that MnSMH NPs could convert H2O2 to •OH and 1O2 to increase oxidation pressure. The appropriate fluorescence intensity in the MnSMH group was credited to lower intracellular H2O2 levels ( Supporting Information Figure S15). The intracellular GSH content decreased along with increasing MnSMH NPs concentrations (Figure 5c). These findings demonstrated that MnSMH NPs could catalyze H2O2 to produce •OH and 1O2 and deplete GSH, which could further amplify intracellular oxidative pressure to enhance the effects of catalytic therapy. Motivated by these results, we further investigated the cytotoxicity of MnSMH NPs. First, we explored the cytotoxicity of the ligand (salen) and nanocarrier (MIL-100). As shown in Supporting Information Figure S16, neither salen (40 μg/mL) nor MIL-100 (100 μg/mL) displayed obvious cytotoxicity at high concentrations. Figure 5d shows that MnSMH NPs exhibited moderate cytotoxicity, which was strengthened by the introduction of exogenous H2O2 (100 μM) due to the more cytotoxic •OH and 1O2 produced and the exhaustion of GSH. Then, the cells were distinguished by live/dead staining, in which the live cells were stained by calcein-AM and dead/late apoptotic cells were stained by propidium iodide ( Supporting Information Figure S17 and Figure 5e). After incubating 4T1 cells with H2O2 (100 μM), the cells were alive and emitted green fluorescence. Notably, significant red fluorescence was detected following the introduction of MnSMH NPs, suggesting the excellent tumor-combating efficiency of the MnSMH NPs. This coincided with the intracellular ROS levels and cytotoxicity assay. Mechanism of oxidative damage To explore the specific mechanism of ROS-induced cell apoptosis, we conducted a plasmid DNA nicking assay to determine the DNA cleavage activity of MnSMH NPs.39,40 Under the trigger of ROS, damaged supercoiled DNA could transform into a nicked form with a single-strand break and a linear form, which could be distinguished by agarose gel electrophoresis due to its different electrophoretic mobilities. As shown in Figure 5f, when the MnSMH NPs concentration was varied from 0 to 2.5 μg/mL by adding extra H2O2, the content of the supercoiled form disappeared, but the nicked form noticeably increased. As the concentration increased further, almost all nicked forms changed to line forms in the gel electrophoretogram under the inducement of large amounts of ROS. These findings implied that the generation of ROS was gradually amplified as MnSMH NPs concentrations were increased in the presence of H2O2 to trigger DNA damage via an oxidative mechanism.2 Next, the DNA double-break marker γ-H2AX was used for immunofluorescence staining to detect cellular DNA damage mediated by the MnSMH NPs.41 MnSMH NPs-treated cells exhibited lower γ-H2AX fluorescence intensity than the MnSMH/H2O2 group (Figure 5g), demonstrating that raising the levels of intracellular H2O2 resulted in more DNA damage. Furthermore, the thiobarbituric acid assay was performed to assess lipid peroxidation following the treatment of 4T1 cells with different concentrations of MnSMH NPs. The malondialdehyde (MDA) level in 4T1 cells pretreated with MnSMH NPs (15 μg/mL) was increased 3.5-fold in comparison with the control group, suggesting that MnSMH NPs induced substantial lipid peroxidation (Figure 5h). This irreversible damage to biological macromolecules in tumor cells was ascribed to the high-efficiency generation of dual ROS (•OH and 1O2) and suggested the possibility of achieving excellent antitumor effects. MRI, pharmacokinetics, biodistribution, and ROS detection Beyond the property of producing ROS, the other properties of MnSMH NPs involved serving as MRI contrast agents. For MRI evaluation in vivo, MnSMH NPs (50 μL, 20 mg/kg) were intratumorally injected into mice. The apparent MRI signals were detected 30 min postinjection and compared with the control group (Figure 6a), which confirmed the excellent MRI ability of the MnSMH NPs. Then, we introduced a Cy5 fluorescent molecule as cargo to track the metabolism of the nanoparticles (CMH NPs) in vivo. According to the pharmacokinetic curve of CMH NPs in mice (Figure 6b), the blood-elimination half-life of CMH NPs was calculated to be 4.0 h through a two-compartment pharmacokinetic model, suggesting that CMH NPs were capable of blood circulation. Furthermore, we investigated the Fe content in organs and tumors by inductively coupled plasma mass spectrometry (ICP-MS) to analyze the biodistribution of MnSMH NPs at different time points. In Figure 6c, the MnSMH NPs were principally accumulated in the liver after intravenous injection owing to capture by the reticuloendothelial system.11 The iron content in the liver reached a maximum at 12 h postinjection, and then diminished significantly at 24 h, confirming that the MnSMH NPs were effectively cleared by the liver. The iron amount within the tumors was 3.3% ID/g at 6 h, which gradually improved with time and reached a peak at 24 h postinjection (8.1% ID/g), implying that MnSMH NPs could efficiently accumulate in tumors. Subsequently, DCFH-DA was performed as a fluorescent probe to explore ROS generation in vivo. The strongest green fluorescence was observed in mice intravenously injected with MnSMH NPs compared with the other groups, which was attributed to the catalytic conversion of endogenous H2O2 to •OH and 1O2 by MnSMH NPs (Figure 6d). Notably, the fluorescence intensity was sharply decreased when mice were administrated 1,3-Dimethylthiourea (DMTU) to remove H2O2 from the tumor site, confirming that ROS production required the involvement of H2O2. In the MH group, mild fluorescence intensity was attributed to the production of only •OH. Furthermore, Supporting Information Figure S18 showed that the MnSMH NPs group significantly consumed GSH levels compared with the PBS group. Therefore, MnSMH NPs still possessed the capability of GSH depletion in vivo, which could further amplify oxidative stress. These results signified that MnSMH NPs possessed the potential to provide catalytic tumor therapy. Figure 6 | (a) MR images of 4T1 xenografted tumor mice at 0 and 30 min after the intratumoral injection of MnSMH NPs. (b) The pharmacokinetic curve of CMH NPs intravenously injected by the two-compartment model, λex/em 640 nm/680 nm. (c) Determination of iron content at different times in heart, liver, spleen, lung, kidney, and tumor after the intravenous injection of MnSMH NPs. (d) ROS production in 4T1 xenografted tumor mice with various treatments was evaluated by fluorescence microscopy. Scale bar: 200 μm. Download figure Download PowerPoint Catalytic therapy of tumors in vivo The noteworthy high-efficiency production of ROS-induced noticeable cytotoxicity and effective tumor accumulation of MnSMH NPs motivated us to carry out a tumor ablation assessment in vivo. We investigated the antitumor capability of MnSMH NPs in 4T1 xenografted tumor mice. When the tumor volume reached nearly 100 mm3, the mice were randomly divided into four groups, the PBS, MnSMH/DMTU, MH, and MnSMH groups. MnSMH NPs or MH NPs were administrated intravenously every 3 days for a total of five times, and only the MnSMH/DMTU group was injected with DMTU 24 h before administration (Figure 7a). As exhibited in Figure 7b, tumor growth was considerably diminished in mice after treatment with MnSMH NPs for 14 days, indicating that MnSMH NPs catalyzed endogenous H2O2 to produce extremely cytotoxic •OH and 1O2. In contrast, the tumor growth curve was comparable with that of the PBS group with rapid growth when the mice were administrated DMTU to deplete H2O2. These findings showed that the production of ROS by the nanoplatform was dependent upon H2O2, and the significant tumor growth inhibition achieved in the MnSMH group was attributed to the generation of •OH and 1O2. Meanwhile, the overexpression of H2O2 in the TME endowed the nanoplatform with tumor specificity. Expectedly, tumor volume was appropriately reduced by withdrawing the function of MnS (MH group), eliminating the contribution of 1O2, further confirming the necessity of dual ROS. This was also verified by the tumor weights and tumor images (Figures 7c and 7d). To investigate pathological changes in the tumor