Engineering Nanoenzymes Integrating Iron-based MOFs with Pt NPs for Enhanced PDT-Ferroptosis Therapy

Engineering Nanoenzymes Integrating Iron-based MOFs with Pt NPs for Enhanced PDT-Ferroptosis Therapy | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineering Nanoenzymes Integrating Iron-based MOFs with Pt NPs for Enhanced PDT-Ferroptosis Therapy Yuyun Ye, Hongli Yu, Yifan Zhao, Lv Bai, Guanghe Xue, Yong Sun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2381256/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Photodynamic therapy (PDT), as a promising strategy in cancer treatment that utilizes photosensitizers (PSs) to produce reactive oxygen species (ROS), has been widely used for eliminating cancer cells under specific wavelength light irradiation. However, the low aqueous solubility of PSs, and special tumor environments (TME), such as high glutathione (GSH) and tumor hypoxia remain challenges towards PDT for hypoxic tumor treatment. Results To address these problems, we constructed a novel nanoenzyme (HMPC) for enhanced PDT-ferroptosis therapy by integrating small Pt nanoparticles (Pt NPs) and near-infrared photosensitizer CyI into iron-based metal organic frameworks (MOFs). In addition, hyaluronic acid (HA) was adhered to the surface of the nanoenzymes to enhance the targeting ability. In this design, MOFs act not only as a delivery vector for PSs, but also a ferroptosis inducer. Pt NPs stabilized into MOFs were functioned as an oxygen (O 2 ) generator by catalyzing hydrogen peroxide (H 2 O 2 ) into O 2 to relieve tumor hypoxia and increase 1 O 2 generation. I n vitro and in vivo results demonstrated that under NIR irradiation, HMPC could effectively relive the tumor hypoxia and decrease the level of GSH in TME, resulting in enhanced PDT-ferroptosis therapy against hypoxic tumor. Conclusion The proposed nanoenzymes represent an important advance in altering TME for improved clinical PDT-ferroptosis therapy, as well as their potential as effective theranostic agents for hypoxic tumors. Photodynamic therapy Iron-based MOFs Pt nanoparticles Ferroptosis therapy Hypoxic tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Photodynamic therapy (PDT), with advantages of spatiotemporal specificity, low systemic toxicity, and non-invasiveness, have been used as a potent treatment method against malignant tumors during the past decades[ 1 , 2 ]. With the laser irradiation of specific wavelength, oxygen (O 2 ) is converted into cytotoxic reactive oxygen species (ROS) through photosensitizer-mediated photochemical reaction, and the excessive intracellular ROS cause biomolecular peroxidation, while leading to the dysregulation of intracellular redox homeostasis, eventually resulting in cell necrosis and apoptosis[ 3 ]. However, the shortcomings of the photosensitizers (PSs) such as low aqueous solubility, lake of tumor-targeting, easy aggregation and light penetration depth are obstacles for deep tumor clinical application. With the development of nanotechnology, numerous drug delivery carriers have been explored to deliver PSs, such as liposomes[ 4 ], polymeric micelles[ 5 ], inorganic nanoparticles[ 6 ], and so on. However, most nanocarriers show shortcomings such as poor drug loading[ 7 ], poor physical stability[ 8 ], and non-repeatability. Metal-organic framework (MOF) is a highly ordered porous heterogeneous material formed by coordination between metal ions and organic ligands with the advantages of adjustable pore size, high loading capacity, easy functionalization, and biodegradability[ 9 ]. Compared with traditional nanocarriers, MOFs have showed high loading properties, biodegradation and low systemic toxicity. Nanoscale MOFs have been explored as drug delivery for proteins[ 10 ], nucleic acids[ 11 , 12 ], and small molecule drugs, such as curcumin[ 13 ], and doxorubicin. In particularly, iron-based MOFs, as a kind of active MOFs, can catalyze hydrogen peroxide (H 2 O 2 ) to generate hydroxyl radical (∙OH) through Fenton reaction, which can further deplete glutathione (GSH) in tumor cells, resulting in the inactivation of glutathione peroxidase (GPX4), the accumulation of lipid peroxides (LPO), and ultimately ferroptosis in tumor cells[ 14 – 16 ]. Therefore, iron-based MOFs can not only be used as carriers for PSs, but also as inducers to implement iron-induced ferroptosis. However, due to the hypoxic tumor microenvironment (TME), and the oxygen consumption in PDT process, which would further aggravate the tumor hypoxia, PDT efficacy are generally limited. Up to now, multiple strategies have been developed to relieve tumor hypoxia[ 17 ]. For instance, nonreactive oxygen carriers, such as hemoglobin (Hb) and perfluorocarbon carbide, as well as reactive oxygen supply materials, such as manganese dioxide (MnO 2 ), hydrogen peroxide enzymes are used to increase O 2 content during antitumor therapy. However, these strategies have showed disadvantages: Hb can only deliver a limited amount of O 2 , and MnO 2 is constantly consumed in redox reaction, which cannot meet the requirements for prolonged O 2 supplementation. Among them, nanoenzymes are a class of nanomaterials with enzymatic catalytic activity where small-sized Pt nanoparticles (Pt NPs) possess catalase-like activity[ 18 ]. Compared with other oxygen carriers and O 2 -producing materials, Pt NPs with continuous catalytic capacity of H 2 O 2 are able to provide O 2 for long time to alleviate hypoxia. Liu et al. [ 19 ]encapsulated Pt NPs in the water chamber of liposomes to overcome the limitation of tumor hypoxia in the efficacy of PDT. Qian et al. [ 20 ]encapsulated Pt NPs in the amphiphilic polymers, distearyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), to form polymer micelles that ameliorated hypoxia and enhanced PDT. However, due to the catalytic activity of Pt NPs is related to size, which Pt NPs with ultra-small size (3 ~ 5nm) have higher catalytic activity, the reported Pt NPs exhibit the defects of easy aggregation, low stability and premature clearance when size decreases below 5 nm[ 21 ]. Therefore, it is essential to develop novel nanosystems to stabilize Pt NPs. Hence, in this study, we developed a novel nanoenzyme (HMPC) for enhanced PDT-ferroptosis therapy by integrating iron-based MOF, MIL-100 (Fe), with Pt NPs, and uploading near-infrared (NIR) photosensitizer CyI into the inner core (Figure. 1). Hyaluronic acid (HA) was adhered to MOF by electrostatic interaction, which endowed the nanoenzymes with active targeting capability. Pt NPs in HMPC nanoenzymes, as a “machine” for O 2 generation by catalytic reaction to relieve tumor hypoxia, were stabilized by MOF through physical adsorption, which can diminish premature clearance by the mononuclear phagocyte system (MPS) and increase the safety in systemic circulation. CyI, as an iodinated-cyanine dye, has been explored and investigated in our previous study, which exhibit improved 1 O 2 yield, effective photothermal and NIR fluorescence imaging properties (Ex/Em: 756/822nm, fluorescence quantum yield is 0.48, the singlet oxygen yield is up to 0.75)[ 22 ]. After the HMPC nanoenzymes are taken up by tumor cells through high expression of CD44 receptors, due to the high concentration of H 2 O 2 in tumor cells, MOF undergoes Fenton reaction to generate Fe 2+ and ·OH, resulting in ferroptosis which could deplete GSH and the collapse of MIL-100 frame. Under NIR irradiation, the released Pt NPs can catalyze H 2 O 2 to offer O 2 , which could improve PDT efficacy of CyI. In addition, depleted GSH caused by ferroptosis therapy also enhance ROS retention in TME. Moreover, CyI was in possession of photothermal effect to implement photothermal therapy (PTT). We presume that by integrating Pt NPs and PSs into iron-based MOF, the nanoenzymes exhibit enhanced PDT-ferroptosis therapy, which may represent a promising novel regimen for hypoxic tumor treatment. 2. Experimental Section 2.1 Materials. CyI (Mw 776.5) was synthesized according to our previously reported protocal. 1, 3, 5-benzenetricarboxylic acid (BTC), Iron chloride (FeCl 3 ), Chloroplatinic acid hexahydrate (H 2 PtCl 6 ·6H 2 O) and 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB) were purchased from Aladdin (Shanghai, China). HA and Deferoxamine mesylate salt (DFO) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Hydrogen peroxide aqueous solution (H 2 O 2 ) was bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glutathione (GSH) and Vitamin C (Vc) was purchased from Macklin (Shanghai, China). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) and SOSG singlet oxygen fluorescence probe was bought from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). ROS Green™ H 2 O 2 Probe was purchased from Maokang Co., Ltd. (Shanghai, China). Image-iT™ Green Hypoxia Reagent was bought from Thermo Fisher Scientific (New York, USA). C11-BODIPY 581/591 was bought from Wuhan Anjiekai Biological Medicine Technology Co., Ltd. (Wuhan, China). GPX4 antibody was purchased from Beyotime (Shanghai, China). 2.2 Preparation of HMPC nanoenzymes. 8 ml of FeCl 3 (40 mM) glyoxal solution was mixed with 8 ml BTC (45 mM) DMF solution for 10 min at room temperature. The obtained solution reacted at 100°C for 2 h. The product (MOF) was collected by centrifugation and washed with DMF, ethanol, deionized water for 3 times. 5 ml of H 2 PtCl 6 (0.60 mM) solution was added dropwise to 45 ml ethanol solution containing polyvinyl pyrrolidone (PVP) stirred at room temperature for 10 min, and refluxed at 70°C for 3 h to obtain Pt NPs solution. 20 ml Pt NPs solution was added dropwise to the MOF (8 mg/ml) DMF solution, and stirred for 3 h at room temperature. The product (MP) was collected by centrifugation and washed with ethanol, deionized water for 3 times. CyI was added into the MP methanol solution and stirred for 24 h at room temperature in the dark. The mixture (MPC) collected by centrifugation and washed with deionized water for three times to remove free CyI. HA aqueous solutions were added dropwise to MPC aqueous solutions, and stirred for 30 min at room temperature in the dark. Then, the precipitate was collected and washed with deionized water to remove free HA. 2.3 Characterization. The morphologies of MOF, Pt NPs, MP and HMPC were examined via TEM. The EDS was utilized to analyze the elements of HMPC. The hydrated particle size and zeta potential of MOF, MP, MPC and HMPC were measured by DLS. UV–vis absorbance spectra of HA, CyI, MP, MPC and HMPC were acquired using a UV–vis absorption spectrophotometer. The X-ray diffraction signal of MOF and MP were obtained by an X-ray diffractometer. 2.4 The Catalase-like activity. The following experiments were conducted to verify that the catalase-like activity of Pt NPs was not impaired after loading CyI and modifying HA. The catalase-like activity of HMPC was assayed by observing the generation of O 2 through the catalytic decomposition of hydrogen peroxide. Firstly, the PBS solution was sealed with liquid paraffin to prevent combination of oxygen with the solution. Next, 20 mM H 2 O 2 PBS solution was prepared by adding H 2 O 2 solution to PBS with a syringe. Then, a probe of the dissolved oxygen meter was inserted under the surface of H 2 O 2 PBS solution, the HMPC, MOF or Pt solution was added with a syringe, and were reacted at room temperature for 12 min. Finally, the changes of dissolved oxygen level in the solution were recorded. 2.5 Detection of hydroxyl radicals for Fenton reaction. MIL-100 catalyzes H 2 O 2 to produce hydroxyl radicals (·OH) through Fenton reactions. This experiment was used to verify whether HMPC was still capable of undergoing Fenton reactions. 3,3',5,5'-tetramethylbenzidine (TMB) could be able to react with ·OH, creating a new significant absorption peak at 652 nm. TMB was added in PBS, MOF or HMPC solutions with or without H 2 O 2 . All samples were measured by UV-Vis after reaction at room temperature for 30 min. 2.6 Cell and animal model. CT26 cell lines were incubated in 1640 culture medium (5% CO 2 ) that was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Incubator temperature was kept at 37°C. Female BALB/c mice were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), which were 4–6 weeks old and weighed about 18–20 g. The tumor model was developed by subcutaneously injecting 3 × 10 6 CT26 cells (suspended in PBS) into the axilla of each BALB/c mouse. When the tumor volumes reach to 100–200 mm 3 , the mice are ready for treatment. All animal procedures were implemented in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document no. 55, 2001) and approved by the Animal Care Ethics Committee of Qingdao University (Qingdao, China). 2.7 Cytotoxicity assays. CT26 Cells and L929 Cells were seeded in 96-well plates for 24 h incubation. After that, different concentrations of HMPC were added to replace the medium and incubated with cells for 24 h. Finally, 100 µl MTT solution was added to each well, incubated for 4 h and measured at 492 nm by enzyme standard instrument. 2.8 In vitro therapeutic effect. CT26 Cells were seeded in 96-well plates for 24 h incubation. After that, different solutions were added to replace the medium, laser (808 nm, 0.96 W/cm 2 ) for 5 min after 4 h, and incubated with cells for 24 h. Finally, 100 µl MTT solution was added to each well, incubated for 4 h and measured at 492 nm by enzyme standard instrument. 2.9 Cell uptake and targeting property. FCM and CLSM were used to investigate the cell uptake of HMPC and their targeting property. To conduct CLSM observation, CT26 cells were seeded into confocal dishes and cultured overnight. Following removal of the culture medium, cells were treated with HMPC and MPC. Subsequently, DAPI fluorescent probe was used to stain the nuclei. After solution removal and washed with PBS twice, the cells were quickly observed under CLSM. In the blocking group, the operation was the same as above, except that cells were treated with HA. CT26 cells were seeded into 6-well plates and cultured overnight. Following removal of the culture medium, cells were treated with HMPC and MPC. After solution removal and washed with PBS twice, the cells were collected and resuspended in PBS for FCM. In the blocking group, the operation was the same as above, except that cells were treated with HA. 2.10 In vitro and in vivo ROS generation. In vitro ROS generation was detected by CLSM and FCM using DCFH-DA (10 µM) as a probe. To conduct CLSM observation, CT26 cells were seeded into confocal dishes and cultured overnight. Following removal of the culture medium, cells were treated with PBS, HMPC, and HMC for another 4 h. Subsequently, DCFH-DA was loaded into cells and incubated for 20 min. The cells in the laser treatment groups were irradiated with 880 nm laser for 5 min (0.96 W/cm 2 ). DAPI fluorescent probe was used to stain the nuclei. After solution removal and washed with PBS twice, the cells were quickly observed under CLSM. In the hypoxic group, the operation was the same as above, except that cells were treated with sodium disulfite (1 mM) for 12 h in advance to simulate hypoxia. Cells were incubated with different reagents for 4 h. After that, the laser group was irradiated. Next, the solution in the well was removed, then the cells were incubated with DCFH-DA for 20 min. After the probe was removed, the cells were washed with PBS for 3 times, and the cells were collected and resuspended in PBS for FCM. SOSG singlet oxygen fluorescence probe acted as a probe to detect in vivo ROS generation. 200 µl of normal saline, CyI, HMC and HMPC were injected into the tail vessel of mice. After 12 h, the tumors were injected 50 µl SOSG (25 µM) and the tumors of the laser treatment groups were exposed to 808 nm laser irradiation for 5 min. The mice were sacrificed and tumors were collected for cryosection. Finally, tumor sections were visualized by CLSM. 2.11 Intercellular H 2 O 2 and hypoxia detection. The level of intercellular H 2 O 2 or hypoxia was detected by CLSM. CT26 cells were prepared in confocal dishes and cultured overnight. The cells in the hypoxic group were treated with sodium disulfite (1 mM) for 12 h in advance to simulate hypoxia. The cells were treated with RPMI 1640, HMC, HMPC, CyI + laser, HMC + laser, HMPC + laser, separately. Then, each of these groups were added ROS Green™ H 2 O 2 Probe (5 µM). After incubation at room temperature for 30 min, 4% paraformaldehyde fix solution was added into all groups equally after removal of the medium and washing the cells three times with PBS. After 20 min, the cells were washed with PBS 2 times. Finally, DAPI staining was performed for observation. Image-iT™ Green Hypoxia Reagent (5 µg/ml) was used for hypoxia detection, and the incubation time is 16 min. Other operations were the same as above. 2.12 In vitro PTT. The performance of PTT is measured by thermocouple thermometer and infrared thermal imaging camera. The cells were incubated with different solutions for 4 h. After the solution removal, the cells were washed with PBS for 3 times. Next, the cells were collected and resuspended in PBS. All the groups were irradiated for 10min, and the temperature was measured every 30 s using thermocouple thermometer. Finally, the final temperature of every group were recorded by infrared thermal imaging camera. 2.13 GSH assay. With various treatments (PBS, HMPC, HMPC + laser, HMPC + H 2 O 2 , HMPC + H 2 O 2 + laser or HMPC + H 2 O 2 + DFO (1 mM) + laser), GSH content was measured with reduced GSH content detection kit. The cells were collected and washed with PBS three times. Subsequently, cells were cleaved by ultrasound (200 W, ultrasound 3 s, pause for 10 s, repeat 30 times). The supernatant was collected by centrifugation at 8000×g for 10 min. According to the instruction of kit, the absorbance of each samples was detected at 412 nm by enzyme standard instrument. According to the standard curve, the content of GSH was calculated. 2.14 MDA assay. With various treatments as above (GSH Assay), MDA content was measured with a MDA determination kit to monitor the level of lipid peroxidation. The cells were collected and washed with PBS three times. cells were cleaved by cell lysis buffer and ultrasound (200 W, ultrasound 3 s, pause for 10 s, repeat 30 times). The supernatant was collected by centrifugation at 1000×g for 10 min and used for MDA detection. Subsequently, according to the instruction of kit, the absorbance of each groups was detected at 532 nm by UV-vis. According to the formula provided by the kit, the content of MDA was calculated. 2.15 Expression of GPX4 evaluation. The expression level of GPX4 in vitro was detected by CLSM. CT26 cells were prepared in confocal dishes and cultured overnight. The cells were treated with RPMI1640, HMPC, HMPC + H 2 O 2 , HMPC + laser, HMPC + H 2 O 2 + laser and HMPC + H 2 O 2 + laser with DFO added, separately. Then, the cells were fixed with 4% paraformaldehyde fix solution for 20 min, each of these groups added the primary antibody of GPX4 after wash with PBS. After incubation at room temperature for 1 h, the second antibody with FITC labeling was added into all groups equally after removal of the medium and washing the cells three times with PBS. The time of secondary antibody incubation is 1 h. After 1 h, the cells were washed with PBS 3 times to remove the second antibody. Finally, DAPI staining was performed for CLSM observation. 2.16 In vitro and in vivo detection of LPO. LPO was typically detected by a C11-BODIPY 581/591 fluorescence probe, which can be observed by CLSM. CT26 cells were treated with various treatments (PBS, HMPC, HMPC + laser, HMPC + H 2 O 2 , HMPC + H 2 O 2 + laser or HMPC + H 2 O 2 + DFO (1mM) + laser) for 4 h. The cells were washed with PBS three times and incubated with the medium containing C11-BODIPY 581/591 (5 µM) for 20 min. The cells in the laser treatment groups were irradiated with 880 nm laser for 5 min (0.96 W/cm 2 ). DAPI fluorescent probe was used to stain the nuclei. Next, cells were washed with PBS then subjected to CLSM observation for in vitro LPO accumulation. To evaluate the production of LPO histologically, 200 µl of normal saline, CyI, HMC and HMPC were injected into the tail vessel of mice. After 12 h, the tumors in the laser treatment groups were exposed to 808 nm laser irradiation for 5min. The mice were sacrificed and tumors were collected for cryosection. Tumor sections were stained with C11-BODIPY 581/591 (2.5 µM) at 37 ℃ for 30 min, and fixed with 10% paraformaldehyde at room temperature for 10 min. Then the tissue sections were stained with DAPI for 10 min. Finally, oxidized C11-BODIPY 581/591 was observed under a confocal microscope to evaluate in vivo LPO accumulation. 2.17 In vivo antitumor study. Seven groups of CT26 tumor-bearing mice were randomly assigned in our experiment for evaluating the curative effect of the combination therapy. When the tumor reached approximately 100 mm 3 , respective treatment (saline, laser, HMPC, CyI + laser, HMC + laser, HMPC + laser, and HMPC + laser + DFO (dose: CyI 1.5 mg/kg, DFO 20 mg/kg) ) was executed separately to the seven groups. The mice in the laser treatment groups were subjected to laser (0.96 W/cm 2 for 5 min) at 12 h post-intravenous injection. The tumor size and body weight were monitored every 2 days. Tumor volume was calculated as width 2 × length/2. After treatment, the mice were sacrificed, and the tumors were collected for photographing and hematoxylin-eosin (HE) staining. Morphological changes of the tumors were observed by fluorescence microscope. To further understand the tumor inhibition mechanism of HMPC, immunofluorescence analysis of Ki67, TUNEL, and GPX4 was performed. 2.18 In vivo imaging. Free CyI and HMPC were administered by intravenous injection separately for two mice bearing about 100 mm 3 tumors. Fluorescence images of the mice were acquired by the IVIS® spectrum in vivo imaging system at different time intervals post-injection. Two mice were sacrificed, with the tumor, heart, spleen, liver, lung, and kidney excised for the observation of the biodistribution of HMPC and free CyI via imaging. 2.19 Statistical analysis. Data were expressed as mean ± SD (n = 3). Statistical analysis was calculated by Students’ t-test with statistical significance assigned for P values of < 0.05. 3. Results And Discussion 3.1 Preparation and characterization of HMPC nanoenzymes. MOF NPs were synthesized using a solvothermal method[ 23 ]. The MOF NPs were mixed with Pt NPs prepared by ethanol reduction method[ 24 ] and stirred for 3 h to obtain Pt NPs-loaded MOF (MOF@Pt, MP). Subsequently, MP NPs or MOF NPs were further loaded with the photosensitizer CyI, to produce CyI-loaded MP (MOF@Pt@CyI, MPC) and CyI-loaded MOF (MOF@CyI, MC). Finally, MPC NPs or MC NPs were coated with hyaluronic acid (HA) to endow active targeting properties. The prepared nanoenzymes were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figure. 2A and S1, MOF and MP NPs with uniformly distributed granular diameters (~ 140 nm and ~ 150 nm, respectively) are regularly coronal spherical. It should be noted that uploading of Pt NPs to MOF NPs (size below 5 nm) did not significantly affect the morphology of MOF NPs. HMPC nanoenzymes were monodisperse nanospheres with ~ 160 nm in diameter and small-size Pt NPs were uniformly distributed on the surface of nanoparticles (Figure. 2B and C). The hydrodynamic diameters of the prepared nanoparticles measured by DLS revealed that the average diameters of particles (all less than 200 nm) (Figure. S2), implying that HMPC nanoenzymes can accumulate and remain at the tumor site through enhanced permeability and retention (EPR) effect. Elemental distribution of the HMPC nanoenzymes according to the mapping images of energy dispersive spectroscopy (EDS) revealed Fe was uniformly distributed within the core region, while most Pt was distributed on the periphery, implying the formation of Pt NPs (Figure. 2D and S3). The crystal phase and composition of nanoparticles was further confirmed by X-ray diffraction (XRD) (Figure. 2E). The diffraction pattern of MOF confirmed the successful synthesis of MIL-100. MP NPs possessed obvious new diffraction peaks as compared to MOF NPs, matching well with the standard XRD data for Pt (JCPDS card, file No.65-2868). The zeta potential of HMPC nanoenzymes in aqueous solution was − 24.23 ± 0.84 mV, implying good stability under physiological conditions (Figure. 2F). Absorption spectra showed that compared with MP NPs, HMPC nanoenzymes have a new absorption peak in the near infrared region of 650–850 nm, confirming the successful uploading of CyI (Figure. 2G). To evaluate the catalase-like activity of the HMPC nanoenzymes, the decomposition of H 2 O 2 was investigated using a fluorescence probe, ROS Green™ H 2 O 2 . This probe can emit fluorescence in the presence of H 2 O 2 , and the fluorescence intensity would raise with the increasing H 2 O 2 concentration. As shown in Figure. S4, the decomposition of H 2 O 2 is about 99% in the presence of HMPC nanoenzymes, much more than that of HMC group, implying that HMPC nanoenzymes have catalase-like activity after incorporation of Pt NPs. Then, to further verify the catalase-like activity of HMPC nanoenzymes, O 2 production was detected and quantitated with dissolved oxygen analyzer. As shown in Figure. 2H, O 2 concentration increased rapidly after adding HMPC nanoenzymes or Pt NPs, while in the absence of Pt NPs, the O 2 concentration did not change significantly. The 1 O 2 generation by CyI under laser irradiation was also explored by SOSG, a specific singlet oxygen probe. The results shown in Figure. S5 confirmed that HMPC nanoenzymes with Pt NPs could increase 1 O 2 production. Hydroxy radical (·OH), as one of the products of Fenton reaction, can undergo redox reaction with tetramethyl benzidine (TMB) and yields strong absorption peak at 652 nm. Therefore, TMB was used to detect the generation of ·OH. As shown in Figure. 2I, MOF NPs and HMPC nanoenzymes did not generate ·OH, and only in the presence of H 2 O 2 , a new absorption peak appeared at 652 nm, indicating the generation of ·OH and the occurrence of Fenton reaction. 3.2 O 2 -induced enhanced PDT ability of HMPC nanoenzymes in vitro. To evaluate PDT efficacy in vitro , the safety and dark toxicity of HMPC nanoenzymes were firstly investigated by MTT assay (Figure. 3A). As shown, no significant cell death was observed in both tumor cells (CT26 cells) and normal cells (L929 cells) by increasing the concentration of CyI for 24 h, which demonstrated that HMPC nanoenzymes has good safety and biocompatibility. Then, flow cytometry (FCM) and confocal microscopy (CLSM) were used to investigate the cell uptake of HMPC nanoenzymes by CT26 cells (Figure. 3B and S6). As the time prolonged, the fluorescence intensity of CyI was increased, and comparable at 4 and 8 h, indicating that HMPC nanoenzymes could be effectively internalized and accumulated in CT26 cells. In addition, the targeting performance of HMPC nanoenzymes towards CT26 cells was verified. As shown in Figure. 3C, the fluorescence intensity of cells incubated with HMPC nanoenzymes was stronger than that of MPC NPs, indicating that the coating of HA increased cellular uptake. To further investigate whether the increased cellular uptake is related to HA-mediated active targeting, blocking experiment was carried out by pretreating the cells with HA. As expected, the fluorescence intensity decreased in the blocking group, suggesting that HMPC nanoenzymes could active target and accumulate to CT26 cells via CD44 receptors (Fig. 3 D). Next, to explore whether Pt NPs-mediated catalytic reaction would relieve hypoxia in vitro , the level of intercellular H 2 O 2 and hypoxia in CT26 cells after various treatments were detected by H 2 O 2 and hypoxia fluorescence probes. As shown in Figure. 3E and F, both the green fluorescence (intercellular H 2 O 2 ) and red fluorescence (intercellular hypoxia) of HMPC group were much weaker than that of CyI + laser group and HMC group with or without laser irradiation. Specifically, the red fluorescence intensity of HMPC with laser irradiation was almost comparable to the control group under normoxic condition, indicating that HMPC nanoenzymes could offset hypoxia aggravated by PDT in a simulated anoxic condition. The evidences confirmed HMPC nanoenzymes with Pt NPs could catalyze H 2 O 2 into O 2 and alleviate tumor hypoxia. Then, the ROS generation in tumor cells was observed by CLSM (Figure. 3G) and FCM (Figure. 3H) using DCFH-DA as a ROS probe. As shown, the control group, as well as HMC and HMPC group without laser irradiation under normoxic conditions exhibited negligible green fluorescence, indicating low or none ROS generation. In comparison, HMPC group under laser irradiation showed enhanced green fluorescence intensity, indicating that HMPC nanoenzymes with Pt NPs to tumor cells where H 2 O 2 is constantly being formed could catalyzed H 2 O 2 into O 2 , thus promoted ROS generation by CyI[ 25 ]. It should be noted that under either normoxic or hypoxic conditions, the fluorescence intensity of cells treated with HMPC under laser irradiation was almost identical, indicating that HMPC nanoenzymes could constantly relieve tumor hypoxia. On the contrary, the fluorescence intensity of cells treated with HMC + laser group under normoxic condition was stronger than that of hypoxic condition. Those experimental results showed that oxygen-dependent PDT efficacy was hindered to some extent in anoxic environment, and demonstrated that HMPC nanoenzymes could relive tumor hypoxia and increase ROS generation. Our previous study has demonstrated that CyI possesses good photothermal effect. To confirm the photothermal effect of HMPC nanoenzymes, infrared thermal imaging camera and thermocouple thermometer were used. As shown in Figure. 3I and J, compared with MP and MOF, significant temperature change, from room temperature (26 ℃) to thermal ablation temperature (47 ℃), was observed in the HMPC group under laser irradiation, which demonstrated the photothermal effect of HMPC nanoenzymes. Finally, the PDT efficacy of HMPC nanoenzymes was examined by MTT assay in CT26 cells in either normoxic or hypoxic conditions. As shown in Figure. 3K, both HMC and HMPC showed concentration-dependent cell inhibition profile. In either normoxic or hypoxic conditions, HMPC under laser irradiation showed better cell inhibition rate compared to that of HMC. Furthermore, as expected, the cell viability of HMPC in either normoxic or hypoxic conditions had no significant difference, while HMC in normoxic conditions exhibited much better cell inhibition rate than in hypoxic conditions. These results further demonstrated that HMPC nanoenzymes could enhance the cell inhibition ability of HMPC in hypoxic conditions. 3.3 Ability of HMPC nanoenzymes in inducing Ferroptosis in vitro. Ferroptosis is a kind of iron- and ROS-dependent regulatory cell death (RCD) forms differed from apoptosis, necrosis and autophagy[ 26 ]. It has been reported that iron-based nanomaterial can induce cell ferroptosis through Fenton reaction, which causes peroxidation of unsaturated fatty acids, leading to lethal accumulation of LPO on the cell membrane[ 27 , 28 ]. Ferroptosis can be prevented by the enzymatic reaction of GSH-GPX4 antioxidant systems where GPX4 catalyzes the reduction of LPO in a GSH-dependent reaction[ 29 ]. Therefore, to explore whether HMPC nanoenzymes could induce ferroptosis, GSH content, the expression of GPX4 and LPO accumulation were explored. Because cytotoxic ·OH produced from Fenton reaction in ferroptosis would oxidize GSH diminishing the level of the intracellular GSH, the level of GSH was generally regarded as the marker of strength in the evaluation of ferroptosis. As shown in Figure. 4A, compared with control, HMPC, and HMPC + laser group, and HMPC + H 2 O 2 group showed lower GSH content in CT26 cells, implying that Fenton reaction would generate ROS which can be reacted with GSH, resulting in ferroptosis. Moreover, under laser irradiation, the GSH content further decreased in HMPC + H 2 O 2 group, indicating that more generated ROS are accumulated in tumor cells by the combination of PDT and Fenton reaction, which resulted in the balance disorder of ROS and GSH in tumor cells to induce ferroptosis. Deferoxamine (DFO) is an iron chelating agent, which is applied as an ferroptosis inhibitor[ 30 ]. As shown, after adding DFO to HMPC + H 2 O 2 + laser group, the GSH content was greatly boosted, mainly because the intervention of DFO has decreased the intracellular free iron content, thus inhibiting Fenton reaction, and slowing down the process of iron-induced ferroptosis. GPX4, as the fourth member of the selenium-containing GPX family, exhibits a scavenging capacity to the membrane lipid hydrogen peroxide products. As a cofactor for GPX4 catalyzing LPO to lipid alcohol, lack of GSH would trigger cysteine deficiency, which directly inactivate GPX4 and induce ferroptosis[ 29 ]. Thus, GPX4, as the core regulator of ferroptosis, has been considered as the "star molecule" of ferroptosis studies. The immunofluorescent staining was used for the investigation of the expression of GPX4, as shown in Figure. 4B and C. Consistent with the above results of GSH content, compared with other groups, HMPC + H 2 O 2 + laser group showed the lowest green fluorescence, indicating that Fenton reaction of HMPC nanoenzymes would restrain the expression of GPX4, resulting in ferroptosis. As expected, the fluorescence of HMPC + H 2 O 2 + laser group was enhanced by the addition of DFO. The above experiments proved that HMPC nanoenzymes could deplete GSH and reduce GPX4 activity, thus inducing ferroptosis. Lipid inside cells is recognized as a crucial biomarker of ferroptosis, of which the accumulation contributes to the lethality of ferroptosis[ 31 , 32 ]. A lipid peroxidation sensor, C11-BODIPY, was utilized to detect the LPO generation accumulation level in tumor cells by CLSM (Figure. 4D and E). In CLSM observation, the red fluorescence represents the non-oxidation state, and the green fluorescence represents the oxidation state. As shown, HMPC + H 2 O 2 + laser group exhibited the strongest green fluorescence, implying that HMPC nanoenzymes caused the lipid oxidation under laser irradiation, and triggered ferroptosis. While the fluorescence was weakened after addition of DFO, which confirmed the accumulation of LPO was related to iron, implying the iron-dependent ferroptosis. In comparison, the HMPC + laser or + H 2 O 2 group showed lower fluorescence intensity, further indicating that the combination of PDT and Fenton reaction would induce more preferable ferroptosis due to iron and ROS. Malondialdehyde (MDA), as one of products of membrane lipid peroxidation, is a toxic aldehyde substance, which is widely used as an index of intracellular LPO. As shown in Figure. 4F, HMPC + laser or + H 2 O 2 group showed elevated MDA content due to PDT or Fenton reaction, while the combination of both contributed to the further increased MDA content. However, this trend was reversed by DFO. The above results again indicated that the HMPC + H 2 O 2 + laser had preferable advantages to induce ferroptosis due to the combination of PDT and Fenton reaction. Finally, MTT assay was performed for assessment of the in vitro effect of HMPC. As shown in Figure. 4G, HMPC + laser group exhibited the best tumor inhibition, compared with PDT (CyI + laser) or ferroptosis therapy (HMPC). In addition, DFO and two antioxidant (Vc and GSH) were used to block ferroptosis. As Figure. 4H indicated, the addition of DFO could reduce the cell toxicity, demonstrating that iron was involved in the cell death. Similarly, the co-incubation with Vc or GSH also improved cell survival, further confirmed that HMPC nanoenzymes could inhibit cell viability by iron- and ROS-mediated ferroptosis. 3.4 Tumor-targeting ability and safety assay of HMPC nanoenzymes in vivo . In order to evaluate the tumor-targeting ability of HMPC nanoenzymes, HMPC nanoenzymes and free CyI were injected intravenously into tumor-bearing mice respectively, observing in vivo distribution of mice at different time periods under IVIS® spectrum in vivo imaging system (PerkinElmer) (Figure. 5A and B). In the free CyI-injected group, the tumor site of the mouse did not show fluorescence during 24 h post-injection, indicating that free CyI lacked tumor-targeting and tumor tissue retention capacity. In contrast, for HMPC-injected group, the fluorescence appeared at tumor tissue at 2 h post-injection, peaked at 10 h, and gradually decreased at 24 h, indicating that HMPC nanoenzymes was metabolized. It should be noted that the fluorescence intensity of free CyI is higher in the liver than in other major organs, mainly due to the fact that the hydrophobicity of CyI increased metabolism in the liver. Obviously, compared to free CyI-injected group, the fluorescence of tumor tissue in HMPC–injected group was still retained after 24 h due to targeting ability. All the evidence showed that compared with the early removal of CyI by the liver, HMPC nanoenzymes achieved the targeting and retention of tumor site by active targeting and nanoscale, and avoided the early accumulation and removal by liver and kidney. To determine the hemocompatibility of HMPC nanoenzymes, the hemolytic activity of the HMPC nanoenzymes was evaluated by hemolysis assay (Figure. S7). No significant hemolytic toxicity was observed in the photograph and hemolysis ratios are within the safe range (less than 4%), indicating the hemocompatibility of HMPC nanoenzymes. H&E staining for major organ of mice was performed, and saline-injected mice were used as control (Figure. 5C). No pathological characteristics were observed in HMPC-injected group, confirming the safety of HMPC. Moreover, the in vivo biocompatibility of HMPC nanoenzymes was investigated. The blood samples of two groups were taken after the treatment and used to test by a hematology analyzer for evaluating the toxicity of HMPC nanoenzymes (Figure. S8). The nine indexes of blood, including mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), red blood cell specific volume (HCT), platelet count (PLT) and red cell volume distribution width (RDW) in HMPC-injected group showed no obvious abnormal, compared to the saline group, implying no blood adverse reactions induced by HMPC nanoenzymes. Due to in vivo distribution, more accumulation of HMPC nanoenzymes was observed in liver and kidney compared with other organs. Hence, liver toxicity indicators (AST and ALT) and kidney indicators (BUN and CREA) were chosen for serum biochemical parameter detection (Figure. 5D-G). As shown, no significant difference was observed between control group and HMPC-injected group, which consistently suggested that HMPC nanoenzymes had the advantages of good hemocompatibility, biocompatibility and safety. 3.5 Combination treatment efficacy of HMPC nanoenzymes. To explore whether HMPC nanoenzymes would exert O 2 -enhanced PDT and ferroptosis in vivo , the alleviation situation of tumor hypoxia was firstly explored. After various administration, the tumors were collected, sliced and stained with hypoxia-inducible factor 1α (HIF-1α), a protein which the expression level could indicate the degree of hypoxia in the tumor. Immunofluorescent staining (Figure. 6A) showed that the tumor in HMPC-injected group exhibited significant lower fluorescence of HIF-1𝛼 compared to other groups, indicating that HMPC nanoenzymes with catalytic effect were contributed to the alleviation of hypoxia. In particular, the group injected with HMPC nanoenzymes exhibited lower hypoxia than free CyI or HMC under laser irradiation, implying HMPC nanoenzymes can still effectively relieve hypoxia under intensified hypoxic condition by PDT. ROS generation of HMPC nanoenzymes under laser irradiation was then investigated by staining the tumors with SOSG. As shown in Figure. 6A and B, the green fluorescence intensity in HMPC group was much higher than other groups, demonstrating that under the catalytic effect of HMPC nanoenzymes, the combination of PDT and ferroptosis would result in enhanced ROS generation. The LPO accumulation level in tumor tissues was assessed using C11-BODIPY, a lipid peroxidation probe, which could indicate the level of ferroptosis. When ferroptosis occurs, the emission of the probe will change from 590 nm (red fluorescence) to 510 nm (green fluorescence). As shown in Figure. 6C and D, the group treated with saline or laser irradiation showed no green fluorescence, while HMPC-treated group showed strong green fluorescence, indicating in vivo ferroptosis occurred. In addition, HMPC-treated group with laser irradiation showed much brighter green fluorescence, indicating that ROS generated by PDT increased the green fluorescence intensity, implying that PDT has a certain promoting effect on ferroptosis[ 33 ]. Green fluorescence intensity in HMPC-treated group under laser irradiation was decreased after adding DFO, demonstrating in vivo iron-related ferroptosis. The above results demonstrated that HMPC nanoenzymes exert enhanced PDT and ferroptosis therapy in vivo . The combination treatment efficacy of HMPC nanoenzymes was evaluated in CT26 tumor-bearing mice. Mice were randomly divided into seven groups (Saline, Laser, HMPC, CyI + laser, HMC + laser, HMPC + laser, HMPC + laser + DFO) for the observation of tumor volumes and body weights following 14-day treatment. The group intravenously injected with saline was defined as the negative control. As shown in Figure. 6E, compared with control and laser group, the tumor growth of HMPC, CyI + laser, HMC + laser, HMPC + laser group showed obviously inhibited. Among them, HMC + laser had better tumor inhibition rate than CyI + laser, due to that HMC held extra targeting property and exerted ferroptosis therapy. HMC + laser had better tumor inhibition than CyI + laser, because HMC held extra targeting property and exerted ferroptosis therapy. Compared with HMC-treated group, HMPC-treated group exhibited better tumor inhibitory effect under laser irradiation due to the contribution of Pt NPs. The group treated with HMPC nanoenzymes under laser irradiation implemented the combination treatment of PDT and ferroptosis therapy, which exhibited the best antitumor effect. To further verify the effect of ferroptosis on the combination treatment, DFO was intraperitoneally injected into mice to block iron-induced ferroptosis. The tumor inhibition efficacy was decreased, implying iron-induced ferroptosis did play a role in tumor treatment. The body weight of all groups exhibited no obvious decrease, indicating that the treatments had no severe side effects on mice (Figure. S9). After 14-days treatment, the mice were sacrificed. Figure. 6F and S10 showed the photos of mice and tumors isolated from mice after 14-day treatments, which were consistent with the trend of tumor volumes. The major organs and the tumors were collected for H&E staining. It was shown that no pathological abnormalities were observed in the major organs (heart, liver, spleen, lung and kidney), implying that treatments had no obvious organ toxicity towards mice (Figure. S11). The H&E staining results of tumors shown in Figure. 6G indicated that no tissue damage was observed in saline and laser group. However, group treated with HMPC nanoenzymes with laser irradiation exhibited an obvious tissue damage, nuclear shrinkage, and nuclear lysis. These results further confirmed the antitumor effect of HMPC nanoenzymes with laser irradiation. In order to explore the tumor inhibition mechanism of HMPC nanoenzymes, immunofluorescence analysis by TUNEL, Ki67 and GPX4 assays were conducted. As shown in Figure. 6G and S12, no obvious apoptosis, inactivation of GPX4 and obvious cell proliferation was observed in saline or laser group, while HMPC + laser group displayed obvious apoptosis, low or none cell proliferation, and the decreased level of GPX4, further confirming that HMPC + laser realized effective tumor inhibition by inducing apoptosis, inhibiting proliferation, as well as triggering ferroptosis. 4. Conclusion In conclusion, we have successfully developed nanoenzymes integrating Pt NPs and CyI into MIL-100 (Fe) capable of relieving hypoxia and inducing ferroptosis, for achieving enhanced PDT-ferroptosis therapy. First, HMPC nanoenzymes improved the hydrophility of CyI and efficiently deliver CyI to tumor cells by EPR effect and active targeting, thus enhancing their accumulation in target cells. Next, Fenton reaction caused by MIL-100 (Fe) made depleted GSH, inactivation of GPX4 and accumulation of LPO, triggering ferroptosis in tumor cells. Then, the released Pt NPs with catalase-like activity constantly catalyzed H 2 O 2 into O 2 to enhance PDT. The experiment results showed the nanoenzymes indeed enhanced ROS generation in the hypoxic condition by catalytic reaction, alleviating the hypoxia aggravated by PDT, and the nanoenzymes implemented ferroptosis therapy. Both in vitro and in vivo therapeutic results demonstrated that the nanoenzymes exhibit potent anticancer efficacy. Hence, the developed nanoenzymes can serve as a regimen combining PDT with ferroptosis therapy, displaying potent tumor inhibition with a good in vivo safety. HMPC nanoenzymes overcame some of key challenges in PDT antitumor treatment by integrating iron-based MOFs with Pt NPs. Such nanoenzymes provided a new model for enhanced PDT-ferroptosis therapy, which may represent a promising novel regimen for hypoxic tumor treatment. Declarations Supporting Information TEM image of MOF; The hydrodynamic size distribution and PDI of MOF, MP, MPC and HMPC; EDS spectrum of HMPC; The H 2 O 2 degradation of different formulations, PBS was used as blank control group, and H 2 O 2 solution was defined as positive group; The 1 O 2 generation of HMC and HMPC under laser irradiation; CLSM images of CT26 cells incubated with HMPC for different time; Hemolysis analysis; Blood routine index analysis for CT26 tumor-bearing mice injected with saline and HMPC; The body weight changes of mice form different groups during 14 d treatments; Photographs of harvested tumor from mice after 14 d treatments; H&E staining of major organs form mice incubated with different treatments; Ki67 immunofluorescent staining of the tumor tissues from different groups of mice. Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 32171362) and Natural Science Foundation of Shandong Province (No. ZR2022YQ73, ZR2021MH087). Author contributions YY and HY: Investigation, Methodology, Data curation. YZ: Methodology, Visualization. BL: Methodology. GX: Methodology. YS: Supervision. JC: Conceptualization, Project administration, Writing – review & editing, Supervision. JC and YY were the major contributors in writing the manuscript. YY and HY performed all experiments. All authors reviewed the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate All animal experiments were carried out according to the protocol approved by the Ethics Committee of Qingdao University . Consent for publication Not applicable. Competing interests The authors declare that they have no conflict of interest. References Li L, Song D, Qi L, Jiang M, Wu Y, Gan J, Cao K, Li Y, Bai Y, Zheng T. Photodynamic therapy induces human esophageal carcinoma cell pyroptosis by targeting the PKM2/caspase-8/caspase-3/GSDME axis. Cancer Lett. 2021;520:143–59. Xie J, Wang Y, Choi W, Jangili P, Ge Y, Xu Y, Kang J, Liu L, Zhang B, Xie Z, et al. Overcoming barriers in photodynamic therapy harnessing nano-formulation strategies. Chem Soc Rev. 2021;50:9152–201. Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer. 2003;3:380–7. Almeida-Marrero V, Bethlehem F, Longo S, Bertolino MC, Torres T, Huskens J, de la Escosura A. Tailored Multivalent Targeting of Siglecs with Photosensitizing Liposome Nanocarriers. Angew Chem Int Ed Engl. 2022;61:e202206900. Ji Y, Sun Y, Hei M, Cheng D, Wang B, Tang Y, Fu Y, Zhu W, Xu Y, Qian X. NIR Activated Upper Critical Solution Temperature Polymeric Micelles for Trimodal Combinational Cancer Therapy. Biomacromolecules. 2022;23:937–47. Wu Z, Tang Y, Chen L, Liu L, Huo H, Ye J, Ge X, Su L, Chen Z, Song J. In-Situ Assembly of Janus Nanoprobe for Cancer Activated NIR-II Photoacoustic Imaging and Enhanced Photodynamic Therapy. Anal Chem. 2022;94:10540–8. Obuobi S, Julin K, Fredheim EGA, Johannessen M, Škalko-Basnet N. Liposomal delivery of antibiotic loaded nucleic acid nanogels with enhanced drug loading and synergistic anti-inflammatory activity against S. aureus intracellular infections. J Control Release. 2020;324:620–32. Cheng X, Gao J, Ding Y, Lu Y, Wei Q, Cui D, Fan J, Li X, Zhu E, Lu Y, et al. Multi-Functional Liposome: A Powerful Theranostic Nano-Platform Enhancing Photodynamic Therapy. Adv Sci (Weinh). 2021;8:e2100876. Wu M, Yang Y. Metal-Organic Framework (MOF)-Based Drug/Cargo Delivery and Cancer Therapy. Adv Mater. 2017;29:201606134. Nadar SS, Vaidya L, Rathod VK. Enzyme embedded metal organic framework (enzyme-MOF): De novo approaches for immobilization. Int J Biol Macromol. 2020;149:861–76. Peng S, Bie B, Jia H, Tang H, Zhang X, Sun Y, Wei Q, Wu F, Yuan Y, Deng H, Zhou X. Efficient Separation of Nucleic Acids with Different Secondary Structures by Metal-Organic Frameworks. J Am Chem Soc. 2020;142:5049–59. Zhuang J, Gong H, Zhou J, Zhang Q, Gao W, Fang RH, Zhang L. Targeted gene silencing in vivo by platelet membrane-coated metal-organic framework nanoparticles. Sci Adv. 2020;6:eaaz6108. Zhang Y, Wang L, Liu L, Lin L, Liu F, Xie Z, Tian H, Chen X. Engineering Metal-Organic Frameworks for Photoacoustic Imaging-Guided Chemo-/Photothermal Combinational Tumor Therapy. ACS Appl Mater Interfaces. 2018;10:41035–45. Zhu J, Wang X, Su Y, Shao J, Song X, Wang W, Zhong L, Gan L, Zhao Y, Dong X. Multifunctional nanolocks with GSH as the key for synergistic ferroptosis and anti-chemotherapeutic resistance. Biomaterials. 2022;288:121704. Cao F, Sang Y, Liu C, Bai F, Zheng L, Ren J, Qu X. Self-Adaptive Single-Atom Catalyst Boosting Selective Ferroptosis in Tumor Cells. ACS Nano. 2022;16:855–68. Winterbourn CC. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett. 1995;82–83:969–74. Shen Z, Ma Q, Zhou X, Zhang G, Hao G, Sun Y, Cao J. Strategies to improve photodynamic therapy efficacy by relieving the tumor hypoxia environment. NPG Asia Materials. 2021;13:39. Wei H, Wang E. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev. 2013;42:6060–93. Liu X, Dong X, Yang S, Lai X, Liu H, Gao Y, Feng H, Zhu M, Yuan Y, Lu Q, et al. Biomimetic Liposomal Nanoplatinum for Targeted Cancer Chemophototherapy. Adv Sci (Weinh). 2021;8:2003679. Hao Y, Chen Y, He X, Yu Y, Han R, Li Y, Yang C, Hu D, Qian Z. Polymeric Nanoparticles with ROS-Responsive Prodrug and Platinum Nanozyme for Enhanced Chemophotodynamic Therapy of Colon Cancer. Adv Sci (Weinh). 2020;7:2001853. Abed A, Derakhshan M, Karimi M, Shirazinia M, Mahjoubin-Tehran M, Homayonfal M, Hamblin MR, Mirzaei SA, Soleimanpour H, Dehghani S, et al. Platinum Nanoparticles in Biomedicine: Preparation, Anti-Cancer Activity, and Drug Delivery Vehicles. Front Pharmacol. 2022;13:797804. Cao J, Chi J, Xia J, Zhang Y, Han S, Sun Y. Iodinated Cyanine Dyes for Fast Near-Infrared-Guided Deep Tissue Synergistic Phototherapy. ACS Appl Mater Interfaces. 2019;11:25720–9. Xu B, Chen Z, Han B, Li C. Glycol assisted synthesis of MIL-100(Fe) nanospheres for photocatalytic oxidation of benzene to phenol. Catal Commun. 2017;98:112–5. Teranishi T, Tanaka MH,T, Miyake M. Size Control of Monodispersed Pt Nanoparticles and Their 2D Organization by Electrophoretic Deposition. J Phys Chem B. 1999;103:3818–27. Lennicke C, Rahn J, Lichtenfels R, Wessjohann LA, Seliger B. Hydrogen peroxide - production, fate and role in redox signaling of tumor cells. Cell Commun Signal. 2015;13:39. Li N, Jiang W, Wang W, Xiong R, Wu X, Geng Q. Ferroptosis and its emerging roles in cardiovascular diseases. Pharmacol Res. 2021;166:105466. Zhang K, Ma Z, Li S, Wu Y, Zhang J, Zhang W, Zhao Y, Han H. Disruption of dual homeostasis by a metal-organic framework nanoreactor for ferroptosis-based immunotherapy of tumor. Biomaterials. 2022;284:121502. Lei G, Zhuang L, Gan B. Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer. 2022;22:381–96. Yan H, Zou T, Tuo Q, Xu S, Li H, Belaidi AA, Lei P. Ferroptosis: mechanisms and links with diseases. Signal Transduct Target Ther. 2021;6:49. Li Z, Dai H, Huang X, Feng J, Deng J, Wang Z, Yang X, Liu Y, Wu Y, Chen P, et al. Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma. Acta Pharmacol Sin. 2021;42:301–10. Poon JF, Pratt DA. Recent Insights on Hydrogen Atom Transfer in the Inhibition of Hydrocarbon Autoxidation. Acc Chem Res. 2018;51:1996–2005. Zou Y, Palte MJ, Deik AA, Li H, Eaton JK, Wang W, Tseng YY, Deasy R, Kost-Alimova M, Dančík V, et al. A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis. Nat Commun. 2019;10:1617. Meng X, Deng J, Liu F, Guo T, Liu M, Dai P, Fan A, Wang Z, Zhao Y. Triggered All-Active Metal Organic Framework: Ferroptosis Machinery Contributes to the Apoptotic Photodynamic Antitumor Therapy. Nano Lett. 2019;19:7866–76. Additional Declarations No competing interests reported. Supplementary Files supprotinginformationF.docx Cite Share Download PDF Status: Posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-2381256","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":160670606,"identity":"893b4fcf-862e-437d-bd16-58ea330e498c","order_by":0,"name":"Yuyun Ye","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuyun","middleName":"","lastName":"Ye","suffix":""},{"id":160670607,"identity":"039e2aed-2170-4eb9-b1dc-f4bd2ca06aef","order_by":1,"name":"Hongli Yu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongli","middleName":"","lastName":"Yu","suffix":""},{"id":160670608,"identity":"0decefa7-d531-4966-ade0-2ec78535f2eb","order_by":2,"name":"Yifan Zhao","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Zhao","suffix":""},{"id":160670609,"identity":"29c1a4e9-d805-450d-8e91-eb6d59b6bdec","order_by":3,"name":"Lv Bai","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lv","middleName":"","lastName":"Bai","suffix":""},{"id":160670610,"identity":"71e88778-7422-428d-83a4-167b8197f492","order_by":4,"name":"Guanghe Xue","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guanghe","middleName":"","lastName":"Xue","suffix":""},{"id":160670611,"identity":"d1f20772-934e-489b-adf7-668ef837be0b","order_by":5,"name":"Yong Sun","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Sun","suffix":""},{"id":160670612,"identity":"413d3d07-068c-4f72-aa5e-49f06d366e35","order_by":6,"name":"Jie Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYJACgwQGCTl+9uZjYB4bO3FabIwle46lMTAkALUwE2dRWuKGGzlmYC0MhLTwtx8+UPCg4rAxw42cbw8+/tgmz8fMwPjhYw5uLRJn0hIMEs4clmPsebvdcEbCbcM2ZgZmyZnb8PiDIcfAILHtsDEze+42aZ6E24xALWzMvPi08L8Bavl3OLGNIecZSIs9YS0SIFsa0hJ7OHLYQFoSCWqRuPEM6JdjNsYSPMfMJGek3U5uY2ZsxusX/v7kY4Y/aiTk7I83P5P4YHPbdn5788EPH/FoAQI2AzQBxga86oGA+QEhFaNgFIyCUTDCAQBwo09wRw2vmwAAAABJRU5ErkJggg==","orcid":"","institution":"Qingdao University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2022-12-15 10:44:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2381256/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2381256/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30583353,"identity":"d8a4686f-8aa4-4f1a-94a1-ab8973c63705","added_by":"auto","created_at":"2022-12-20 17:59:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":268334,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration showing the synthesis process of HMPC nanoenzymes; (B) Schematic diagram displaying the nanoenzymes for enhanced PDT-ferroptosis therapy of hypoxic tumor.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/d08c9f1a57f25f83d4919fc5.jpg"},{"id":30583351,"identity":"06afdb75-26e6-480c-abd3-3aa38e2e28ef","added_by":"auto","created_at":"2022-12-20 17:59:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":344476,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TEM images of MOF, Pt and MP NPs; (B)TEM image of HMPC nanoenzymes, insert image is size distribution of HMPC nanoenzymes; (C) Enlarged TEM image of the dotted area of Figure 2B, insert is a further enlarged image of HMPC nanoenzymes; (D) EDS mapping images of HMPC nanoenzymes; (E) XRD patterns of MOF and MP NPs; (F) Zeta potential diagram of different nanoparticles (MOF, MP, MPC and HMPC nanoenzymes); (G) UV–vis spectra of free CyI, MP, MPC, HMPC and HA; (H) O\u003csub\u003e2\u003c/sub\u003e production profiles of MOF, catalase-like Pt NPs and HMPC with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 720 s; (I) Hydroxyl radical production of PBS, MOF, HMPC with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e determined by TMB. Insert shows the color change of TMB oxidation with various treatments.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/976111e3d9d5693b9126431c.jpg"},{"id":30583356,"identity":"27354428-3285-4c18-8f28-a458368a1260","added_by":"auto","created_at":"2022-12-20 17:59:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413587,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cytotoxicity studies by MTT assay for CT26 and L929 cells after incubation with HMPC nanoenzymes with different concentrations of CyI, data are presented as means ± SD (n=3); (B) FCM histogram profiles of CT26 cells incubated with HMPC nanoenzymes for different time; (C) FCM histogram profiles and (D) CLSM images of CT26 cells incubated with different treatments; (E) CLSM images of CT26 cells after treated with different treatments for intercellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and hypoxia detection; (F) Mean fluorescence intensity of hypoxia level of CT26 cells with different treatments, data are presented as means ± SD (n=3); (G) CLSM images and (H) FCM histogram profiles of CT26 cells after treated with different treatments for ROS detection; (I) Photothermal effects profiles and (J) Representative thermal images of CT26 cells under laser irradiation, data are presented as means ± SD (n=3); (K) \u003cem\u003ein vitro\u003c/em\u003e PDT treatment of CT26 cells by HMC and HMPC nanoenzymes under laser irradiation in hypoxic or normoxic conditions, data are presented as means ± SD (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001; (laser: 0.96 W/cm\u003csup\u003e2\u003c/sup\u003e, 5 min).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/61aa59acddf7de6cf5efd775.jpg"},{"id":30583358,"identity":"3db54053-e920-4437-9e95-d6618906cbcb","added_by":"auto","created_at":"2022-12-20 17:59:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401999,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Intracellular GSH level of CT26 cells after treated with different formulas for 4 h, data are presented as means ± SD (n=3); (B) CLSM image of the expression of GPX4 in CT26 cells after different treatments (from I to VI); (C) Mean fluorescence intensity of GPX4 level in CT26 cells after different treatments (from I to VI), data are presented as means ± SD (n=3); (D) CLSM observation of the accumulation of LPO in CT26 cells with C11-BODIPY probe during different treatments (from I to VI); (E) Mean fluorescence intensity of LPO in CT26 cells after different treatments (from I to VI), data are presented as means ± SD (n=3); (F) Intracellular MDA level of CT26 cells after treated with different formulas for 24 h, data are presented as means ± SD (n=3); (G)\u003cstrong\u003e \u003c/strong\u003eCytotoxicity studies by MTT assay for CT26 cells after incubation with different treatments in hypoxic condition, under hypoxic conditions (laser: 0.96 W/cm\u003csup\u003e2\u003c/sup\u003e, 5 min), data are presented as means ± SD (n=3); (H) Cell viability after treated with Vc (200 μM), GSH (5 mM), DFO (100 μM), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 μM) and HMPC nanoenzymes under laser irradiation for 24 h, data are presented as means ± SD (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/6df7b5fd4b62d24f1c3b998d.jpg"},{"id":30583369,"identity":"754eb14b-4c2f-4e45-856a-607f66b7c9b7","added_by":"auto","created_at":"2022-12-20 17:59:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":373720,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence imaging of HMPC and free CyI at 1 h, 2 h, 4 h, 6 h, 10 h, and 24 h after intravenous injection; (B) \u003cem\u003eEx vivo\u003c/em\u003efluorescence images of major organs and tumors dissected from mice injected with HMPC and free CyI; (C) H\u0026amp;E slices of major organs for safety determination by comparing the HMPC-injected group to the saline-injected group; (D–G) The AST, ALT, BUN, and CREA level in BALB/c mice after injected with HMPC and saline, data are presented as means ± SD (n=3). The dotted lines indicate the normal value range of the biochemical parameters in healthy mice.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/5347343e4d6ccd2e236d05b3.jpg"},{"id":30583387,"identity":"528e4ee5-6b60-43ec-8908-7b0926a3ac6c","added_by":"auto","created_at":"2022-12-20 17:59:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":494892,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e level and hypoxia level in tumor tissue from mice after different treatments; (B) Mean fluorescence intensity of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e level in tumor tissue after different treatments, data are presented as means ± SD (n=3); (C) LPO accumulation level in tumor tissues using C11-BODIPY after different treatments; (D) Fluorescence ratio of probe indicating the LPO accumulation level in tumor tissue after different treatments, data are presented as means ± SD (n=3); (E) Tumor growth curves of mice during 14-d treatment, data are presented as means ± SD (n=5); (F) Photographs of mice after 14-d treatments; (G) Histological observation of the tumor tissues with H\u0026amp;E staining, and TUNEL immunofluorescence staining of the tumor from different groups of mice, as well as the level of GPX4 expression in tumor tissues from mice after different treatments, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/25abe1fdee9cedf798fb038f.jpg"},{"id":31396134,"identity":"ea5dc258-1f48-4120-908c-a6f2a946c7ee","added_by":"auto","created_at":"2023-01-11 00:44:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1785185,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/137724df-1add-4811-9790-df7fe16087b4.pdf"},{"id":30583354,"identity":"bc567baa-89d8-45c3-accd-d7b51e0d8688","added_by":"auto","created_at":"2022-12-20 17:59:20","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6469464,"visible":true,"origin":"","legend":"","description":"","filename":"supprotinginformationF.docx","url":"https://assets-eu.researchsquare.com/files/rs-2381256/v1/8254e17be08ed4fe30b56f34.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineering Nanoenzymes Integrating Iron-based MOFs with Pt NPs for Enhanced PDT-Ferroptosis Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePhotodynamic therapy (PDT), with advantages of spatiotemporal specificity, low systemic toxicity, and non-invasiveness, have been used as a potent treatment method against malignant tumors during the past decades[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With the laser irradiation of specific wavelength, oxygen (O\u003csub\u003e2\u003c/sub\u003e) is converted into cytotoxic reactive oxygen species (ROS) through photosensitizer-mediated photochemical reaction, and the excessive intracellular ROS cause biomolecular peroxidation, while leading to the dysregulation of intracellular redox homeostasis, eventually resulting in cell necrosis and apoptosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the shortcomings of the photosensitizers (PSs) such as low aqueous solubility, lake of tumor-targeting, easy aggregation and light penetration depth are obstacles for deep tumor clinical application.\u003c/p\u003e \u003cp\u003eWith the development of nanotechnology, numerous drug delivery carriers have been explored to deliver PSs, such as liposomes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], polymeric micelles[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], inorganic nanoparticles[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and so on. However, most nanocarriers show shortcomings such as poor drug loading[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], poor physical stability[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and non-repeatability. Metal-organic framework (MOF) is a highly ordered porous heterogeneous material formed by coordination between metal ions and organic ligands with the advantages of adjustable pore size, high loading capacity, easy functionalization, and biodegradability[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared with traditional nanocarriers, MOFs have showed high loading properties, biodegradation and low systemic toxicity. Nanoscale MOFs have been explored as drug delivery for proteins[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], nucleic acids[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and small molecule drugs, such as curcumin[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and doxorubicin. In particularly, iron-based MOFs, as a kind of active MOFs, can catalyze hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to generate hydroxyl radical (∙OH) through Fenton reaction, which can further deplete glutathione (GSH) in tumor cells, resulting in the inactivation of glutathione peroxidase (GPX4), the accumulation of lipid peroxides (LPO), and ultimately ferroptosis in tumor cells[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, iron-based MOFs can not only be used as carriers for PSs, but also as inducers to implement iron-induced ferroptosis.\u003c/p\u003e \u003cp\u003eHowever, due to the hypoxic tumor microenvironment (TME), and the oxygen consumption in PDT process, which would further aggravate the tumor hypoxia, PDT efficacy are generally limited. Up to now, multiple strategies have been developed to relieve tumor hypoxia[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For instance, nonreactive oxygen carriers, such as hemoglobin (Hb) and perfluorocarbon carbide, as well as reactive oxygen supply materials, such as manganese dioxide (MnO\u003csub\u003e2\u003c/sub\u003e), hydrogen peroxide enzymes are used to increase O\u003csub\u003e2\u003c/sub\u003e content during antitumor therapy. However, these strategies have showed disadvantages: Hb can only deliver a limited amount of O\u003csub\u003e2\u003c/sub\u003e, and MnO\u003csub\u003e2\u003c/sub\u003e is constantly consumed in redox reaction, which cannot meet the requirements for prolonged O\u003csub\u003e2\u003c/sub\u003e supplementation. Among them, nanoenzymes are a class of nanomaterials with enzymatic catalytic activity where small-sized Pt nanoparticles (Pt NPs) possess catalase-like activity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared with other oxygen carriers and O\u003csub\u003e2\u003c/sub\u003e-producing materials, Pt NPs with continuous catalytic capacity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are able to provide O\u003csub\u003e2\u003c/sub\u003e for long time to alleviate hypoxia. Liu \u003cem\u003eet al.\u003c/em\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]encapsulated Pt NPs in the water chamber of liposomes to overcome the limitation of tumor hypoxia in the efficacy of PDT. Qian \u003cem\u003eet al.\u003c/em\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]encapsulated Pt NPs in the amphiphilic polymers, distearyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), to form polymer micelles that ameliorated hypoxia and enhanced PDT. However, due to the catalytic activity of Pt NPs is related to size, which Pt NPs with ultra-small size (3\u0026thinsp;~\u0026thinsp;5nm) have higher catalytic activity, the reported Pt NPs exhibit the defects of easy aggregation, low stability and premature clearance when size decreases below 5 nm[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, it is essential to develop novel nanosystems to stabilize Pt NPs.\u003c/p\u003e \u003cp\u003eHence, in this study, we developed a novel nanoenzyme (HMPC) for enhanced PDT-ferroptosis therapy by integrating iron-based MOF, MIL-100 (Fe), with Pt NPs, and uploading near-infrared (NIR) photosensitizer CyI into the inner core (Figure. 1). Hyaluronic acid (HA) was adhered to MOF by electrostatic interaction, which endowed the nanoenzymes with active targeting capability. Pt NPs in HMPC nanoenzymes, as a \u0026ldquo;machine\u0026rdquo; for O\u003csub\u003e2\u003c/sub\u003e generation by catalytic reaction to relieve tumor hypoxia, were stabilized by MOF through physical adsorption, which can diminish premature clearance by the mononuclear phagocyte system (MPS) and increase the safety in systemic circulation. CyI, as an iodinated-cyanine dye, has been explored and investigated in our previous study, which exhibit improved \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e yield, effective photothermal and NIR fluorescence imaging properties (Ex/Em: 756/822nm, fluorescence quantum yield is 0.48, the singlet oxygen yield is up to 0.75)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After the HMPC nanoenzymes are taken up by tumor cells through high expression of CD44 receptors, due to the high concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in tumor cells, MOF undergoes Fenton reaction to generate Fe\u003csup\u003e2+\u003c/sup\u003e and \u0026middot;OH, resulting in ferroptosis which could deplete GSH and the collapse of MIL-100 frame. Under NIR irradiation, the released Pt NPs can catalyze H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to offer O\u003csub\u003e2\u003c/sub\u003e, which could improve PDT efficacy of CyI. In addition, depleted GSH caused by ferroptosis therapy also enhance ROS retention in TME. Moreover, CyI was in possession of photothermal effect to implement photothermal therapy (PTT). We presume that by integrating Pt NPs and PSs into iron-based MOF, the nanoenzymes exhibit enhanced PDT-ferroptosis therapy, which may represent a promising novel regimen for hypoxic tumor treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Materials.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCyI (Mw 776.5) was synthesized according to our previously reported protocal. 1, 3, 5-benzenetricarboxylic acid (BTC), Iron chloride (FeCl\u003csub\u003e3\u003c/sub\u003e), Chloroplatinic acid hexahydrate (H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) and 3,3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine dihydrochloride (TMB) were purchased from Aladdin (Shanghai, China). HA and Deferoxamine mesylate salt (DFO) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Hydrogen peroxide aqueous solution (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glutathione (GSH) and Vitamin C (Vc) was purchased from Macklin (Shanghai, China). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) and SOSG singlet oxygen fluorescence probe was bought from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). ROS Green\u0026trade; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Probe was purchased from Maokang Co., Ltd. (Shanghai, China). Image-iT\u0026trade; Green Hypoxia Reagent was bought from Thermo Fisher Scientific (New York, USA). C11-BODIPY 581/591 was bought from Wuhan Anjiekai Biological Medicine Technology Co., Ltd. (Wuhan, China). GPX4 antibody was purchased from Beyotime (Shanghai, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Preparation of HMPC nanoenzymes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e8 ml of FeCl\u003csub\u003e3\u003c/sub\u003e (40 mM) glyoxal solution was mixed with 8 ml BTC (45 mM) DMF solution for 10 min at room temperature. The obtained solution reacted at 100\u0026deg;C for 2 h. The product (MOF) was collected by centrifugation and washed with DMF, ethanol, deionized water for 3 times. 5 ml of H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e (0.60 mM) solution was added dropwise to 45 ml ethanol solution containing polyvinyl pyrrolidone (PVP) stirred at room temperature for 10 min, and refluxed at 70\u0026deg;C for 3 h to obtain Pt NPs solution. 20 ml Pt NPs solution was added dropwise to the MOF (8 mg/ml) DMF solution, and stirred for 3 h at room temperature. The product (MP) was collected by centrifugation and washed with ethanol, deionized water for 3 times. CyI was added into the MP methanol solution and stirred for 24 h at room temperature in the dark. The mixture (MPC) collected by centrifugation and washed with deionized water for three times to remove free CyI. HA aqueous solutions were added dropwise to MPC aqueous solutions, and stirred for 30 min at room temperature in the dark. Then, the precipitate was collected and washed with deionized water to remove free HA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Characterization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphologies of MOF, Pt NPs, MP and HMPC were examined via TEM. The EDS was utilized to analyze the elements of HMPC. The hydrated particle size and zeta potential of MOF, MP, MPC and HMPC were measured by DLS. UV\u0026ndash;vis absorbance spectra of HA, CyI, MP, MPC and HMPC were acquired using a UV\u0026ndash;vis absorption spectrophotometer. The X-ray diffraction signal of MOF and MP were obtained by an X-ray diffractometer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 The Catalase-like activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following experiments were conducted to verify that the catalase-like activity of Pt NPs was not impaired after loading CyI and modifying HA. The catalase-like activity of HMPC was assayed by observing the generation of O\u003csub\u003e2\u003c/sub\u003e through the catalytic decomposition of hydrogen peroxide. Firstly, the PBS solution was sealed with liquid paraffin to prevent combination of oxygen with the solution. Next, 20 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e PBS solution was prepared by adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution to PBS with a syringe. Then, a probe of the dissolved oxygen meter was inserted under the surface of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e PBS solution, the HMPC, MOF or Pt solution was added with a syringe, and were reacted at room temperature for 12 min. Finally, the changes of dissolved oxygen level in the solution were recorded.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Detection of hydroxyl radicals for Fenton reaction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMIL-100 catalyzes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce hydroxyl radicals (\u0026middot;OH) through Fenton reactions. This experiment was used to verify whether HMPC was still capable of undergoing Fenton reactions. 3,3',5,5'-tetramethylbenzidine (TMB) could be able to react with \u0026middot;OH, creating a new significant absorption peak at 652 nm. TMB was added in PBS, MOF or HMPC solutions with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. All samples were measured by UV-Vis after reaction at room temperature for 30 min.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Cell and animal model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT26 cell lines were incubated in 1640 culture medium (5% CO\u003csub\u003e2\u003c/sub\u003e) that was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Incubator temperature was kept at 37\u0026deg;C. Female BALB/c mice were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), which were 4\u0026ndash;6 weeks old and weighed about 18\u0026ndash;20 g. The tumor model was developed by subcutaneously injecting 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CT26 cells (suspended in PBS) into the axilla of each BALB/c mouse. When the tumor volumes reach to 100\u0026ndash;200 mm\u003csup\u003e3\u003c/sup\u003e, the mice are ready for treatment. All animal procedures were implemented in compliance with the Animal Management Rules of the Ministry of Health of the People\u0026rsquo;s Republic of China (document no. 55, 2001) and approved by the Animal Care Ethics Committee of Qingdao University (Qingdao, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Cytotoxicity assays.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT26 Cells and L929 Cells were seeded in 96-well plates for 24 h incubation. After that, different concentrations of HMPC were added to replace the medium and incubated with cells for 24 h. Finally, 100 \u0026micro;l MTT solution was added to each well, incubated for 4 h and measured at 492 nm by enzyme standard instrument.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 \u003cem\u003eIn vitro\u003c/em\u003e therapeutic effect.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT26 Cells were seeded in 96-well plates for 24 h incubation. After that, different solutions were added to replace the medium, laser (808 nm, 0.96 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min after 4 h, and incubated with cells for 24 h. Finally, 100 \u0026micro;l MTT solution was added to each well, incubated for 4 h and measured at 492 nm by enzyme standard instrument.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Cell uptake and targeting property.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFCM and CLSM were used to investigate the cell uptake of HMPC and their targeting property. To conduct CLSM observation, CT26 cells were seeded into confocal dishes and cultured overnight. Following removal of the culture medium, cells were treated with HMPC and MPC. Subsequently, DAPI fluorescent probe was used to stain the nuclei. After solution removal and washed with PBS twice, the cells were quickly observed under CLSM. In the blocking group, the operation was the same as above, except that cells were treated with HA. CT26 cells were seeded into 6-well plates and cultured overnight. Following removal of the culture medium, cells were treated with HMPC and MPC. After solution removal and washed with PBS twice, the cells were collected and resuspended in PBS for FCM. In the blocking group, the operation was the same as above, except that cells were treated with HA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e ROS generation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e ROS generation was detected by CLSM and FCM using DCFH-DA (10 \u0026micro;M) as a probe. To conduct CLSM observation, CT26 cells were seeded into confocal dishes and cultured overnight. Following removal of the culture medium, cells were treated with PBS, HMPC, and HMC for another 4 h. Subsequently, DCFH-DA was loaded into cells and incubated for 20 min. The cells in the laser treatment groups were irradiated with 880 nm laser for 5 min (0.96 W/cm\u003csup\u003e2\u003c/sup\u003e). DAPI fluorescent probe was used to stain the nuclei. After solution removal and washed with PBS twice, the cells were quickly observed under CLSM. In the hypoxic group, the operation was the same as above, except that cells were treated with sodium disulfite (1 mM) for 12 h in advance to simulate hypoxia. Cells were incubated with different reagents for 4 h. After that, the laser group was irradiated. Next, the solution in the well was removed, then the cells were incubated with DCFH-DA for 20 min. After the probe was removed, the cells were washed with PBS for 3 times, and the cells were collected and resuspended in PBS for FCM. SOSG singlet oxygen fluorescence probe acted as a probe to detect \u003cem\u003ein vivo\u003c/em\u003e ROS generation. 200 \u0026micro;l of normal saline, CyI, HMC and HMPC were injected into the tail vessel of mice. After 12 h, the tumors were injected 50 \u0026micro;l SOSG (25 \u0026micro;M) and the tumors of the laser treatment groups were exposed to 808 nm laser irradiation for 5 min. The mice were sacrificed and tumors were collected for cryosection. Finally, tumor sections were visualized by CLSM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Intercellular H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003eand hypoxia detection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of intercellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or hypoxia was detected by CLSM. CT26 cells were prepared in confocal dishes and cultured overnight. The cells in the hypoxic group were treated with sodium disulfite (1 mM) for 12 h in advance to simulate hypoxia. The cells were treated with RPMI 1640, HMC, HMPC, CyI\u0026thinsp;+\u0026thinsp;laser, HMC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;laser, separately. Then, each of these groups were added ROS Green\u0026trade; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Probe (5 \u0026micro;M). After incubation at room temperature for 30 min, 4% paraformaldehyde fix solution was added into all groups equally after removal of the medium and washing the cells three times with PBS. After 20 min, the cells were washed with PBS 2 times. Finally, DAPI staining was performed for observation. Image-iT\u0026trade; Green Hypoxia Reagent (5 \u0026micro;g/ml) was used for hypoxia detection, and the incubation time is 16 min. Other operations were the same as above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eIn vitro\u003c/span\u003e \u003cstrong\u003ePTT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe performance of PTT is measured by thermocouple thermometer and infrared thermal imaging camera. The cells were incubated with different solutions for 4 h. After the solution removal, the cells were washed with PBS for 3 times. Next, the cells were collected and resuspended in PBS. All the groups were irradiated for 10min, and the temperature was measured every 30 s using thermocouple thermometer. Finally, the final temperature of every group were recorded by infrared thermal imaging camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 GSH assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith various treatments (PBS, HMPC, HMPC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser or HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DFO (1 mM)\u0026thinsp;+\u0026thinsp;laser), GSH content was measured with reduced GSH content detection kit. The cells were collected and washed with PBS three times. Subsequently, cells were cleaved by ultrasound (200 W, ultrasound 3 s, pause for 10 s, repeat 30 times). The supernatant was collected by centrifugation at 8000\u0026times;g for 10 min. According to the instruction of kit, the absorbance of each samples was detected at 412 nm by enzyme standard instrument. According to the standard curve, the content of GSH was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 MDA assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith various treatments as above (GSH Assay), MDA content was measured with a MDA determination kit to monitor the level of lipid peroxidation. The cells were collected and washed with PBS three times. cells were cleaved by cell lysis buffer and ultrasound (200 W, ultrasound 3 s, pause for 10 s, repeat 30 times). The supernatant was collected by centrifugation at 1000\u0026times;g for 10 min and used for MDA detection. Subsequently, according to the instruction of kit, the absorbance of each groups was detected at 532 nm by UV-vis. According to the formula provided by the kit, the content of MDA was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15 Expression of GPX4 evaluation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression level of GPX4 \u003cem\u003ein vitro\u003c/em\u003e was detected by CLSM. CT26 cells were prepared in confocal dishes and cultured overnight. The cells were treated with RPMI1640, HMPC, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, HMPC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser and HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser with DFO added, separately. Then, the cells were fixed with 4% paraformaldehyde fix solution for 20 min, each of these groups added the primary antibody of GPX4 after wash with PBS. After incubation at room temperature for 1 h, the second antibody with FITC labeling was added into all groups equally after removal of the medium and washing the cells three times with PBS. The time of secondary antibody incubation is 1 h. After 1 h, the cells were washed with PBS 3 times to remove the second antibody. Finally, DAPI staining was performed for CLSM observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.16\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eIn vitro\u003c/span\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003ein vivo\u003c/span\u003e \u003cstrong\u003edetection of LPO.\u003c/strong\u003e LPO was typically detected by a C11-BODIPY 581/591 fluorescence probe, which can be observed by CLSM. CT26 cells were treated with various treatments (PBS, HMPC, HMPC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser or HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;DFO (1mM)\u0026thinsp;+\u0026thinsp;laser) for 4 h. The cells were washed with PBS three times and incubated with the medium containing C11-BODIPY 581/591 (5 \u0026micro;M) for 20 min. The cells in the laser treatment groups were irradiated with 880 nm laser for 5 min (0.96 W/cm\u003csup\u003e2\u003c/sup\u003e). DAPI fluorescent probe was used to stain the nuclei. Next, cells were washed with PBS then subjected to CLSM observation for \u003cem\u003ein vitro\u003c/em\u003e LPO accumulation. To evaluate the production of LPO histologically, 200 \u0026micro;l of normal saline, CyI, HMC and HMPC were injected into the tail vessel of mice. After 12 h, the tumors in the laser treatment groups were exposed to 808 nm laser irradiation for 5min. The mice were sacrificed and tumors were collected for cryosection. Tumor sections were stained with C11-BODIPY 581/591 (2.5 \u0026micro;M) at 37 ℃ for 30 min, and fixed with 10% paraformaldehyde at room temperature for 10 min. Then the tissue sections were stained with DAPI for 10 min. Finally, oxidized C11-BODIPY 581/591 was observed under a confocal microscope to evaluate \u003cem\u003ein vivo\u003c/em\u003e LPO accumulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.17\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eIn vivo\u003c/span\u003e \u003cstrong\u003eantitumor study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven groups of CT26 tumor-bearing mice were randomly assigned in our experiment for evaluating the curative effect of the combination therapy. When the tumor reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, respective treatment (saline, laser, HMPC, CyI\u0026thinsp;+\u0026thinsp;laser, HMC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;laser, and HMPC\u0026thinsp;+\u0026thinsp;laser\u0026thinsp;+\u0026thinsp;DFO (dose: CyI 1.5 mg/kg, DFO 20 mg/kg) ) was executed separately to the seven groups. The mice in the laser treatment groups were subjected to laser (0.96 W/cm\u003csup\u003e2\u003c/sup\u003e for 5 min) at 12 h post-intravenous injection. The tumor size and body weight were monitored every 2 days. Tumor volume was calculated as width\u003csup\u003e2\u003c/sup\u003e \u0026times; length/2. After treatment, the mice were sacrificed, and the tumors were collected for photographing and hematoxylin-eosin (HE) staining. Morphological changes of the tumors were observed by fluorescence microscope. To further understand the tumor inhibition mechanism of HMPC, immunofluorescence analysis of Ki67, TUNEL, and GPX4 was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.18\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eIn vivo\u003c/span\u003e \u003cstrong\u003eimaging.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFree CyI and HMPC were administered by intravenous injection separately for two mice bearing about 100 mm\u003csup\u003e3\u003c/sup\u003e tumors. Fluorescence images of the mice were acquired by the IVIS\u0026reg; spectrum \u003cem\u003ein vivo\u003c/em\u003e imaging system at different time intervals post-injection. Two mice were sacrificed, with the tumor, heart, spleen, liver, lung, and kidney excised for the observation of the biodistribution of HMPC and free CyI via imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.19 Statistical analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Statistical analysis was calculated by Students\u0026rsquo; t-test with statistical significance assigned for P values of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Preparation and characterization of HMPC nanoenzymes.\u003c/h2\u003e\n\u003cp\u003eMOF NPs were synthesized using a solvothermal method[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The MOF NPs were mixed with Pt NPs prepared by ethanol reduction method[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] and stirred for 3 h to obtain Pt NPs-loaded MOF (MOF@Pt, MP). Subsequently, MP NPs or MOF NPs were further loaded with the photosensitizer CyI, to produce CyI-loaded MP (MOF@Pt@CyI, MPC) and CyI-loaded MOF (MOF@CyI, MC). Finally, MPC NPs or MC NPs were coated with hyaluronic acid (HA) to endow active targeting properties. The prepared nanoenzymes were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Figure. 2A and S1, MOF and MP NPs with uniformly distributed granular diameters (~\u0026thinsp;140 nm and ~\u0026thinsp;150 nm, respectively) are regularly coronal spherical. It should be noted that uploading of Pt NPs to MOF NPs (size below 5 nm) did not significantly affect the morphology of MOF NPs. HMPC nanoenzymes were monodisperse nanospheres with ~\u0026thinsp;160 nm in diameter and small-size Pt NPs were uniformly distributed on the surface of nanoparticles (Figure. 2B and C). The hydrodynamic diameters of the prepared nanoparticles measured by DLS revealed that the average diameters of particles (all less than 200 nm) (Figure. S2), implying that HMPC nanoenzymes can accumulate and remain at the tumor site through enhanced permeability and retention (EPR) effect.\u003c/p\u003e\n\u003cp\u003eElemental distribution of the HMPC nanoenzymes according to the mapping images of energy dispersive spectroscopy (EDS) revealed Fe was uniformly distributed within the core region, while most Pt was distributed on the periphery, implying the formation of Pt NPs (Figure. 2D and S3). The crystal phase and composition of nanoparticles was further confirmed by X-ray diffraction (XRD) (Figure. 2E). The diffraction pattern of MOF confirmed the successful synthesis of MIL-100. MP NPs possessed obvious new diffraction peaks as compared to MOF NPs, matching well with the standard XRD data for Pt (JCPDS card, file No.65-2868). The zeta potential of HMPC nanoenzymes in aqueous solution was \u0026minus;\u0026thinsp;24.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 mV, implying good stability under physiological conditions (Figure. 2F). Absorption spectra showed that compared with MP NPs, HMPC nanoenzymes have a new absorption peak in the near infrared region of 650\u0026ndash;850 nm, confirming the successful uploading of CyI (Figure. 2G).\u003c/p\u003e\n\u003cp\u003eTo evaluate the catalase-like activity of the HMPC nanoenzymes, the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was investigated using a fluorescence probe, ROS Green\u0026trade; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. This probe can emit fluorescence in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the fluorescence intensity would raise with the increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration. As shown in Figure. S4, the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is about 99% in the presence of HMPC nanoenzymes, much more than that of HMC group, implying that HMPC nanoenzymes have catalase-like activity after incorporation of Pt NPs. Then, to further verify the catalase-like activity of HMPC nanoenzymes, O\u003csub\u003e2\u003c/sub\u003e production was detected and quantitated with dissolved oxygen analyzer. As shown in Figure. 2H, O\u003csub\u003e2\u003c/sub\u003e concentration increased rapidly after adding HMPC nanoenzymes or Pt NPs, while in the absence of Pt NPs, the O\u003csub\u003e2\u003c/sub\u003e concentration did not change significantly. The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation by CyI under laser irradiation was also explored by SOSG, a specific singlet oxygen probe. The results shown in Figure. S5 confirmed that HMPC nanoenzymes with Pt NPs could increase \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e production.\u003c/p\u003e\n\u003cp\u003eHydroxy radical (\u0026middot;OH), as one of the products of Fenton reaction, can undergo redox reaction with tetramethyl benzidine (TMB) and yields strong absorption peak at 652 nm. Therefore, TMB was used to detect the generation of \u0026middot;OH. As shown in Figure. 2I, MOF NPs and HMPC nanoenzymes did not generate \u0026middot;OH, and only in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a new absorption peak appeared at 652 nm, indicating the generation of \u0026middot;OH and the occurrence of Fenton reaction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 O\u003csub\u003e2\u003c/sub\u003e-induced enhanced PDT ability of HMPC nanoenzymes \u003cem\u003ein vitro.\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eTo evaluate PDT efficacy \u003cem\u003ein vitro\u003c/em\u003e, the safety and dark toxicity of HMPC nanoenzymes were firstly investigated by MTT assay (Figure. 3A). As shown, no significant cell death was observed in both tumor cells (CT26 cells) and normal cells (L929 cells) by increasing the concentration of CyI for 24 h, which demonstrated that HMPC nanoenzymes has good safety and biocompatibility. Then, flow cytometry (FCM) and confocal microscopy (CLSM) were used to investigate the cell uptake of HMPC nanoenzymes by CT26 cells (Figure. 3B and S6). As the time prolonged, the fluorescence intensity of CyI was increased, and comparable at 4 and 8 h, indicating that HMPC nanoenzymes could be effectively internalized and accumulated in CT26 cells. In addition, the targeting performance of HMPC nanoenzymes towards CT26 cells was verified. As shown in Figure. 3C, the fluorescence intensity of cells incubated with HMPC nanoenzymes was stronger than that of MPC NPs, indicating that the coating of HA increased cellular uptake. To further investigate whether the increased cellular uptake is related to HA-mediated active targeting, blocking experiment was carried out by pretreating the cells with HA. As expected, the fluorescence intensity decreased in the blocking group, suggesting that HMPC nanoenzymes could active target and accumulate to CT26 cells via CD44 receptors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eNext, to explore whether Pt NPs-mediated catalytic reaction would relieve hypoxia \u003cem\u003ein vitro\u003c/em\u003e, the level of intercellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and hypoxia in CT26 cells after various treatments were detected by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and hypoxia fluorescence probes. As shown in Figure. 3E and F, both the green fluorescence (intercellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and red fluorescence (intercellular hypoxia) of HMPC group were much weaker than that of CyI\u0026thinsp;+\u0026thinsp;laser group and HMC group with or without laser irradiation. Specifically, the red fluorescence intensity of HMPC with laser irradiation was almost comparable to the control group under normoxic condition, indicating that HMPC nanoenzymes could offset hypoxia aggravated by PDT in a simulated anoxic condition. The evidences confirmed HMPC nanoenzymes with Pt NPs could catalyze H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e and alleviate tumor hypoxia.\u003c/p\u003e\n\u003cp\u003eThen, the ROS generation in tumor cells was observed by CLSM (Figure. 3G) and FCM (Figure. 3H) using DCFH-DA as a ROS probe. As shown, the control group, as well as HMC and HMPC group without laser irradiation under normoxic conditions exhibited negligible green fluorescence, indicating low or none ROS generation. In comparison, HMPC group under laser irradiation showed enhanced green fluorescence intensity, indicating that HMPC nanoenzymes with Pt NPs to tumor cells where H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is constantly being formed could catalyzed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e, thus promoted ROS generation by CyI[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. It should be noted that under either normoxic or hypoxic conditions, the fluorescence intensity of cells treated with HMPC under laser irradiation was almost identical, indicating that HMPC nanoenzymes could constantly relieve tumor hypoxia. On the contrary, the fluorescence intensity of cells treated with HMC\u0026thinsp;+\u0026thinsp;laser group under normoxic condition was stronger than that of hypoxic condition. Those experimental results showed that oxygen-dependent PDT efficacy was hindered to some extent in anoxic environment, and demonstrated that HMPC nanoenzymes could relive tumor hypoxia and increase ROS generation.\u003c/p\u003e\n\u003cp\u003eOur previous study has demonstrated that CyI possesses good photothermal effect. To confirm the photothermal effect of HMPC nanoenzymes, infrared thermal imaging camera and thermocouple thermometer were used. As shown in Figure. 3I and J, compared with MP and MOF, significant temperature change, from room temperature (26 ℃) to thermal ablation temperature (47 ℃), was observed in the HMPC group under laser irradiation, which demonstrated the photothermal effect of HMPC nanoenzymes. Finally, the PDT efficacy of HMPC nanoenzymes was examined by MTT assay in CT26 cells in either normoxic or hypoxic conditions. As shown in Figure. 3K, both HMC and HMPC showed concentration-dependent cell inhibition profile. In either normoxic or hypoxic conditions, HMPC under laser irradiation showed better cell inhibition rate compared to that of HMC. Furthermore, as expected, the cell viability of HMPC in either normoxic or hypoxic conditions had no significant difference, while HMC in normoxic conditions exhibited much better cell inhibition rate than in hypoxic conditions. These results further demonstrated that HMPC nanoenzymes could enhance the cell inhibition ability of HMPC in hypoxic conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Ability of HMPC nanoenzymes in inducing Ferroptosis \u003cem\u003ein vitro.\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eFerroptosis is a kind of iron- and ROS-dependent regulatory cell death (RCD) forms differed from apoptosis, necrosis and autophagy[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. It has been reported that iron-based nanomaterial can induce cell ferroptosis through Fenton reaction, which causes peroxidation of unsaturated fatty acids, leading to lethal accumulation of LPO on the cell membrane[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Ferroptosis can be prevented by the enzymatic reaction of GSH-GPX4 antioxidant systems where GPX4 catalyzes the reduction of LPO in a GSH-dependent reaction[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, to explore whether HMPC nanoenzymes could induce ferroptosis, GSH content, the expression of GPX4 and LPO accumulation were explored.\u003c/p\u003e\n\u003cp\u003eBecause cytotoxic \u0026middot;OH produced from Fenton reaction in ferroptosis would oxidize GSH diminishing the level of the intracellular GSH, the level of GSH was generally regarded as the marker of strength in the evaluation of ferroptosis. As shown in Figure. 4A, compared with control, HMPC, and HMPC\u0026thinsp;+\u0026thinsp;laser group, and HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group showed lower GSH content in CT26 cells, implying that Fenton reaction would generate ROS which can be reacted with GSH, resulting in ferroptosis. Moreover, under laser irradiation, the GSH content further decreased in HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, indicating that more generated ROS are accumulated in tumor cells by the combination of PDT and Fenton reaction, which resulted in the balance disorder of ROS and GSH in tumor cells to induce ferroptosis. Deferoxamine (DFO) is an iron chelating agent, which is applied as an ferroptosis inhibitor[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. As shown, after adding DFO to HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser group, the GSH content was greatly boosted, mainly because the intervention of DFO has decreased the intracellular free iron content, thus inhibiting Fenton reaction, and slowing down the process of iron-induced ferroptosis.\u003c/p\u003e\n\u003cp\u003eGPX4, as the fourth member of the selenium-containing GPX family, exhibits a scavenging capacity to the membrane lipid hydrogen peroxide products. As a cofactor for GPX4 catalyzing LPO to lipid alcohol, lack of GSH would trigger cysteine deficiency, which directly inactivate GPX4 and induce ferroptosis[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, GPX4, as the core regulator of ferroptosis, has been considered as the \"star molecule\" of ferroptosis studies. The immunofluorescent staining was used for the investigation of the expression of GPX4, as shown in Figure. 4B and C. Consistent with the above results of GSH content, compared with other groups, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser group showed the lowest green fluorescence, indicating that Fenton reaction of HMPC nanoenzymes would restrain the expression of GPX4, resulting in ferroptosis. As expected, the fluorescence of HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser group was enhanced by the addition of DFO. The above experiments proved that HMPC nanoenzymes could deplete GSH and reduce GPX4 activity, thus inducing ferroptosis.\u003c/p\u003e\n\u003cp\u003eLipid inside cells is recognized as a crucial biomarker of ferroptosis, of which the accumulation contributes to the lethality of ferroptosis[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. A lipid peroxidation sensor, C11-BODIPY, was utilized to detect the LPO generation accumulation level in tumor cells by CLSM (Figure. 4D and E). In CLSM observation, the red fluorescence represents the non-oxidation state, and the green fluorescence represents the oxidation state. As shown, HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser group exhibited the strongest green fluorescence, implying that HMPC nanoenzymes caused the lipid oxidation under laser irradiation, and triggered ferroptosis. While the fluorescence was weakened after addition of DFO, which confirmed the accumulation of LPO was related to iron, implying the iron-dependent ferroptosis. In comparison, the HMPC\u0026thinsp;+\u0026thinsp;laser or +\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group showed lower fluorescence intensity, further indicating that the combination of PDT and Fenton reaction would induce more preferable ferroptosis due to iron and ROS.\u003c/p\u003e\n\u003cp\u003eMalondialdehyde (MDA), as one of products of membrane lipid peroxidation, is a toxic aldehyde substance, which is widely used as an index of intracellular LPO. As shown in Figure. 4F, HMPC\u0026thinsp;+\u0026thinsp;laser or +\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group showed elevated MDA content due to PDT or Fenton reaction, while the combination of both contributed to the further increased MDA content. However, this trend was reversed by DFO. The above results again indicated that the HMPC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;laser had preferable advantages to induce ferroptosis due to the combination of PDT and Fenton reaction.\u003c/p\u003e\n\u003cp\u003eFinally, MTT assay was performed for assessment of the \u003cem\u003ein vitro\u003c/em\u003e effect of HMPC. As shown in Figure. 4G, HMPC\u0026thinsp;+\u0026thinsp;laser group exhibited the best tumor inhibition, compared with PDT (CyI\u0026thinsp;+\u0026thinsp;laser) or ferroptosis therapy (HMPC). In addition, DFO and two antioxidant (Vc and GSH) were used to block ferroptosis. As Figure. 4H indicated, the addition of DFO could reduce the cell toxicity, demonstrating that iron was involved in the cell death. Similarly, the co-incubation with Vc or GSH also improved cell survival, further confirmed that HMPC nanoenzymes could inhibit cell viability by iron- and ROS-mediated ferroptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Tumor-targeting ability and safety assay of HMPC nanoenzymes \u003cem\u003ein vivo\u003c/em\u003e.\u003c/h2\u003e\n\u003cp\u003eIn order to evaluate the tumor-targeting ability of HMPC nanoenzymes, HMPC nanoenzymes and free CyI were injected intravenously into tumor-bearing mice respectively, observing \u003cem\u003ein vivo\u003c/em\u003e distribution of mice at different time periods under IVIS\u0026reg; spectrum \u003cem\u003ein vivo\u003c/em\u003e imaging system (PerkinElmer) (Figure. 5A and B). In the free CyI-injected group, the tumor site of the mouse did not show fluorescence during 24 h post-injection, indicating that free CyI lacked tumor-targeting and tumor tissue retention capacity. In contrast, for HMPC-injected group, the fluorescence appeared at tumor tissue at 2 h post-injection, peaked at 10 h, and gradually decreased at 24 h, indicating that HMPC nanoenzymes was metabolized. It should be noted that the fluorescence intensity of free CyI is higher in the liver than in other major organs, mainly due to the fact that the hydrophobicity of CyI increased metabolism in the liver. Obviously, compared to free CyI-injected group, the fluorescence of tumor tissue in HMPC\u0026ndash;injected group was still retained after 24 h due to targeting ability. All the evidence showed that compared with the early removal of CyI by the liver, HMPC nanoenzymes achieved the targeting and retention of tumor site by active targeting and nanoscale, and avoided the early accumulation and removal by liver and kidney.\u003c/p\u003e\n\u003cp\u003eTo determine the hemocompatibility of HMPC nanoenzymes, the hemolytic activity of the HMPC nanoenzymes was evaluated by hemolysis assay (Figure. S7). No significant hemolytic toxicity was observed in the photograph and hemolysis ratios are within the safe range (less than 4%), indicating the hemocompatibility of HMPC nanoenzymes. H\u0026amp;E staining for major organ of mice was performed, and saline-injected mice were used as control (Figure. 5C). No pathological characteristics were observed in HMPC-injected group, confirming the safety of HMPC. Moreover, the \u003cem\u003ein vivo\u003c/em\u003e biocompatibility of HMPC nanoenzymes was investigated. The blood samples of two groups were taken after the treatment and used to test by a hematology analyzer for evaluating the toxicity of HMPC nanoenzymes (Figure. S8). The nine indexes of blood, including mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), red blood cell specific volume (HCT), platelet count (PLT) and red cell volume distribution width (RDW) in HMPC-injected group showed no obvious abnormal, compared to the saline group, implying no blood adverse reactions induced by HMPC nanoenzymes. Due to \u003cem\u003ein vivo\u003c/em\u003e distribution, more accumulation of HMPC nanoenzymes was observed in liver and kidney compared with other organs. Hence, liver toxicity indicators (AST and ALT) and kidney indicators (BUN and CREA) were chosen for serum biochemical parameter detection (Figure. 5D-G). As shown, no significant difference was observed between control group and HMPC-injected group, which consistently suggested that HMPC nanoenzymes had the advantages of good hemocompatibility, biocompatibility and safety.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Combination treatment efficacy of HMPC nanoenzymes.\u003c/h2\u003e\n\u003cp\u003eTo explore whether HMPC nanoenzymes would exert O\u003csub\u003e2\u003c/sub\u003e-enhanced PDT and ferroptosis \u003cem\u003ein vivo\u003c/em\u003e, the alleviation situation of tumor hypoxia was firstly explored. After various administration, the tumors were collected, sliced and stained with hypoxia-inducible factor 1\u0026alpha; (HIF-1\u0026alpha;), a protein which the expression level could indicate the degree of hypoxia in the tumor. Immunofluorescent staining (Figure. 6A) showed that the tumor in HMPC-injected group exhibited significant lower fluorescence of HIF-1𝛼 compared to other groups, indicating that HMPC nanoenzymes with catalytic effect were contributed to the alleviation of hypoxia. In particular, the group injected with HMPC nanoenzymes exhibited lower hypoxia than free CyI or HMC under laser irradiation, implying HMPC nanoenzymes can still effectively relieve hypoxia under intensified hypoxic condition by PDT. ROS generation of HMPC nanoenzymes under laser irradiation was then investigated by staining the tumors with SOSG. As shown in Figure. 6A and B, the green fluorescence intensity in HMPC group was much higher than other groups, demonstrating that under the catalytic effect of HMPC nanoenzymes, the combination of PDT and ferroptosis would result in enhanced ROS generation.\u003c/p\u003e\n\u003cp\u003eThe LPO accumulation level in tumor tissues was assessed using C11-BODIPY, a lipid peroxidation probe, which could indicate the level of ferroptosis. When ferroptosis occurs, the emission of the probe will change from 590 nm (red fluorescence) to 510 nm (green fluorescence). As shown in Figure. 6C and D, the group treated with saline or laser irradiation showed no green fluorescence, while HMPC-treated group showed strong green fluorescence, indicating \u003cem\u003ein vivo\u003c/em\u003e ferroptosis occurred. In addition, HMPC-treated group with laser irradiation showed much brighter green fluorescence, indicating that ROS generated by PDT increased the green fluorescence intensity, implying that PDT has a certain promoting effect on ferroptosis[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Green fluorescence intensity in HMPC-treated group under laser irradiation was decreased after adding DFO, demonstrating \u003cem\u003ein vivo\u003c/em\u003e iron-related ferroptosis. The above results demonstrated that HMPC nanoenzymes exert enhanced PDT and ferroptosis therapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe combination treatment efficacy of HMPC nanoenzymes was evaluated in CT26 tumor-bearing mice. Mice were randomly divided into seven groups (Saline, Laser, HMPC, CyI\u0026thinsp;+\u0026thinsp;laser, HMC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;laser\u0026thinsp;+\u0026thinsp;DFO) for the observation of tumor volumes and body weights following 14-day treatment. The group intravenously injected with saline was defined as the negative control. As shown in Figure. 6E, compared with control and laser group, the tumor growth of HMPC, CyI\u0026thinsp;+\u0026thinsp;laser, HMC\u0026thinsp;+\u0026thinsp;laser, HMPC\u0026thinsp;+\u0026thinsp;laser group showed obviously inhibited. Among them, HMC\u0026thinsp;+\u0026thinsp;laser had better tumor inhibition rate than CyI\u0026thinsp;+\u0026thinsp;laser, due to that HMC held extra targeting property and exerted ferroptosis therapy. HMC\u0026thinsp;+\u0026thinsp;laser had better tumor inhibition than CyI\u0026thinsp;+\u0026thinsp;laser, because HMC held extra targeting property and exerted ferroptosis therapy. Compared with HMC-treated group, HMPC-treated group exhibited better tumor inhibitory effect under laser irradiation due to the contribution of Pt NPs. The group treated with HMPC nanoenzymes under laser irradiation implemented the combination treatment of PDT and ferroptosis therapy, which exhibited the best antitumor effect. To further verify the effect of ferroptosis on the combination treatment, DFO was intraperitoneally injected into mice to block iron-induced ferroptosis. The tumor inhibition efficacy was decreased, implying iron-induced ferroptosis did play a role in tumor treatment. The body weight of all groups exhibited no obvious decrease, indicating that the treatments had no severe side effects on mice (Figure. S9).\u003c/p\u003e\n\u003cp\u003eAfter 14-days treatment, the mice were sacrificed. Figure. 6F and S10 showed the photos of mice and tumors isolated from mice after 14-day treatments, which were consistent with the trend of tumor volumes. The major organs and the tumors were collected for H\u0026amp;E staining. It was shown that no pathological abnormalities were observed in the major organs (heart, liver, spleen, lung and kidney), implying that treatments had no obvious organ toxicity towards mice (Figure. S11). The H\u0026amp;E staining results of tumors shown in Figure. 6G indicated that no tissue damage was observed in saline and laser group. However, group treated with HMPC nanoenzymes with laser irradiation exhibited an obvious tissue damage, nuclear shrinkage, and nuclear lysis. These results further confirmed the antitumor effect of HMPC nanoenzymes with laser irradiation. In order to explore the tumor inhibition mechanism of HMPC nanoenzymes, immunofluorescence analysis by TUNEL, Ki67 and GPX4 assays were conducted. As shown in Figure. 6G and S12, no obvious apoptosis, inactivation of GPX4 and obvious cell proliferation was observed in saline or laser group, while HMPC\u0026thinsp;+\u0026thinsp;laser group displayed obvious apoptosis, low or none cell proliferation, and the decreased level of GPX4, further confirming that HMPC\u0026thinsp;+\u0026thinsp;laser realized effective tumor inhibition by inducing apoptosis, inhibiting proliferation, as well as triggering ferroptosis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, we have successfully developed nanoenzymes integrating Pt NPs and CyI into MIL-100 (Fe) capable of relieving hypoxia and inducing ferroptosis, for achieving enhanced PDT-ferroptosis therapy. First, HMPC nanoenzymes improved the hydrophility of CyI and efficiently deliver CyI to tumor cells by EPR effect and active targeting, thus enhancing their accumulation in target cells. Next, Fenton reaction caused by MIL-100 (Fe) made depleted GSH, inactivation of GPX4 and accumulation of LPO, triggering ferroptosis in tumor cells. Then, the released Pt NPs with catalase-like activity constantly catalyzed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e to enhance PDT. The experiment results showed the nanoenzymes indeed enhanced ROS generation in the hypoxic condition by catalytic reaction, alleviating the hypoxia aggravated by PDT, and the nanoenzymes implemented ferroptosis therapy. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e therapeutic results demonstrated that the nanoenzymes exhibit potent anticancer efficacy. Hence, the developed nanoenzymes can serve as a regimen combining PDT with ferroptosis therapy, displaying potent tumor inhibition with a good \u003cem\u003ein vivo\u003c/em\u003e safety. HMPC nanoenzymes overcame some of key challenges in PDT antitumor treatment by integrating iron-based MOFs with Pt NPs. Such nanoenzymes provided a new model for enhanced PDT-ferroptosis therapy, which may represent a promising novel regimen for hypoxic tumor treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEM image of MOF; The hydrodynamic size distribution and PDI of MOF, MP, MPC and HMPC; EDS spectrum of HMPC; The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e degradation of different formulations, PBS was used as blank control group, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution was defined as positive group; The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation of HMC and HMPC under laser irradiation; CLSM images of CT26 cells incubated with HMPC for different time; Hemolysis analysis; Blood routine index analysis for CT26 tumor-bearing mice injected with saline and HMPC; The body weight changes of mice form different groups during 14 d treatments; Photographs of harvested tumor from mice after 14 d treatments; H\u0026amp;E staining of major organs form mice incubated with different treatments; Ki67 immunofluorescent staining of the tumor tissues from different groups of mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by National Natural Science Foundation of China (No. 32171362) and Natural Science Foundation of Shandong Province (No. ZR2022YQ73, ZR2021MH087).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYY and HY: Investigation, Methodology, Data curation. YZ: Methodology, Visualization. BL: Methodology. GX: Methodology. YS: Supervision. JC: Conceptualization, Project administration, Writing \u0026ndash; review \u0026amp; editing, Supervision. JC and YY were the major contributors in writing the manuscript. YY and HY performed all experiments. All authors reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were carried out according to the protocol approved by the Ethics Committee of Qingdao University\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi L, Song D, Qi L, Jiang M, Wu Y, Gan J, Cao K, Li Y, Bai Y, Zheng T. Photodynamic therapy induces human esophageal carcinoma cell pyroptosis by targeting the PKM2/caspase-8/caspase-3/GSDME axis. Cancer Lett. 2021;520:143\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie J, Wang Y, Choi W, Jangili P, Ge Y, Xu Y, Kang J, Liu L, Zhang B, Xie Z, et al. Overcoming barriers in photodynamic therapy harnessing nano-formulation strategies. Chem Soc Rev. 2021;50:9152\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer. 2003;3:380\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmeida-Marrero V, Bethlehem F, Longo S, Bertolino MC, Torres T, Huskens J, de la Escosura A. Tailored Multivalent Targeting of Siglecs with Photosensitizing Liposome Nanocarriers. Angew Chem Int Ed Engl. 2022;61:e202206900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi Y, Sun Y, Hei M, Cheng D, Wang B, Tang Y, Fu Y, Zhu W, Xu Y, Qian X. NIR Activated Upper Critical Solution Temperature Polymeric Micelles for Trimodal Combinational Cancer Therapy. Biomacromolecules. 2022;23:937\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Z, Tang Y, Chen L, Liu L, Huo H, Ye J, Ge X, Su L, Chen Z, Song J. In-Situ Assembly of Janus Nanoprobe for Cancer Activated NIR-II Photoacoustic Imaging and Enhanced Photodynamic Therapy. Anal Chem. 2022;94:10540\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObuobi S, Julin K, Fredheim EGA, Johannessen M, Škalko-Basnet N. Liposomal delivery of antibiotic loaded nucleic acid nanogels with enhanced drug loading and synergistic anti-inflammatory activity against S. aureus intracellular infections. J Control Release. 2020;324:620\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Gao J, Ding Y, Lu Y, Wei Q, Cui D, Fan J, Li X, Zhu E, Lu Y, et al. Multi-Functional Liposome: A Powerful Theranostic Nano-Platform Enhancing Photodynamic Therapy. Adv Sci (Weinh). 2021;8:e2100876.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, Yang Y. Metal-Organic Framework (MOF)-Based Drug/Cargo Delivery and Cancer Therapy. Adv Mater. 2017;29:201606134.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNadar SS, Vaidya L, Rathod VK. Enzyme embedded metal organic framework (enzyme-MOF): De novo approaches for immobilization. Int J Biol Macromol. 2020;149:861\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng S, Bie B, Jia H, Tang H, Zhang X, Sun Y, Wei Q, Wu F, Yuan Y, Deng H, Zhou X. Efficient Separation of Nucleic Acids with Different Secondary Structures by Metal-Organic Frameworks. J Am Chem Soc. 2020;142:5049\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuang J, Gong H, Zhou J, Zhang Q, Gao W, Fang RH, Zhang L. Targeted gene silencing in vivo by platelet membrane-coated metal-organic framework nanoparticles. Sci Adv. 2020;6:eaaz6108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang L, Liu L, Lin L, Liu F, Xie Z, Tian H, Chen X. Engineering Metal-Organic Frameworks for Photoacoustic Imaging-Guided Chemo-/Photothermal Combinational Tumor Therapy. ACS Appl Mater Interfaces. 2018;10:41035\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Wang X, Su Y, Shao J, Song X, Wang W, Zhong L, Gan L, Zhao Y, Dong X. Multifunctional nanolocks with GSH as the key for synergistic ferroptosis and anti-chemotherapeutic resistance. Biomaterials. 2022;288:121704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao F, Sang Y, Liu C, Bai F, Zheng L, Ren J, Qu X. Self-Adaptive Single-Atom Catalyst Boosting Selective Ferroptosis in Tumor Cells. ACS Nano. 2022;16:855\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinterbourn CC. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett. 1995;82\u0026ndash;83:969\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Z, Ma Q, Zhou X, Zhang G, Hao G, Sun Y, Cao J. Strategies to improve photodynamic therapy efficacy by relieving the tumor hypoxia environment. NPG Asia Materials. 2021;13:39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei H, Wang E. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev. 2013;42:6060\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Dong X, Yang S, Lai X, Liu H, Gao Y, Feng H, Zhu M, Yuan Y, Lu Q, et al. Biomimetic Liposomal Nanoplatinum for Targeted Cancer Chemophototherapy. Adv Sci (Weinh). 2021;8:2003679.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao Y, Chen Y, He X, Yu Y, Han R, Li Y, Yang C, Hu D, Qian Z. Polymeric Nanoparticles with ROS-Responsive Prodrug and Platinum Nanozyme for Enhanced Chemophotodynamic Therapy of Colon Cancer. Adv Sci (Weinh). 2020;7:2001853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbed A, Derakhshan M, Karimi M, Shirazinia M, Mahjoubin-Tehran M, Homayonfal M, Hamblin MR, Mirzaei SA, Soleimanpour H, Dehghani S, et al. Platinum Nanoparticles in Biomedicine: Preparation, Anti-Cancer Activity, and Drug Delivery Vehicles. Front Pharmacol. 2022;13:797804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao J, Chi J, Xia J, Zhang Y, Han S, Sun Y. Iodinated Cyanine Dyes for Fast Near-Infrared-Guided Deep Tissue Synergistic Phototherapy. ACS Appl Mater Interfaces. 2019;11:25720\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, Chen Z, Han B, Li C. Glycol assisted synthesis of MIL-100(Fe) nanospheres for photocatalytic oxidation of benzene to phenol. Catal Commun. 2017;98:112\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeranishi T, Tanaka MH,T, Miyake M. Size Control of Monodispersed Pt Nanoparticles and Their 2D Organization by Electrophoretic Deposition. J Phys Chem B. 1999;103:3818\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLennicke C, Rahn J, Lichtenfels R, Wessjohann LA, Seliger B. Hydrogen peroxide - production, fate and role in redox signaling of tumor cells. Cell Commun Signal. 2015;13:39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi N, Jiang W, Wang W, Xiong R, Wu X, Geng Q. Ferroptosis and its emerging roles in cardiovascular diseases. Pharmacol Res. 2021;166:105466.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang K, Ma Z, Li S, Wu Y, Zhang J, Zhang W, Zhao Y, Han H. Disruption of dual homeostasis by a metal-organic framework nanoreactor for ferroptosis-based immunotherapy of tumor. Biomaterials. 2022;284:121502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei G, Zhuang L, Gan B. Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer. 2022;22:381\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan H, Zou T, Tuo Q, Xu S, Li H, Belaidi AA, Lei P. Ferroptosis: mechanisms and links with diseases. Signal Transduct Target Ther. 2021;6:49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Dai H, Huang X, Feng J, Deng J, Wang Z, Yang X, Liu Y, Wu Y, Chen P, et al. Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma. Acta Pharmacol Sin. 2021;42:301\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoon JF, Pratt DA. Recent Insights on Hydrogen Atom Transfer in the Inhibition of Hydrocarbon Autoxidation. Acc Chem Res. 2018;51:1996\u0026ndash;2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Y, Palte MJ, Deik AA, Li H, Eaton JK, Wang W, Tseng YY, Deasy R, Kost-Alimova M, Danč\u0026iacute;k V, et al. A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis. Nat Commun. 2019;10:1617.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X, Deng J, Liu F, Guo T, Liu M, Dai P, Fan A, Wang Z, Zhao Y. Triggered All-Active Metal Organic Framework: Ferroptosis Machinery Contributes to the Apoptotic Photodynamic Antitumor Therapy. Nano Lett. 2019;19:7866\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photodynamic therapy, Iron-based MOFs, Pt nanoparticles, Ferroptosis therapy, Hypoxic tumor","lastPublishedDoi":"10.21203/rs.3.rs-2381256/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2381256/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePhotodynamic therapy (PDT), as a promising strategy in cancer treatment that utilizes photosensitizers (PSs) to produce reactive oxygen species (ROS), has been widely used for eliminating cancer cells under specific wavelength light irradiation. However, the low aqueous solubility of PSs, and special tumor environments (TME), such as high glutathione (GSH) and tumor hypoxia remain challenges towards PDT for hypoxic tumor treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTo address these problems, we constructed a novel nanoenzyme (HMPC) for enhanced PDT-ferroptosis therapy by integrating small Pt nanoparticles (Pt NPs) and near-infrared photosensitizer CyI into iron-based metal organic frameworks (MOFs). In addition, hyaluronic acid (HA) was adhered to the surface of the nanoenzymes to enhance the targeting ability. In this design, MOFs act not only as a delivery vector for PSs, but also a ferroptosis inducer. Pt NPs stabilized into MOFs were functioned as an oxygen (O\u003csub\u003e2\u003c/sub\u003e) generator by catalyzing hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) into O\u003csub\u003e2\u003c/sub\u003e to relieve tumor hypoxia and increase \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation. I\u003cem\u003en vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results demonstrated that under NIR irradiation, HMPC could effectively relive the tumor hypoxia and decrease the level of GSH in TME, resulting in enhanced PDT-ferroptosis therapy against hypoxic tumor.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe proposed nanoenzymes represent an important advance in altering TME for improved clinical PDT-ferroptosis therapy, as well as their potential as effective theranostic agents for hypoxic tumors.\u003c/p\u003e","manuscriptTitle":"Engineering Nanoenzymes Integrating Iron-based MOFs with Pt NPs for Enhanced PDT-Ferroptosis Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-20 17:59:13","doi":"10.21203/rs.3.rs-2381256/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ea0b063-a454-4126-8e76-0fe450938948","owner":[],"postedDate":"December 20th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-11T00:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-20 17:59:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2381256","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2381256","identity":"rs-2381256","version":["v1"]},"buildId":"afDZ1USd8LLqUjqjB8QhT","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}