Metal-organic nanostructures based on sono/chemo-nanodynamic synergy of TixOy/Ru reaction units: for ultrasound-induced dynamic cancer therapy

Metal-organic nanostructures based on sono/chemo-nanodynamic synergy of TixOy/Ru reaction units: for ultrasound-induced dynamic cancer 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metal-organic nanostructures based on sono/chemo-nanodynamic synergy of TixOy/Ru reaction units: for ultrasound-induced dynamic cancer therapy Tao Jiang, Zixiang Tang, Shumiao Tian, Haitian Tang, Zhekun Jia, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6273421/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jul, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 13 You are reading this latest preprint version Abstract Sonodynamic therapy (SDT) has demonstrated significant clinical potential in malignant tumor treatment due to its deep tissue penetration and spatiotemporal controllability. Its core mechanism relies on ultrasound-activated sonosensitizers to generate reactive oxygen species (ROS), thereby inducing tumor cell apoptosis. However, conventional sonosensitizers face limitations in ROS yield and tumor-targeting efficiency. In this study, we innovatively designed a multifunctional metal-organic nanosheet (TiZrRu-MON) by hydrothermal coordination of [Ru(bpy) 3 ] 2+ photosensitizing units with TiZr-O clusters, while incorporating Fe 3+ to construct a cascade catalytic system. Experimental results demonstrated that: ( 1 ) Fe 3+ lattice doping significantly enhanced charge carrier mobility and ultrasound-triggered 1 O 2 quantum yield via the formation charge transfer channels; ( 2 ) The acidic tumor microenvironment activated Fe 3+ -mediated Fenton reactions, establishing a positive feedback loop with SDT to synergistically amplify ROS generation; ( 3 ) Hyaluronic acid functionalization improved nanosheet internalization in HepG2 tumor cells through CD44 receptor-mediated endocytosis. Remarkably, ultrasound irradiation induced substantial oxidative stress and immunogenic cell death, promoting the release of damage-associated molecular patterns (DAMPs), which elevated the maturation rate of tumor-infiltrating dendritic cells (DCs) and significantly increased the proportion of CD8 + T cells. In a mouse subcutaneous tumor model, the system achieved effective tumor suppression with manageable systemic toxicity. This work proposes a metal-ligand coordination strategy to advance the development of high-performance sonosensitizers and immunomodulatory antitumor technologies. Metal-organic nanostructure Sonodynamic therapy Chemodynamic therapy Magnetic resonance imaging cancer therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Cancer remains one of the most severe threats to global human health ( 1 , 2 ). Despite emerging therapeutic approaches, current treatments demonstrate suboptimal efficacy and severe side effects, highlighting the urgent need to develop more effective and safer cancer therapies ( 3 ). With the rapid advancement of nanotechnology, ultrasound (US)-triggered SDT has emerged as a promising non-invasive antitumor strategy, characterized by deep tissue penetration and high spatiotemporal precision ( 4 – 6 ). Through ultrasonic irradiation, SDT precisely activates sonosensitizers accumulated in deep tumor tissues to generate cytotoxic ROS, thereby effectively eliminating malignant cells ( 7 – 11 ). Notably, elevated ROS accumulation facilitates robust antitumor immunity through immunogenic cell death (ICD)-based mechanisms ( 12 – 16 ). SDT-induced tumor cells release substantial tumor-associated antigens (TAAs) and DAMPs, which promote antigen presentation by DCs. This process activates and recruits cytotoxic T lymphocytes (CTLs), ultimately triggering systemic antitumor immune responses ( 17 , 18 ). Over the past decades, organic sonosensitizers with high ROS-generation efficiency—including porphyrins, 5-aminolevulinic acid, phthalocyanines, oxazines, and indocyanines—have shown promise. However, their applications in cancer therapy remain constrained by poor water solubility, low chemical stability, limited tumor-targeting capability, and significant phototoxicity ( 9 , 19 , 20 ). In contrast, inorganic sonosensitizers represented by titanium dioxide (TiO 2 ) nanomaterials exhibit advantages such as superior chemical stability, low phototoxicity, and facile surface modification, making them attractive for oncological SDT applications ( 8 , 21 – 23 ). Nevertheless, challenges including high immunogenicity, prolonged in vivo retention, and insufficient tumor accumulation continue to impede their clinical translation ( 22 , 23 ). The cytotoxic effects of sonosensitizers are primarily mediated by ROS generated under ultrasound irradiation. A key mechanism of SDT involves the ultrasound-triggered excitation of sonosensitizers, which promotes electron transitions from the valence band (VB) to the conduction band (CB). This process generates reactive electron-hole pairs (e − /h + ) that subsequently convert surrounding water and oxygen molecules into ROS ( 7 , 10 , 21 , 24 , 25 ). However, conventional sonosensitizers often suffer from low electron-hole pair separation efficiency and rapid recombination, resulting in suboptimal ROS generation and compromised SDT efficacy ( 26 – 29 ). To address these limitations, titanium-oxo clusters (TOCs) derived from the hybridization of TiO 2 with organic molecules have been engineered to enhance photo/ultrasonic catalytic reduction activity by optimizing charge carrier separation and migration ( 30 – 33 ). This improvement is attributed to four synergistic effects: 1) modulated electronic structures, 2) enhanced charge carrier separation, 3) improved light absorption capacity, and 4) optimized surface chemical properties. A growing body of research highlights the potential of constructing multifunctional nanoscale metal-organic frameworks (MOFs) as ideal platforms for advancing cancer therapeutics, owing to their exceptional biocompatibility, tunable pore structures, superior chemical stability, and efficient endosomal escape capacity ( 11 , 24 , 34 , 35 ). Our study demonstrates that [Ti 8 Zr 2 O 12 (COO) 16 ] clusters, featuring highly stable bimetallic units, serve as critical building blocks for creating photoactive MOFs ( 36 ). Upon activation, these TiZr clusters effectively trap photoexcited electrons, thereby suppressing rapid e − /h + recombination. Furthermore, we discovered that [Ru(bpy) 3 ] 2+ can coordinate with photocatalytically active clusters through organic ligands to form structurally stable metal-organic nanosheets. This novel architecture expands the light absorption spectrum, facilitates photoinduced charge carrier separation/migration, enhances reactive site activity/stability, and ultimately achieves high-efficiency synergistic photo-/sono-catalytic performance. The bandgap of sonosensitizers serves as a critical parameter governing e − /h + separation efficiency. Studies demonstrate that doping with transition metal ions (e.g., Fe 3+ , Mn 2+ ) significantly enhances charge carrier separation dynamics under ultrasound irradiation through band structure reconstruction ( 14 , 24 – 26 ). For instance, Shang et al. ( 37 ) achieved bandgap narrowing via Fe 3+ doping, while Yang et al. ( 38 ) optimized both band alignment and ROS generation pathways through Mn 3+ incorporation. Notably, MOFs, with their high porosity and tunable channel structures, can effectively chelate high-concentration Fe 3+ ions. This introduces intermediate energy levels to reduce bandgap width while enabling directional migration of photogenerated carriers for the production of more 1 O 2 . The integration of Fe 3+ and Mn 3+ leverages their intrinsic Fenton-like catalytic activity, establishing a synergistic SDT-CDT (chemodynamic therapy) mechanism. Under ultrasound irradiation, piezoelectric electrons generated on the sensitizer surface accelerate valence cycling of metal ions (Fe 3+ /Fe 2+ , Mn 2+ /Mn 3+ ) via interfacial charge transfer, dramatically enhancing Fenton-like reaction kinetics. This process induces instantaneous burst generation of highly cytotoxic hydroxyl radicals (·OH). This SDT-CDT coupling mechanism achieves dual therapeutic outcomes through oxidative stress cascades that directly induce tumor cell apoptosis while concurrently triggering DAMPs release. The liberated DAMPs enhance DCs infiltration into tumor sites, facilitate antigen capture, and potentiate cross-presentation processes. Through systematic reprogramming of the immunosuppressive microenvironment, cytotoxic T lymphocytes (CTLs) become activated, leading to reconstructed antitumor immune responses that synergistically coordinate ICD with the establishment of systemic immune memory. Based on the [Ru(bpy) 3 ] 2+ photosensitizing unit and Fe 3+ doping strategy, this study developed a multifunctional metal-organic nanostructure (denoted as MF, TiZrRuFe-MON) that integrates precise tumor theranostics through synergistic SDT and CDT mechanisms (Scheme 1 ). MF nanoparticles, synthesized via a controlled hydrothermal method, exhibited excellent monodispersity and crystalline integrity. Their sonocatalytic performance under US irradiation significantly surpassed that of Fe 3+ -undoped TiZrRu-MON (M), attributed to the intrinsic efficient photo/sonocatalytic properties of TiZrRu-MON and the Fe 3+ doping-induced suppression of e − /h + recombination kinetics coupled with bandgap narrowing. Furthermore, Fe 3+ incorporation enabled efficient SDT/CDT synergy by catalyzing H 2 O 2 decomposition into highly cytotoxic·OH through Fenton-like reactions. To enhance tumor-specific accumulation, MF was functionalized with hyaluronic acid (HA). In vitro and in vivo T1-weighted magnetic resonance imaging (MRI) demonstrated HA-mediated significant improvement in tumor-targeted enrichment. In murine tumor models, HA-modified MF (MFH) combined with US irradiation achieved marked tumor growth suppression. Notably, SDT-induced ROS bursts enhanced tumor-associated antigen (TAA) exposure, triggering effector T cell-mediated antitumor immunity via DC-dependent antigen uptake and presentation. This work not only provides new insights into metal-organic framework design but also establishes a critical foundation for clinical translation of SDT/CDT synergistic therapeutic strategies. 2. Materials and methods 2.1 Materials All chemicals were of analytical purity and used without further purification. Bis(2,2'-bipyridyl) (4,4'-dicarboxy1-2,2'bipyridyl) ruthenium(II) dichloride was purchased from Suna Technology Inc (Suzhou, China). Trifluoroacetic acid and acetic acid were purchased from Macklin. Titanium tetraisopropanolate, Absolute ethanol, NaOH, 3,3′,5,5′-Tetramethylbenzidine dihydrochloride hydrate (TMB), FeCl 3 ·6H 2 O, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-Tetramethyl-4-piperidone hydrochloride (TEMP) were purchased from Aladdin (Shanghai, China). 1,3-diphenylisobenzofuran (DPBF), Cell Counting Kit-8 (CCK-8) and 2,7-dichlorofluorescein diacetate (DCFH-DA) by GLPBIO Technology Inc. Calcein AM/PI Cell Viability/Cytotoxicity Assay Kit was purchased from KeyGEN BioTech Inc (Suzhou, China). Annexin V-FITC/PI Kit and ATP test kit were bought from Beyotime Biotechnology (Shanghai, China). Rhodamine B was purchased from Sigma-Aldrich (California, USA). Calreticulin Rabbit pAb, HMGB1 Rabbit pAb, Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) and Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) were purchased from Zen-Biosciencer Inc (Chengdu, China). Anti-CD80-PE, anti-CD86-421, anti-CD11-650, anti-CD3-APC, anti-CD4-PE and anti-CD8-FITC antibodies were obtained from Biolegend. Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Hyclone (USA). HepG2 cells and Hepa1-6 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Nude mice and C57BL/6J mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). 2.2 Characterization Transmission electron microscopy (TEM) images were acquired on a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Scanning electron microscope (SEM) images and element mapping were taken on an FEI Talos F200S transmission electron microscope. X-ray photoelectron spectroscopy analyses were carried out using an X-ray photoelectron spectroscopy (XPS). The size of the particles and zeta potential were measured by dynamic light scattering (DLS; Malvern Ltd., UK). The fluorescence images of cells were taken on a Fluorescent Inverted microscope (Nikon., Japan). Magnetic resonance (MR) properties of materials were measured using a 1.5 T NMR relaxation measurement system (HT-MICNMR-60, Huanyu., China). The MR images were acquired using an MRI system (Bruker 9.4 T Micro-MRI equipment). The concentration of the material was measured with a Inductively coupled plasma-Optical emission spectrometry(ICP-OES)(SPECTRO Co. German). 2.3 Synthesis of TiZrRuFe-MON@HA (MFH) For the synthesis of TiZrRu-MON, the [Ti 8 Zr 2 O 12 (COO) 16 ] cluster was firstly synthesized. The ZrCl 4 (20 mg) was dispersed in DMF (5 mL) using ultrasonic agitation. Subsequently, Ti(OiPr)₄ (0.04 mL) and acetic acid (0.2 mL) were added to the reaction kettle, which was then heated in an oven at 100°C for 24 hours. After cooling of the reactor, the obtained clusters were stored in centrifuge tubes. 2,2‘-Bipyridine-4,4’-dicarboxylic acid (5.25 mg) and Ru-COOH (16mg) were dissolved in DMF, respectively. Both were then mixed with [Ti 8 Zr 2 O 12 (COO) 16 ] cluster (10mg), trifluoroacetic acid (1.5 mL) was added and finally stirred well using vortex. The reaction mixture was heated in a 140°C oven for 24 h. The claybank crystalline sediment was harvested by centrifugation and washed with ethanol. FeCl 3 solution (10% Zr concentration) was added to the resulting TiZrRu-MON solution, and then the mixture stirred in the dark for 12 hours at room temperature. The resulting TiZrRuFe-MON solution was collected by centrifugation and washed with DI water. Finally, the HA solution was added into TiZrRu-MON solution or TiZrRuFe-MON solution respectively, and the reaction was carried out in water bath ultrasonication for 30 min. The resulting TiZrRu-MON@HA (MH) and TiZrRuFe-MON@HA (MFH) solution was collected by centrifugation and washed with deionised water (DI water). 2.4 Electron Spin Resonance (ESR) Spectra Test in Vitro Utilizing electron spin resonance (ESR), MF during US or H 2 O 2 activation produced different forms of ROS. The activity of ·OH was investigated by ESR using a spin-trapping agent precisely (DMPO). For purpose of measuring the ESR, solutions containing DI water, M or MF ([Zr] 100 µM), 100 mM H 2 O 2 , and 100 mM DMPO were combined. The solutions were reacting for 5 minutes. Additionally, the production of 1 O 2 was confirmed using the TEMP (100 mM). DI water, M or MF ([Zr] 100 µM), and TEMP were combined, and were subjected to US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm 2 ) for 5 min. The 1 O 2 or ·OH signal were picked up by the ESR spectrometer, respectively. 2.5 In vitro detection of ROS generation To detect 1 O 2 generated by SDT, DPBF was dissolved in DMSO (1 mg/mL). 1 mL of MFH solution ([Zr] 100 uM) was mixed with 20 µL of DPBF. The mixed solutions were tested for UV-visible absorption after different periods of time of US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm 2 ) in dark. Similarly, results were obtained under US irradiation using DI water and M solution as controls. To detect ▪OH generated by the reaction with H 2 O 2 , 5 µL of TMB (20 mg/mL in DMSO) was added into 1 mL of MFH consisting of 100 µM H 2 O 2 . After reaction for 10 minutes, the absorbance change of TMB at 600 nm reflected the generation of ROS by the Fenton reaction of MFH. In addition, DI water, FeCl 3 solution and M solution were used for comparison. Meanwhile, the absorbance changes of the TMB working solution with or without US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm 2 ) were recorded after 0, 1, 2, 3, 4 or 5 minutes at the same concentration of MF ([Zr] 100 µM). Detection of in vitro cellular ROS generation was conducted with the 2′,7′-dichlorodihy-drofluorescein diacetate (DCFH-DA). Specifically, 5 µL DCFH-DA (10 mM) was added to 20 µL NaOH solution (10 mM), and the mixture was incubated for 30 min at room temperature away from light to prepare 2′,7′-dichlorodihy-drofluorescein (DCFH). Afterwards, 1 mL PBS (pH 7.4) was added to neutralize the reaction. Then, 100 µL of DCFH solution was mixed with an equal volume of different groups of solutions. After US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm 2 ), ROS were detected by fluorescence intensity scanning (Ex: 480 nm, Em: 525 nm) in multifunctional enzyme marker. 2.6 Cell culture Mouse hepatocellular carcinoma cells (Hepa1-6) and human hepatocellular carcinoma cells (HepG2) were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). All cells were cultured at 37°C with 5% CO 2 . 2.7 Cellular uptake For cellular uptake study, HepG2 cells were seeded in 12-well plates (1 × 10 5 cells/well) and incubated at 37 ℃ overnight. Then the cells were treated with Rh-b-labeled MF or MFH at the Zr concentration of 100 µM for 1 h, 4 h, 8 h or 12 h respectively. After being washed with cold PBS for three times, the cell was fixed with 4% paraformaldehyde and cell nuclei was stained with Hoechst 33342. Subsequently, images were collected by CLSM (High Sensitivity Laser Confocal Microscope. Evident FV4000, Japan). To quantify the cellular uptake, HepG2 cells were seeded into 6-well plates (1 × 10 6 cells/well) and cultured for 12 h. After the culture media was removed, the fresh media containing MF or MFH at a Zr concentration of 100 µM was added. Then, the cells were collected and counted with automated cell counter after incubation for 1, 4, 8 and 12 h. The obtained samples were digested by aqua regia (hydrochloric acid (HCl) and nitric acid (HNO 3 ) in a molar ratio of 3:1) for 48 hours and the concentrations of Zr in cells were measured by ICP-OES. 2.8 In vitro ROS generation in cells by SDT Intracellular ROS level was investigated by using DCFH-DA as a fluorescent probe. Briefly, 1 mL of HepG2 cells (1× 10 5 ) were added to a confocal dish and incubated overnight at 37°C. Following apposition, cells were treated with MH, MF, and MFH (20 uM) at the same Zr concentration. After 12 h of incubation, the cells were washed with PBS for 3 times to eliminate free agents, and then added serum-free medium containing DCFH-DA (10 µM) for 30 min. Then, the cells were treated by US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm 2 ). Subsequently, images were collected with CLSM. Furthermore, the intracellular ROS level was further detected and quantified by FCM. Firstly, cells were seeded in 12-well plate at a density of 2 × 10 5 per well and incubated at 37°C overnight. Afterward, the cells were treated with the same approach and conditions as the above CLSM analysis. Finally, the cells were harvested immediately to examine the intracellular ROS by FCM. 2.9 Cytotoxicity assay in vitro HepG2 cells were seeded in 96-well plates (1×10 4 per well) and incubated with complete medium (10% FBS) at 37°C overnight. Then, the culture media was removed and replenished with fresh media containing different concentrations of MH, MF or MFH. After incubating for another 24 h, the cells viability was determined using cck-8 assay. To study the cytotoxicity of SDT treatment. Cells were seeded in 96-well plates and incubated overnight, then the different concentrations of MH, MF or MFH was added and incubated for another 12 h. Cells were washed three times with PBS and treated with ultrasonic irradiation. Afterwards, the cells were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm 2 ) and further incubated for 24 h. The relative cytotoxicity was assessed by cck-8 assay. The cytotoxicity of SDT treatment was also evaluated by calcein-AM/PI experiment. The Hepa 1–6 cells were incubated with MH, MF or MFH (100 µM) in 12-well plates for 12 h and washed with PBS three times. Then, the cells were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm 2 ). Finally, the cells were stained with calcein-AM and PI, and the images were acquired by an Inverted fluorescence microscope. 2.10 Colony formation assay The HepG2 cells were seeded into 6-well plates at a density of 2×10 5 cells/well for 12 h. The cells were incubated with PBS, MH, MF or MFH ([Zr] 100 µM) for 12 h, and then they were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm 2 ). Then, the cells were replanted into 6-well plates (1000/well, 3 wells in each group) and cultured for 7–10 days. Finally, the cells were fixed with 4% paraformaldehyde and stained with 0.5% Gentian Violet. Colonies of exceeding 50 cells were counted to calculate survival fractions. Surviving fraction = (surviving colonies)/ (cells seeded × plating efficiency). 2.11 Apoptosis analysis in vitro Cellular apoptosis was assessed with an Annexin V-FITC apoptosis detection kit according to the manufacturer’s instruction. HepG2 cells were cultured in 12-well plates (2×10 5 cells per well) overnight. Afterwards, the cells were treated with PBS, MFH, PBS + US, MH + US, MF + US and MFH + US ([Zr] 100 µM) for 12 h. The cells in the US groups were irradiated with US (1.0 MHz, 50% duty cycle, 1 W/cm 2 ) and cultured for another 12 h. Finally, the cells were incubated with Annexin/PI reagent in the dark for 20 min and immediately measured with FCM. 2.12 ICD induced by MFH + US in vitro To determine different formulations-induced immunogenic cell death (ICD) of the tumor cells, the secretion of adenosine triphosphate (ATP), calreticulin (CRT) exposure, and extracellular release of high mobility group box 1 (HMGB1) were examined in vitro. In order to study the exposed CRT on the cell surface, the Hepa1-6 cells (1 × 10 5 ) in 1 mL media were added to the confocal dish and incubated at 37°C overnight, and then the cells were treated with PBS, MFH, PBS + US, MH + US, MF + US and MFH + US for 12 h (at the Zr concentration of 100 µM). Further incubated with primary CRT antibody for overnight at 4 ℃ and then incubated with the 488-conjugated secondary antibody. Finally, the cells were stained with DAPI and observed by CLSM. For FCM analysis, cells were seeded in the 12-wells plate at a density of 2×10 5 cells/well overnight. Then the cell treatment was the same as CLSM analysis. Next, the cells were washed with PBS and incubated with CRT antibody. After that, the cells were incubated with secondary antibody for 30 min and then analyzed by FCM. For evaluating intracellular HMGB1, Hepa1-6 cells (1×10 5 ) in 1 mL media were added to the confocal dish. After 12 h incubation, the cells were treated with different formulations like the CRT analysis for 24 h. Next, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, incubated with 5% bovine serum albumin for 30 min. After that, the cells were incubated with primary HMGB1 antibody overnight at 4 ℃ and then incubated with secondary antibody. Finally, the cells were examined by CLSM. For ATP level assay, the cells were seeded in 12-well plate at a density of 15×10 5 cells per well and incubated overnight. Subsequently, the cells were treated with different formulations like the CRT analysis for 24 h. To measure the release of ATP from treated cancer cells, the supernatant from each group was collected. ATP assay kit (Beyotime, S0026) was used to determine the RLU values for each set of samples using a chemiluminescence meter (luminometer) according to the manufacturer's protocol, and the concentration was defined by the developed calibration curve. 2.13 BMDC mature in vitro To evaluate dendritic cells (DCs) activation in vitro, bone-marrow derived dendritic cells (BMDCs) were obtained from C57BL/6 mice. B16F10 cells were pretreated with PBS, MFH, PBS + US, MH + US, MF + US and MFH + US ([Zr] 100 µM) and then co-incubated with BMDCs for 24 h. After that, DCs were stained with anti-CD11-650, anti-CD80-PE and anti-CD86-421 for DCs mature analysis by FCM. 2.15 Animal welfare and protocols Female BALB/c nude mice and C57BL/6 mice (5–6 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China), and all animal experiments were performed according to the protocol with the Animal Care and Use Committee (CC/ACUCC) of Xiamen University. 2.16 In vivo biosafety evaluation Female BALB/c nude mice were randomly divided into 5 groups (n = 5 mice per group). On day 1, 3, 5, each group mice were injected with PBS, MH, MFH (5 mg/kg) through tail vein, respectively. Then the mice were sacrificed at 15th days after treatments, and the blood samples of the mice were collected for hematological and serum biochemical analysis. Meanwhile, the main organs including heart, liver, spleen, lung and kidney were also obtained for further analysis. All the tissues were paraffin-embedded and tissue sections were prepared. Then H&E staining were performed to observe pathological features, and images were captured using an Microscopic Digital Section Scanning System Motic VM1 (McAuldie, Hong Kong). 2.17 Biodistribution of MFH in vivo HepG2 cells were injected subcutaneously into the right flank of the BALB/c mice. MF or MFH (5 mg/kg) was intravenously injected into the mice when the tumor volume reached 150–200 mm 3 . Afterwards, the MR imaging were acquired at 0, 1, 4, 8, 12 and 24 h post-injection and quantified using the imaging system. 2.18 Antitumor efficacy evaluation in the HepG2 tumor model To evaluate the antitumor effects of MFH in a human-derived tumor model, the female BALB/c nude mice subcutaneous tumor model were constructed by injecting HepG2 cells into the right flank of the mice, respectively. When the tumor volume reached approximately 60–80 mm3, the mice were randomly divided into 5 groups (n = 5/group) and PBS, PBS + US, MH + US(5 mg/kg), MFH (5 mg/kg), and MFH + US (5 mg/kg) were injected intravenously viathe tail veins. The mice of US groups were irradiated with US at intensity of 1 W/cm 2 for 5 min after 12 h injection. All the mice were treated every two days for 3 cycles, and the tumor volume and body weight were monitored every 2 days. The tumor volumes were calculated by a formula: volume = length×width 2 /2. The mice were sacrificed after 15 or 16 days of treatments. 2.19 Immunohistochemical analyses After completion of treatments, the mice were sacrificed and then the major organs and tumors were excised for histological observation by H&E staining and Immunohistochemical (IHC) staining. For H&E staining, excised tumors and organs were fixed in 4% paraformaldehyde solution, embedded in paraffin sections, and stained with hematoxylin and eosin. TUNEL and Ki67 staining were used to assess apoptosis and proliferation of tumor tissues, respectively. The procedures were consistent with the manufacturer's protocol, and finally images were captured using an Microscopic Digital Section Scanning System Motic VM1 (McAuldie, Hong Kong). 2.20 Ultrasound-activated immune effects and metastatic tumor models Hepa 1–6 cells were injected into the right flank of the C57BL/6J female mice. The mice were randomly grouped (n = 5 /group) including PBS, PBS + US, MFH (5 mg/kg), and MFH + US (5 mg/kg) when the tumor volume reached 60–80 mm 3 . The mice of US groups were irradiated with US (1.0 MHz, 50% duty cycle, 1 W/cm 2 ). All the mice were treated every two days for 3 cycles, and the tumor volume and body weight were monitored every 2 days. The mice were sacrificed after 15 or 16 days of treatments. The primary tumors were excised by H&E staining and TUNEL staining and finally images were captured using a Microscopic Digital Section Scanning System Motic VM1. 2.21 Flow cytometry analysis of the animal tissues The mice were sacrificed after treatments. The obtained fresh tumors and tumor-draining lymph modes were used to prepare single-cell suspension. Then the suspensions were further incubated with different antibody against the immune cells. To detect the DCs maturation in tumor and lymph modes, the cell suspensions were stained with antibodies of anti-CD11-650, anti-CD80-PE and anti-CD86-421, respectively. The matured DCs were marked as CD11 + CD80 + CD86 + cells. To detect the infiltration of antitumor T cells and the suspensions of tumor were incubated with anti-CD3-APC, anti-CD8a-FITC and anti-CD4-PE and the CD8 + T cells were denoted as CD3 + CD4 − CD8 + cells. Finally, these cells were for FCM analysis. 2.22 Statistical analysis Experiments were performed at least three times and results were expressed as means ± SD. Statistical significances were analyzed using the one-way ANOVA test or two-way ANOVA test. The difference was regarded as significant when the p value was less than or equal to 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant 3. Results and discussion 3.1 Preparation and characterization of MFH Building upon prior methodologies, the [Ti₈Zr₂O₁₂(COO)₁₆] cluster was integrated with Bpy-COOH and Ru-COOH via hydrothermal coordination, yielding TiZrRu-MON (M). Transmission electron microscopy (TEM) characterization revealed that M exhibited lamellar structures with surface folding, displaying an average particle size of approximately 200 nm (Fig. 1 A, B&S1), and formed a homogeneously dispersed ochre solution in aqueous media. To enhance the photo/sono-catalytic properties of M, Fe³⁺ ions were immobilized within its lattice through chelation with Bpy-COOH groups, generating TiZrRuFe-MON (MF). MF exhibited a maroon coloration in aqueous solution, its structural integrity remained unaltered compared to M as confirmed by TEM (Fig. 1 C&S1). Finally, to improve biosafety and prolong systemic circulation, MF was encapsulated with hyaluronic acid (HA), producing TiZrRuFe-MON@HA (MFH). MFH displayed a lighter hue compared to MF and featured distinct 'nano-coatings' in TEM imaging (Fig. 1 D&S1). The prepared MFH exhibited an increased particle size (≈ 310 nm) compared to M (≈ 230 nm) or MF (≈ 270 nm), likely attributed to HA surface coating (Fig. 1 E & S2). Notably, MFH displayed a higher zeta potential (+ 25.6 mV), suggesting enhanced nanoparticle stability through HA encapsulation (Fig. 1 E). UV-Vis spectra (Fig. 1 F) revealed that M maintained a similar absorption peak to Ru-COOH, indicating new chemical interactions between Ru-COOH and the clusters. Importantly, neither Fe³⁺ nor HA modified the characteristic Ru absorption peaks. The material’s elemental composition and valence band structure was analyzed by X-ray photoelectron spectra (XPS) (Fig. 1 G&S3), revealing significant peak variations at Fe and Ru positions indicative of strong coordination effects. MF nanosheets exhibited Ru characteristic peaks at 458.50 and 459.30 eV in the Ru 3p region, contrasted with Ru-COOH's distinct peak at 280.0 eV in the Ru 3d region, demonstrating higher binding energy in MF (Fig. 1 H). The Fe 2p orbital displayed split peaks at 726.41 and 728.33 eV, deviating significantly from conventional Fe³⁺ binding energies (~ 710 eV), likely due to interfacial Fe-O-Ru charge transfer-induced binding energy elevation (Fig. 1 I). These findings suggest successful Fe³⁺ doping into the nanosheet lattice and Ru local electron structure reorganization, potentially enhancing catalytic activity. HAADF imaging (Fig. 1 J) and EDS mapping (Fig. 1 K) confirmed homogeneous spatial distribution of Ti, Zr, Ru, and Fe throughout the nanostructure. ICP-OES analysis revealed substantially increased Fe content in MF and MFH compared with M (Fig. S4), verifying precise iron chelation within the nanosheets. Finally, the T1-weighted magnetic resonance imaging (MRI) of Fe 3+ , M, and MF solutions was investigated using a 1.5T MRI scanner. As illustrated in Fig. 1 L, the MF solution exhibited a longitudinal relaxation (r1) of 12.5 mM − 1 s − 1 , significantly higher than that of the M solution and Fe 3+ solution, which demonstrated r1 values of merely 1.6 mM − 1 s − 1 and 3.26 mM − 1 s − 1 , respectively. These findings suggest that the lattice structure of M enhances the intrinsic magnetic resonance intensity of Fe 3+ , thereby conferring MRI capability to the MF complex. 3.2 Enhanced ROS Generation Efficiency through CDT-SDT Synergy US-activated sonosensitizers facilitate e⁻/h⁺ separation, with the generated electrons not only enhancing Fenton reactions by reducing Fe 3+ but also suppressing e⁻/h⁺ recombination, thereby improving SDT efficiency. Specifically, Fe 2+ participates in the Fenton reaction to convert into Fe 3+ , which is subsequently reduced back to Fe 2+ by US-generated electrons, establishing a “Fe³⁺ → Fe²⁺ → Fe³⁺” catalytic cycle (Scheme 2 ). To validate this mechanism, the generation of singlet oxygen ( 1 O 2 ) and hydroxyl radicals (·OH) was assessed using electron spin resonance (ESR) spectroscopy and relevant chemical probes, respectively. First, the production of 1 O 2 was validated in terms of the sonodynamic effect. When MF was not subjected to US irradiation or with pure US irradiation alone, the ESR spectra showed no significant 1 O 2 production. However, upon US irradiation, the ESR spectra of MF nanosheets exhibited a substantially higher intensity of 1 O 2 generation compared to M nanosheets (Fig. 2 B). Additionally, 1,3-diphenylisobenzofuran (DPBF) was used as a 1 O 2 probe to detect ROS generated under US irradiation. Briefly, DPBF was mixed with different solutions before US exposure. Comparative results revealed that pure US irradiation alone produced minimal ROS. Notably, the 1 O 2 generation efficiency of MF was significantly higher than that of M, confirming that the incorporation of Fe 3+ markedly enhanced the sonodynamic effect of M (Fig. 2 C). Furthermore, as the US irradiation time increased, the characteristic absorption peak of DPBF at 420 nm gradually decreased, and the solution’s yellow color faded, indicating the continuous production of 1 O 2 (Fig. 2 D). Next, the generation of ·OH was validated in the context of the chemical fenton reaction. The ESR spectra of MF nanosheets exhibited a significantly higher intensity of ·OH production compared to other groups (Fig. 2 E). Additionally, 3,3',5,5'-tetramethylbenzidine (TMB) was employed as an ·OH probe to detect ROS generated via the chemical Fenton reaction. DI water, M, or MF was mixed with H 2 O 2 (100 µM), and the characteristic absorption peak at 600 nm was monitored to evaluate ·OH generation. The results showed that neither pure water nor the MF solution produced any detectable ·OH, as evidenced by the absence of color change in the solutions. In contrast, MF generated a substantial amount of ·OH, turning the solution into a deep blue color, thereby confirming the potent catalytic activity of MF in CDT (Fig. 2 F). Furthermore, as the concentration of the MF system increased, the characteristic absorption peak of TMB at 600 nm gradually intensified, and the solution's color deepened, indicating that higher MF concentrations lead to greater ·OH production efficiency (Fig. 2 G). To verify that US accelerates the catalytic cycle based on the Fenton reaction, ·OH generation was quantitatively measured in systems with the same MF concentration. The results demonstrated that ·OH production gradually increased with prolonged reaction time (Fig. 2 H). Notably, the nanosheets subjected to US irradiation exhibited a more rapid and efficient ·OH generation (Fig. 2 I). These compelling results confirm that the meticulously designed sonosensitizer MF can more effectively produce ROS under US irradiation through a dual ROS generation mechanism. In addition, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed as a probe to assess the total ROS production. DCFH-DA itself is non-fluorescent but can be hydrolyzed by low concentrations of NaOH or intracellular esterases to form DCFH, which is further oxidized by ROS to generate highly fluorescent DCF. This fluorescence can be detected using a microplate reader. As shown on Fig. 2 J, in the absence of US irradiation, no significant ROS production by MF was observed. However, under US irradiation, MF demonstrated concentration-dependent ROS generation. These findings indicate that MF possesses remarkable ROS generation efficiency, making it highly suitable for application in combined SDT-CDT cancer therapy. 3.3 In vitro anticancer activity The first step for MFH to exert its anticancer effects is intracellular internalization. Transmission electron microscopy (TEM) observations revealed that HepG2 cells effectively internalized nanoparticles predominantly through an endocytosis mechanism. This process can be divided into four stages: (Ⅰ) recognition and binding, (Ⅱ) formation of endocytic vesicles, (Ⅲ) maturation of endosomes, and (Ⅳ) lysosomal degradation (Fig. 3 A). 1.5T magnetic resonance (MR) scanning demonstrated that cells exposed to varying concentrations of MF or MFH, MFH exhibited superior relaxation performance and enhanced signal intensity (Fig. 3 B). In summary, MFH is more efficiently internalized by HepG2 cells and exhibits superior capabilities for MR imaging. To further elucidate the uptake efficiency of MFH, HepG2 cells were incubated with rhodamine b-labeled MFH or MF for varying durations, and the intracellular uptake was analyzed via confocal laser scanning microscopy (CLSM). The results demonstrated a time-dependent increase in fluorescence intensity of the rhodamine-labeled nanoparticles, indicating progressive cellular uptake. Notably, MFH exhibited significantly higher uptake efficiency compared to MF (Fig. 3 C). Additionally, cells were incubated with MF or MFH using the same protocol, followed by digestion and centrifugation to collect the cells. After aqua regia digestion, Zr concentrations in each group were measured using ICP-OES. The results demonstrated cellular uptake efficiencies consistent with those observed via CLSM (Fig. 3 D). ROS production was assessed using DCFH-DA as a probe, where stronger green fluorescence correlates with higher ROS levels. CLSM observations of HepG2 cells under different treatments revealed that the pure US group displayed minimal fluorescence, indicating extremely low ROS generation efficiency in the absence of a sonosensitizer. In contrast, the MFH + US group exhibited significantly stronger green fluorescence (Fig. 3 E). Notably, groups without ultrasound irradiation showed no ROS production, indicating that MH, MF, or MFH require external US irradiation to generate ROS. Among the ultrasound-treated groups, the MH + US group exhibited weaker green fluorescence compared to the MFH + US group due to the absence of Fe(III) in MH, which are essential for the synergistic SDT-CDT enhancement effect. Similarly, the MF + US group showed lower fluorescence intensity than the MFH + US group, likely because the latter achieved higher cellular uptake, resulting in increased fluorescence intensity. Quantitative analysis of ROS production using flow cytometry (FCM) further confirmed that cells treated with MFH + US generated significantly higher ROS levels than the other groups (Fig. 3 F&S5). These results collectively demonstrate that the MFH + US group possesses superior ROS generation efficiency, attributed to enhanced cellular uptake and the synergistic effect of combined CDT-SDT. ROS can induce cytotoxic effects by damaging biomolecules such as nucleic acids, proteins, and lipids. The cytotoxicity of MH, MF, and MFH against HepG2 cells was assessed using the CCK-8 assay. The results demonstrated that even after 24 hours of incubation with HepG2 cells, the prepared nanostructures did not exhibit significant cytotoxicity at high concentrations ([Zr], 200 µM) (Fig. 4 A). On the other hand, following US irradiation, MFH group showed significant cytotoxic effects on HepG2 cells, with a 63% cell inhibition rate, which was 2.3 times higher than that of MH group (27%) at a concentration of [Zr] 200 µM (Fig. 4 B). Further, colony formation assays were performed to evaluate the proliferative ability of the treated cells, with results showing that the colony formation of the MFH + US group was significantly suppressed (Fig. S6). Based on this, the effect of different treatments on HepG2 cell apoptosis was investigated. The results revealed that the apoptosis rate of HepG2 cells treated with MFH + US (59.7%) was 9.5 times higher than that of cells treated with PBS (6.28%) (Fig. 4 C). Finally, live/dead cell assays were performed on HepG2 cancer cells, showing that the percentage of dead cells (red) induced by MFH + US was higher compared to MH + US and MF + US groups (Fig. 4 D). Taken together, the above results demonstrated that MFH + US was effective in generating ROS, which subsequently triggered cancer cell death. 3.4 Excessive ROS Triggering ICD Effect and DCs Maturation High levels of ROS are critical and indispensable for ICD, which is characterized by the release of DAMPs, such as calreticulin (CRT) exposure, high mobility group box 1 (HMGB1) secretion, and ATP release ( 13 , 18 , 39 – 41 ). These DAMPs act as "danger signals" that facilitate the recruitment and activation of DCs, thereby bridging innate and adaptive immune responses. Specifically, CRT is exposed on the cell surface which acts as an ‘eat-me’ signal and ATP serves as a ‘find-me’ signal, which together trigger the phagocytosis of dying tumor cells by DCs. Moreover, HMGB1 promotes DCs maturation and antigen presentation. These signals together induce immune responses. To investigate whether the designed nanosheets can induce ICD in cancer cells under ultrasonic irradiation, we conducted a series of experiments. First, immunofluorescence staining was performed to evaluate HMGB1 (red) in Hepa1-6 cells. The results revealed that the red fluorescence intensity in MFH + US-treated cells was significantly higher than in other treatment groups, indicating a greater capacity of MFH + US to promote HMGB1 release (Fig. 5 A&S7). Next, ATP levels were measured across different treatment groups. Hepa1-6 cells treated with MFH + US exhibited the highest ATP release (1.55 µM), which was 7.8-fold and 2.5-fold greater than those treated with PBS (0.2 µM) and MH + US (0.62 µM), respectively (Fig. 5 B). Third, CLSM analysis showed that CRT green fluorescence was markedly elevated in Hepa1-6 cells treated with MFH + US compared to other groups (Fig. 5 C&S8). This observation was further quantified using FCM, revealing that cells treated with MFH + US (3163.7) exhibited fluorescence intensities 4.7-fold and 1.2-fold higher than those treated with PBS (678.7) and MH + US (2717.7), respectively (Fig. 5 D and S9). Collectively, these findings confirm that MFH + US generates substantial ROS, thereby triggering oxidative stress and inducing a potent ICD response in cancer cells. To further evaluate the potential of promoting the maturation of bone marrow-derived dendritic cells (BMDCs), we co-cultured treated cancer cells with BMDCs extracted from C57BL/6J mice. Flow cytometric analysis of DC maturation revealed that MFH + US induced the highest proportion of DC maturation (77%), which was 3.8-fold and 1.1-fold greater than that induced by MH + US (20%) and MF + US (69%), respectively (Fig. 5 E and Fig. 5 F). These results demonstrate that MFH + US not only induces a robust ICD effect but also significantly enhances DC maturation in vitro. 3.5 Biodistribution and Antitumor Effects in HepG2 Tumor-Bearing Nude Mice A subcutaneous tumor model of HepG2 liver cancer was established in BALB/c nude mice to evaluate the in vivo accumulation and biodistribution of the nanosheets (Fig. 6 A). Due to the ability of the nanosheets to enhance magnetic resonance signals, their biodistribution was monitored using a 9.4T magnetic resonance imaging system (Bruke 9.4T MicroMRI) following tail vein injection of MF or MFH. In vivo imaging revealed that the MR signal intensity at the tumor site progressively increased over time, reaching its peak at 12 hours post-injection (Fig. 6 B and 6 C). Compared to MF group, MFH group demonstrated a higher accumulation at the tumor site, resulting in a significantly stronger magnetic resonance signal (Fig. 6 C). This enhanced accumulation can be attributed to the HA coating, which improved tumor uptake of the nanosheets. To evaluate the antitumor efficacy of the treatment regimens, nude mice with subcutaneous HepG2 tumors were treated and monitored. To achieve satisfactory tumor elimination, treatments were administered every two days for a total of three cycles (Fig. 6 A). The body weights and tumor volumes for all groups were recorded every two days. No significant changes in body weight were observed in any group, indicating the low systemic toxicity of the nanomedicine (Fig. 6 D). In addition, vigorous tumor growth inhibition was observed in mice treated with MFH + US group, with a mean tumor volume of 22.3 mm 3 at day 15, compared to 325.4 mm³ in MH + US group and 861.2 mm³ in PBS group (Fig. 6 E, F&S10). Additionally, the average tumor weight in PBS group up to 520 mg, which was 21 times higher than that of mice treated with MFH + US group (25mg) (Fig. S11). Histological analyses were conducted to assess tumor necrosis, apoptosis, and proliferative activity in tumor tissues using H&E staining, TUNEL staining, and Ki-67 staining, respectively. The H&E staining and TUNEL staining results showed extensive tumor necrosis and apoptosis in the MFH + US group. Ki-67 staining revealed that the MFH + US group had the lowest proportion of proliferating cells compared to other groups (Fig. 6 G). These results demonstrate that MFH + US group effectively suppresses tumor growth by inducing significant necrosis and reducing cellular proliferation in tumors. To evaluate the biosafety of all treatments, blood routine tests, biochemical analyses, and organ histology were conducted for all mice. As shown in Fig. S12, the hemolysis rate of MFH at various concentrations was below 4%, indicating that the nanosheets did not cause hemolysis. Blood routine and biochemical parameter analyses further confirmed that all groups exhibited blood values within the normal range (Fig. S13&S14). Additionally, H&E staining of the major organs (heart, liver, spleen, lung and kidney) showed no noticeable pathological changes or toxicity associated with the prepared nanostructures (Fig. S15). These findings demonstrate that MFH + US group not only exhibits excellent biosafety but also provides strong magnetic resonance imaging signals and exceptional tumor growth inhibition capabilities, making it a promising candidate for effective and safe cancer therapy. 3.6 Augmentation of SDT-induced anti-tumor immunity through ICD mechanism To gain deeper insights into the mechanism by which MFH-enhanced SDT combats tumors through immunogenic cell death (ICD) induction by amplifying immunogenicity and alleviating the immunosuppressive microenvironment (ISM), relevant immune markers were further monitored. A subcutaneous tumor model of Hepa1-6 was established to evaluate tumor growth dynamics and immune profiles. When tumor volumes reached 60–80 mm³, mice were randomized into four groups (n = 3 per group) and treated with PBS, MFH, PBS + US, or MFH + US, respectively (Fig. 7 A). The results demonstrated rapid tumor growth in PBS and MFH treated groups. Strikingly, tumors in the MFH + US group exhibited nearly complete suppression (Fig. 7 B, C&S16). No significant body weight differences were observed across groups (Fig. 7 D). To analyze DC maturation in tumors and lymph nodes, mice were euthanized on day 16 for tumor immune profiling via flow cytometry. The results demonstrated that compared to the PBS group, the MFH + US group exhibited increased proportions of mature DCs in tumors and lymph nodes from 3.57–10.3% and 14.5–31.6%, respectively (Fig. 7 E, F&S17), indicating that MFH + US possessed the strongest capacity to activate T cells and enhance CD8 + T cell infiltration. As shown in the figure G, H, MFH + US-treated mice displayed the highest proportion of CD8 + T cells in tumors (15%), representing a 1.82-fold increase over PBS + US-treated mice (8.23%). Notably, the observed clonal expansion of CD8 + T cells in tumor-draining lymph nodes (TDLNs) suggests systemic immune activation, potentially driving the transition from immunologically 'cold' to 'hot' tumor microenvironment (Fig. S18). This lymphoid compartment priming may facilitate subsequent T cell trafficking to tumor sites, as evidenced by enhanced intratumoral CD8 + T cell infiltration - a hallmark feature of hot tumors. In summary, MFH induces immunogenic cell death (ICD) effects via ultrasound-induced high-level ROS generation, thereby potentiating the sonodynamic immunotherapy efficacy. This process encompasses CRT exposure, HMGB1 and ATP release, promotion of DC maturation, activation of CD8⁺ T cells, thereby facilitating antitumor immune responses. 4. Conclusion In summary, we have developed a metal-organic nanostructure (MFH) based on Ti x O y /Ru catalytic units. The synthesized MFH exhibits the following distinctive features: ( 1 ) TiZr-O clusters and Ru-COOH form a uniform and stable metal-organic nanosheet (TiZrRu-MON, M) through metal ion coordination. We demonstrate that the clusters transfer ultrasound-triggered energy to Ru-COOH, subsequently generating ROS; ( 2 ) Fe 3+ is incorporated into the M framework (denoted as MF) via chelation. Experimental results confirm that MF significantly enhances ROS production under US, indicating that Fe 3+ doping promotes SDT efficiency. Furthermore, MF generates Fenton-based ·OH via H 2 O 2 activation, and US irradiation accelerates the CDT process. This synergy between SDT and CDT induces potent oxidative stress, establishing a critical foundation for efficient cancer therapy; ( 3 ) A HA-based "nanocoating" enables tumor-targeted accumulation of nanosheets by binding to CD44 receptors overexpressed on cancer cells, thereby enhancing in vivo retention and improving MR imaging performance; ( 4 ) MFH combined with US irradiation triggers ICD in tumor cells, promotes DCs maturation, and recruits cytotoxic CD8 + T cells into tumors and lymph nodes through antigen-presenting mechanisms, thereby activating robust antitumor immunity. Collectively, our findings validate MFH as a promising sonosensitizer capable of addressing current clinical limitations of SDT. However, further studies are required to optimize its therapeutic window for deep-seated tumor treatment. Declarations Acknowledgements Not applicable. Authors’ contributions Tao Jiang, Zixiang Tang and Shumiao Tian contributed to this work equally. Tao Jiang, Zixiang Tang and Shumiao Tian designed experiments and wrote the manuscript. Tao Jiang, Zixiang Tang, Shumiao Tian, Haitian Tang, Zhekun Jia, Fangjian Li, Chenyue Qiu, Lin Deng, Lang Ke, Pan He conducted all experiments and related analysis. Yongfu Xiong, Chengchao Chu and Gang Liu revised the manuscript and supervised this study. Funding This work was funded by Sichuan Provincial Health Commission special research project (2024HR03), Sichuan Science and Technology Department Science and Technology Ability Improvement Project (2024JDKP0022), Nanchong Science and Technology Bureau Basic Research Platform Project (23JCYJPT0043), National Natural Science Foundation of China (82302403). Data availability No datasets were generated or analysed during the current study. Ethics approval and consent to participate All the animals were raised at Animal Care and Use Committee of Xiamen University (Ethics number XMULAC20190146). Consent for publication Not applicable. 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Li","email":"","orcid":"","institution":"Department of General Surgery, Academician (Expert) Workstation, Sichuan Digestive System Disease Clinical Medical Research Center, Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Fangjian","middleName":"","lastName":"Li","suffix":""},{"id":443958004,"identity":"5c0576ff-edd6-40b6-8295-1316f93fbfa9","order_by":6,"name":"Chenyue Qiu","email":"","orcid":"","institution":"Xiamen University Affiliated Xiamen Eye Center, Eye Institute of Xiamen University, School of Medicine, Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Chenyue","middleName":"","lastName":"Qiu","suffix":""},{"id":443958006,"identity":"2360331f-f92d-42be-a01d-d6d0f7709811","order_by":7,"name":"Lin Deng","email":"","orcid":"","institution":"Department of Clinical Medicine, North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Deng","suffix":""},{"id":443958007,"identity":"d3606831-7ff1-4810-99b5-f3f0a7a68c45","order_by":8,"name":"Lang Ke","email":"","orcid":"","institution":"Xiamen University Affiliated Xiamen Eye Center, Eye Institute of Xiamen University, School of Medicine, Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Lang","middleName":"","lastName":"Ke","suffix":""},{"id":443958008,"identity":"1b8deed7-3820-4ad7-bae1-4591f8067f54","order_by":9,"name":"Pan He","email":"","orcid":"","institution":"Department of Hepatobiliary and Pancreatic Surgery, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"He","suffix":""},{"id":443958009,"identity":"0a407254-fbac-463c-a9b8-048e99034022","order_by":10,"name":"Gang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYLCCBwYgkrGBgaECIiBBUEsCREtjA8MZorVAKMYGxjYitMi7nz38IqHgMAP/tMPtj27OOxxtcID54G0eBrs8XFoMz+SlWSQYHGaQuJ3Y2Jy7LS13wwG2ZGsehuRinFoacswMQFoMpMFabIBaeMykeRgOJDbg0tL/BlnLHAmgFv5veLXIS+QYP0BoaQDbwoZXi4HEGzNgIKfzgPwyO+dYWu7Mw2zGlnMMknHb0p9j/OHDH2s5/tnpDz7n1BzO7Tve/PDGmwo73LYcYGADxQIPQogZLI5DPciWBgbmD7ilR8EoGAWjYBQAAQCBaVlMY7oeXAAAAABJRU5ErkJggg==","orcid":"","institution":"State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, Xiamen University","correspondingAuthor":true,"prefix":"","firstName":"Gang","middleName":"","lastName":"Liu","suffix":""},{"id":443958010,"identity":"2f3c06f0-10ae-4970-ab93-3f9ec39139ac","order_by":11,"name":"Chengchao Chu","email":"","orcid":"","institution":"Xiamen University Affiliated Xiamen Eye Center, Eye Institute of Xiamen University, School of Medicine, Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Chengchao","middleName":"","lastName":"Chu","suffix":""},{"id":443958011,"identity":"058bfb6b-0e32-4119-b1fa-0c8f19831b55","order_by":12,"name":"Yongfu Xiong","email":"","orcid":"","institution":"Department of General Surgery, Academician (Expert) Workstation, Sichuan Digestive System Disease Clinical Medical Research Center, Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yongfu","middleName":"","lastName":"Xiong","suffix":""}],"badges":[],"createdAt":"2025-03-21 02:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6273421/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6273421/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03599-1","type":"published","date":"2025-07-21T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81374449,"identity":"4cacceea-c0e9-468d-9b54-9582036ee4b3","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3408697,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TEM of TiZrRu-MON (M) (Scale bars: 0.5 μm), (B) TiZrRu-MON (M) (Scale bars: 200 nm), (C) TiZrRuFe-MON (MF) (Scale bars: 200 nm), (D) TiZrRuFe-MON@HA (MFH) (Scale bars: 200 nm). (E) Average size and zeta potential of M, MF and MFH. (F) UV–Vis absorption of the TiZr-cluster, Ru-COOH, M, MF and MFH. (G,H,I) XPS spectra of MF. (H) Ru 3p, (I)Fe 2p. (J) HAADF image of MF. (K) EDS elemental mapping images of MF. (L) T1-weighted MR imaging and intensity of M, Fe\u003csup\u003e3+\u003c/sup\u003e and MF at different concentrations.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/76e4a662da91dc9b348e23c9.png"},{"id":81374448,"identity":"19e9b82e-1d79-4422-91f2-baefe9c0454a","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":525660,"visible":true,"origin":"","legend":"\u003cp\u003e(A) ESR signals of generated \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under US irradiation are trapped with TEMP. (B) The SDT performance of the DI-water, M and MF was demonstrated by the change in the UV-Vis-NIR spectra of the DPBF. (C) UV–vis absorbance of MF containing DPBF exposure to US irradiation (power density: 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e, duty cycle: 50%, 5 min) for different durations (0, 1, 2, 3, 4 and 5 min). (D) ESR signals of generated ·OH under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e(100 μM) excitation, which is trapped with DMPO. (E) CDT performance of DI-water, Fe\u003csup\u003e3+\u003c/sup\u003e, M and MF within 5 minutes. (F) UV–vis absorbance of MF containing TMB exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e([Zr]100 μM) excitation for different concentrations ([Zr] 0, 12.5, 25, 50, 100 and 200 μM). （G,H）UV–vis absorbance of TMB to evaluate the ·OH generation of MF without （G） or with （H） US irradiation at same concentration ([Zr] 100 μM). (I) The DCFH fluo­rescence of MF under US irradiation or not. *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 and ns for non-significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/9b02a395e1cda79daa926bed.png"},{"id":81374450,"identity":"389c325b-ab44-48f9-8b50-cdad0b865659","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1066214,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The cellular TEM of HepG2 cells after incubated with MFH for 12h. (B) HepG2 cells were analyzed by 1.5T MR scanning after uptake of MF or MFH. (C) Fluorescence staining of rhodamine b-labeled MF and MFH for different durations (1, 4, 8 and 12 h) to show the cellular uptake (Scale bars: 20 μm). (D) Zr concentration analysis after cellular uptake of MF and MFH for different times (1, 4, 8 and 12 h). (E) Confocal images of HepG2 cells incubated with different treatments and then stained by DCFH-DA to demonstrate the level of ROS generation (Scale bars: 100 μm). (US power density: 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e, duty cycle: 50%, 5 min). (F) Flow cytometric profiles of ROS generation. *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 and ns for non-significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/73164e431f9835a43b286d76.png"},{"id":81374452,"identity":"f1ed8757-c06f-4d10-b33e-4b22a91ac5a5","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1001679,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Relative viability of HepG2 cells incubated with MH, MF and MFH. (B) The viability of HepG2 cells incubated with MH or MFH, and then irradiated with or without US. (C) Flow-cytometry apoptosis assays of HepG2 cells after different treatments followed by staining with Annexin V-FITC and PI. (D) Fluorescence images of HepG2 cells incubated with PBS, MH, MF and MFH after varied treatments by stained with calcein-AM (green fluorescence) and PI (red fluorescence) (Scale bars: 100 μm). *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 and ns for non-significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/44186d321b190f677a1c8477.png"},{"id":81374459,"identity":"bd5288cb-6e72-40b5-979d-ea037b49aec4","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":907393,"visible":true,"origin":"","legend":"\u003cp\u003eMFH resulted in a stronger ICD effect and more DCs maturation in vitro. A) Representative CLSM images of release of HMGB1 in Hepa1-6 cells. Scale bar = 20 μm. B) Extracellular ATP levels in Hepa1-6 cells after various treatments. C) Representative CLSM images of the exposure of CRT in Hepa1-6 cells. Scale bar = 20 μm. D) Representative flow cytometric curves of surface expression of CRT on Hepa1-6 cells. E, F) Semi-quantitative study of the maturation of BMDCs co-cultured with Hepa1-6 cells with various pretreatments by FCM. n = 3. * p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/b81023f95fa382c48fc80b9c.png"},{"id":81374784,"identity":"0639c664-b081-487d-9b69-25cae1735a86","added_by":"auto","created_at":"2025-04-25 11:20:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1906201,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution and antitumor efficacy of MFH on a HepG2 tumor-bearing nude mice model. (A) Schematic illustration of the treatment schedule (US power density: 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e, duty cycle: 50%, 5 min). The in vivo MR (B, C) studies of the nude mice subcutaneous model after i.v. injection MF and MFH at 0, 1, 4, 8, 12, and 24 h. (D) Body weight changes of BALB/c nude mice after various treatments. (E) Tumor growth inhibition curve. (F) Representative tumor images. (G) H\u0026amp;E, Ki-67 staining and TUNEL assay (Scale bar = 100 μm). *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 and ns for non-significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/75757d22d8c92ac7fef76bd3.png"},{"id":81374455,"identity":"2622f420-42c5-447e-b29e-fec906ad86fc","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":474050,"visible":true,"origin":"","legend":"\u003cp\u003eMFH amplified the sonodynamic-immunotherapy on a Hepa1-6 subcutaneous cancer model. (A) Schematic illustration of the treatment schedule. (B) Tumor growth inhibition curve and (C) tumor weight of mice in different groups. (D) Body weight of mice in different groups. (E) Representative FCM plots and (F) the percentages of matured DCs (CD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e) in the tumor. n = 3.(G) Representative FCM plots and (H) the percentages of CD8\u003csup\u003e+\u003c/sup\u003eT cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e) in the tumor. n = 3. *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 and ns for non-significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/f6c1d57f5acb34dec72867b9.png"},{"id":87756730,"identity":"4a2e0ea0-a965-4b26-8d93-e325dba93328","added_by":"auto","created_at":"2025-07-28 16:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10426920,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/b9aef561-e10f-4329-b878-b06abd536c14.pdf"},{"id":81374790,"identity":"b0272708-135e-43cc-9b0b-23457a518ae4","added_by":"auto","created_at":"2025-04-25 11:20:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8788971,"visible":true,"origin":"","legend":"","description":"","filename":"TixOyRureactionunitssupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/32663d9e683b898b34bbc516.docx"},{"id":81374453,"identity":"a3b9eab2-082f-48b5-a230-4e6e2a28c517","added_by":"auto","created_at":"2025-04-25 11:12:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2148353,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-6273421/v1/ae429f31a8a3783a7fffbbca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metal-organic nanostructures based on sono/chemo-nanodynamic synergy of TixOy/Ru reaction units: for ultrasound-induced dynamic cancer therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer remains one of the most severe threats to global human health (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Despite emerging therapeutic approaches, current treatments demonstrate suboptimal efficacy and severe side effects, highlighting the urgent need to develop more effective and safer cancer therapies (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). With the rapid advancement of nanotechnology, ultrasound (US)-triggered SDT has emerged as a promising non-invasive antitumor strategy, characterized by deep tissue penetration and high spatiotemporal precision (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Through ultrasonic irradiation, SDT precisely activates sonosensitizers accumulated in deep tumor tissues to generate cytotoxic ROS, thereby effectively eliminating malignant cells (\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Notably, elevated ROS accumulation facilitates robust antitumor immunity through immunogenic cell death (ICD)-based mechanisms (\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). SDT-induced tumor cells release substantial tumor-associated antigens (TAAs) and DAMPs, which promote antigen presentation by DCs. This process activates and recruits cytotoxic T lymphocytes (CTLs), ultimately triggering systemic antitumor immune responses (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOver the past decades, organic sonosensitizers with high ROS-generation efficiency\u0026mdash;including porphyrins, 5-aminolevulinic acid, phthalocyanines, oxazines, and indocyanines\u0026mdash;have shown promise. However, their applications in cancer therapy remain constrained by poor water solubility, low chemical stability, limited tumor-targeting capability, and significant phototoxicity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In contrast, inorganic sonosensitizers represented by titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) nanomaterials exhibit advantages such as superior chemical stability, low phototoxicity, and facile surface modification, making them attractive for oncological SDT applications (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Nevertheless, challenges including high immunogenicity, prolonged in vivo retention, and insufficient tumor accumulation continue to impede their clinical translation (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe cytotoxic effects of sonosensitizers are primarily mediated by ROS generated under ultrasound irradiation. A key mechanism of SDT involves the ultrasound-triggered excitation of sonosensitizers, which promotes electron transitions from the valence band (VB) to the conduction band (CB). This process generates reactive electron-hole pairs (e\u003csup\u003e\u0026minus;\u003c/sup\u003e/h\u003csup\u003e+\u003c/sup\u003e) that subsequently convert surrounding water and oxygen molecules into ROS (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, conventional sonosensitizers often suffer from low electron-hole pair separation efficiency and rapid recombination, resulting in suboptimal ROS generation and compromised SDT efficacy (\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). To address these limitations, titanium-oxo clusters (TOCs) derived from the hybridization of TiO\u003csub\u003e2\u003c/sub\u003e with organic molecules have been engineered to enhance photo/ultrasonic catalytic reduction activity by optimizing charge carrier separation and migration (\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This improvement is attributed to four synergistic effects: 1) modulated electronic structures, 2) enhanced charge carrier separation, 3) improved light absorption capacity, and 4) optimized surface chemical properties.\u003c/p\u003e \u003cp\u003eA growing body of research highlights the potential of constructing multifunctional nanoscale metal-organic frameworks (MOFs) as ideal platforms for advancing cancer therapeutics, owing to their exceptional biocompatibility, tunable pore structures, superior chemical stability, and efficient endosomal escape capacity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Our study demonstrates that [Ti\u003csub\u003e8\u003c/sub\u003eZr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e(COO)\u003csub\u003e16\u003c/sub\u003e] clusters, featuring highly stable bimetallic units, serve as critical building blocks for creating photoactive MOFs (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Upon activation, these TiZr clusters effectively trap photoexcited electrons, thereby suppressing rapid e\u003csup\u003e\u0026minus;\u003c/sup\u003e/h\u003csup\u003e+\u003c/sup\u003e recombination. Furthermore, we discovered that [Ru(bpy)\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e2+\u003c/sup\u003e can coordinate with photocatalytically active clusters through organic ligands to form structurally stable metal-organic nanosheets. This novel architecture expands the light absorption spectrum, facilitates photoinduced charge carrier separation/migration, enhances reactive site activity/stability, and ultimately achieves high-efficiency synergistic photo-/sono-catalytic performance.\u003c/p\u003e \u003cp\u003eThe bandgap of sonosensitizers serves as a critical parameter governing e\u003csup\u003e\u0026minus;\u003c/sup\u003e/h\u003csup\u003e+\u003c/sup\u003e separation efficiency. Studies demonstrate that doping with transition metal ions (e.g., Fe\u003csup\u003e3+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e) significantly enhances charge carrier separation dynamics under ultrasound irradiation through band structure reconstruction (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). For instance, Shang et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) achieved bandgap narrowing via Fe\u003csup\u003e3+\u003c/sup\u003e doping, while Yang et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) optimized both band alignment and ROS generation pathways through Mn\u003csup\u003e3+\u003c/sup\u003e incorporation. Notably, MOFs, with their high porosity and tunable channel structures, can effectively chelate high-concentration Fe\u003csup\u003e3+\u003c/sup\u003e ions. This introduces intermediate energy levels to reduce bandgap width while enabling directional migration of photogenerated carriers for the production of more \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe integration of Fe\u003csup\u003e3+\u003c/sup\u003e and Mn\u003csup\u003e3+\u003c/sup\u003e leverages their intrinsic Fenton-like catalytic activity, establishing a synergistic SDT-CDT (chemodynamic therapy) mechanism. Under ultrasound irradiation, piezoelectric electrons generated on the sensitizer surface accelerate valence cycling of metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e/Mn\u003csup\u003e3+\u003c/sup\u003e) via interfacial charge transfer, dramatically enhancing Fenton-like reaction kinetics. This process induces instantaneous burst generation of highly cytotoxic hydroxyl radicals (\u0026middot;OH). This SDT-CDT coupling mechanism achieves dual therapeutic outcomes through oxidative stress cascades that directly induce tumor cell apoptosis while concurrently triggering DAMPs release. The liberated DAMPs enhance DCs infiltration into tumor sites, facilitate antigen capture, and potentiate cross-presentation processes. Through systematic reprogramming of the immunosuppressive microenvironment, cytotoxic T lymphocytes (CTLs) become activated, leading to reconstructed antitumor immune responses that synergistically coordinate ICD with the establishment of systemic immune memory.\u003c/p\u003e \u003cp\u003eBased on the [Ru(bpy)\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e2+\u003c/sup\u003e photosensitizing unit and Fe\u003csup\u003e3+\u003c/sup\u003e doping strategy, this study developed a multifunctional metal-organic nanostructure (denoted as MF, TiZrRuFe-MON) that integrates precise tumor theranostics through synergistic SDT and CDT mechanisms (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). MF nanoparticles, synthesized via a controlled hydrothermal method, exhibited excellent monodispersity and crystalline integrity. Their sonocatalytic performance under US irradiation significantly surpassed that of Fe\u003csup\u003e3+\u003c/sup\u003e-undoped TiZrRu-MON (M), attributed to the intrinsic efficient photo/sonocatalytic properties of TiZrRu-MON and the Fe\u003csup\u003e3+\u003c/sup\u003e doping-induced suppression of e\u003csup\u003e\u0026minus;\u003c/sup\u003e/h\u003csup\u003e+\u003c/sup\u003e recombination kinetics coupled with bandgap narrowing. Furthermore, Fe\u003csup\u003e3+\u003c/sup\u003e incorporation enabled efficient SDT/CDT synergy by catalyzing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition into highly cytotoxic\u0026middot;OH through Fenton-like reactions. To enhance tumor-specific accumulation, MF was functionalized with hyaluronic acid (HA). In vitro and in vivo T1-weighted magnetic resonance imaging (MRI) demonstrated HA-mediated significant improvement in tumor-targeted enrichment. In murine tumor models, HA-modified MF (MFH) combined with US irradiation achieved marked tumor growth suppression. Notably, SDT-induced ROS bursts enhanced tumor-associated antigen (TAA) exposure, triggering effector T cell-mediated antitumor immunity via DC-dependent antigen uptake and presentation. This work not only provides new insights into metal-organic framework design but also establishes a critical foundation for clinical translation of SDT/CDT synergistic therapeutic strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eAll chemicals were of analytical purity and used without further purification. Bis(2,2'-bipyridyl) (4,4'-dicarboxy1-2,2'bipyridyl) ruthenium(II) dichloride was purchased from Suna Technology Inc (Suzhou, China). Trifluoroacetic acid and acetic acid were purchased from Macklin. Titanium tetraisopropanolate, Absolute ethanol, NaOH, 3,3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine dihydrochloride hydrate (TMB), FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-Tetramethyl-4-piperidone hydrochloride (TEMP) were purchased from Aladdin (Shanghai, China). 1,3-diphenylisobenzofuran (DPBF), Cell Counting Kit-8 (CCK-8) and 2,7-dichlorofluorescein diacetate (DCFH-DA) by GLPBIO Technology Inc. Calcein AM/PI Cell Viability/Cytotoxicity Assay Kit was purchased from KeyGEN BioTech Inc (Suzhou, China). Annexin V-FITC/PI Kit and ATP test kit were bought from Beyotime Biotechnology (Shanghai, China). Rhodamine B was purchased from Sigma-Aldrich (California, USA). Calreticulin Rabbit pAb, HMGB1 Rabbit pAb, Goat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor 488) and Goat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor 647) were purchased from Zen-Biosciencer Inc (Chengdu, China). Anti-CD80-PE, anti-CD86-421, anti-CD11-650, anti-CD3-APC, anti-CD4-PE and anti-CD8-FITC antibodies were obtained from Biolegend. Fetal bovine serum (FBS) and Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) were purchased from Hyclone (USA). HepG2 cells and Hepa1-6 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Nude mice and C57BL/6J mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) images were acquired on a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Scanning electron microscope (SEM) images and element mapping were taken on an FEI Talos F200S transmission electron microscope. X-ray photoelectron spectroscopy analyses were carried out using an X-ray photoelectron spectroscopy (XPS). The size of the particles and zeta potential were measured by dynamic light scattering (DLS; Malvern Ltd., UK). The fluorescence images of cells were taken on a Fluorescent Inverted microscope (Nikon., Japan). Magnetic resonance (MR) properties of materials were measured using a 1.5 T NMR relaxation measurement system (HT-MICNMR-60, Huanyu., China). The MR images were acquired using an MRI system (Bruker 9.4 T Micro-MRI equipment). The concentration of the material was measured with a Inductively coupled plasma-Optical emission spectrometry(ICP-OES)(SPECTRO Co. German).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Synthesis of TiZrRuFe-MON@HA (MFH)\u003c/h2\u003e \u003cp\u003eFor the synthesis of TiZrRu-MON, the [Ti\u003csub\u003e8\u003c/sub\u003eZr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e(COO)\u003csub\u003e16\u003c/sub\u003e] cluster was firstly synthesized. The ZrCl\u003csub\u003e4\u003c/sub\u003e (20 mg) was dispersed in DMF (5 mL) using ultrasonic agitation. Subsequently, Ti(OiPr)₄ (0.04 mL) and acetic acid (0.2 mL) were added to the reaction kettle, which was then heated in an oven at 100\u0026deg;C for 24 hours. After cooling of the reactor, the obtained clusters were stored in centrifuge tubes. 2,2\u0026lsquo;-Bipyridine-4,4\u0026rsquo;-dicarboxylic acid (5.25 mg) and Ru-COOH (16mg) were dissolved in DMF, respectively. Both were then mixed with [Ti\u003csub\u003e8\u003c/sub\u003eZr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e(COO)\u003csub\u003e16\u003c/sub\u003e] cluster (10mg), trifluoroacetic acid (1.5 mL) was added and finally stirred well using vortex. The reaction mixture was heated in a 140\u0026deg;C oven for 24 h. The claybank crystalline sediment was harvested by centrifugation and washed with ethanol. FeCl\u003csub\u003e3\u003c/sub\u003e solution (10% Zr concentration) was added to the resulting TiZrRu-MON solution, and then the mixture stirred in the dark for 12 hours at room temperature. The resulting TiZrRuFe-MON solution was collected by centrifugation and washed with DI water. Finally, the HA solution was added into TiZrRu-MON solution or TiZrRuFe-MON solution respectively, and the reaction was carried out in water bath ultrasonication for 30 min. The resulting TiZrRu-MON@HA (MH) and TiZrRuFe-MON@HA (MFH) solution was collected by centrifugation and washed with deionised water (DI water).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Electron Spin Resonance (ESR) Spectra Test in Vitro\u003c/h2\u003e \u003cp\u003eUtilizing electron spin resonance (ESR), MF during US or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activation produced different forms of ROS. The activity of \u0026middot;OH was investigated by ESR using a spin-trapping agent precisely (DMPO). For purpose of measuring the ESR, solutions containing DI water, M or MF ([Zr] 100 \u0026micro;M), 100 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 100 mM DMPO were combined. The solutions were reacting for 5 minutes. Additionally, the production of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was confirmed using the TEMP (100 mM). DI water, M or MF ([Zr] 100 \u0026micro;M), and TEMP were combined, and were subjected to US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min. The \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e or \u0026middot;OH signal were picked up by the ESR spectrometer, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In vitro detection of ROS generation\u003c/h2\u003e \u003cp\u003eTo detect \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generated by SDT, DPBF was dissolved in DMSO (1 mg/mL). 1 mL of MFH solution ([Zr] 100 uM) was mixed with 20 \u0026micro;L of DPBF. The mixed solutions were tested for UV-visible absorption after different periods of time of US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) in dark. Similarly, results were obtained under US irradiation using DI water and M solution as controls.\u003c/p\u003e \u003cp\u003eTo detect ▪OH generated by the reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 5 \u0026micro;L of TMB (20 mg/mL in DMSO) was added into 1 mL of MFH consisting of 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. After reaction for 10 minutes, the absorbance change of TMB at 600 nm reflected the generation of ROS by the Fenton reaction of MFH. In addition, DI water, FeCl\u003csub\u003e3\u003c/sub\u003e solution and M solution were used for comparison. Meanwhile, the absorbance changes of the TMB working solution with or without US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) were recorded after 0, 1, 2, 3, 4 or 5 minutes at the same concentration of MF ([Zr] 100 \u0026micro;M).\u003c/p\u003e \u003cp\u003eDetection of in vitro cellular ROS generation was conducted with the 2\u0026prime;,7\u0026prime;-dichlorodihy-drofluorescein diacetate (DCFH-DA). Specifically, 5 \u0026micro;L DCFH-DA (10 mM) was added to 20 \u0026micro;L NaOH solution (10 mM), and the mixture was incubated for 30 min at room temperature away from light to prepare 2\u0026prime;,7\u0026prime;-dichlorodihy-drofluorescein (DCFH). Afterwards, 1 mL PBS (pH 7.4) was added to neutralize the reaction. Then, 100 \u0026micro;L of DCFH solution was mixed with an equal volume of different groups of solutions. After US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm \u003csup\u003e2\u003c/sup\u003e), ROS were detected by fluorescence intensity scanning (Ex: 480 nm, Em: 525 nm) in multifunctional enzyme marker.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell culture\u003c/h2\u003e \u003cp\u003eMouse hepatocellular carcinoma cells (Hepa1-6) and human hepatocellular carcinoma cells (HepG2) were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). All cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cellular uptake\u003c/h2\u003e \u003cp\u003eFor cellular uptake study, HepG2 cells were seeded in 12-well plates (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) and incubated at 37 ℃ overnight. Then the cells were treated with Rh-b-labeled MF or MFH at the Zr concentration of 100 \u0026micro;M for 1 h, 4 h, 8 h or 12 h respectively. After being washed with cold PBS for three times, the cell was fixed with 4% paraformaldehyde and cell nuclei was stained with Hoechst 33342. Subsequently, images were collected by CLSM (High Sensitivity Laser Confocal Microscope. Evident FV4000, Japan).\u003c/p\u003e \u003cp\u003eTo quantify the cellular uptake, HepG2 cells were seeded into 6-well plates (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) and cultured for 12 h. After the culture media was removed, the fresh media containing MF or MFH at a Zr concentration of 100 \u0026micro;M was added. Then, the cells were collected and counted with automated cell counter after incubation for 1, 4, 8 and 12 h. The obtained samples were digested by aqua regia (hydrochloric acid (HCl) and nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e) in a molar ratio of 3:1) for 48 hours and the concentrations of Zr in cells were measured by ICP-OES.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In vitro ROS generation in cells by SDT\u003c/h2\u003e \u003cp\u003eIntracellular ROS level was investigated by using DCFH-DA as a fluorescent probe. Briefly, 1 mL of HepG2 cells (1\u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were added to a confocal dish and incubated overnight at 37\u0026deg;C. Following apposition, cells were treated with MH, MF, and MFH (20 uM) at the same Zr concentration. After 12 h of incubation, the cells were washed with PBS for 3 times to eliminate free agents, and then added serum-free medium containing DCFH-DA (10 \u0026micro;M) for 30 min. Then, the cells were treated by US irradiation (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e). Subsequently, images were collected with CLSM. Furthermore, the intracellular ROS level was further detected and quantified by FCM. Firstly, cells were seeded in 12-well plate at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e per well and incubated at 37\u0026deg;C overnight. Afterward, the cells were treated with the same approach and conditions as the above CLSM analysis. Finally, the cells were harvested immediately to examine the intracellular ROS by FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cytotoxicity assay in vitro\u003c/h2\u003e \u003cp\u003eHepG2 cells were seeded in 96-well plates (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e per well) and incubated with complete medium (10% FBS) at 37\u0026deg;C overnight. Then, the culture media was removed and replenished with fresh media containing different concentrations of MH, MF or MFH. After incubating for another 24 h, the cells viability was determined using cck-8 assay.\u003c/p\u003e \u003cp\u003eTo study the cytotoxicity of SDT treatment. Cells were seeded in 96-well plates and incubated overnight, then the different concentrations of MH, MF or MFH was added and incubated for another 12 h. Cells were washed three times with PBS and treated with ultrasonic irradiation. Afterwards, the cells were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) and further incubated for 24 h. The relative cytotoxicity was assessed by cck-8 assay.\u003c/p\u003e \u003cp\u003eThe cytotoxicity of SDT treatment was also evaluated by calcein-AM/PI experiment. The Hepa 1\u0026ndash;6 cells were incubated with MH, MF or MFH (100 \u0026micro;M) in 12-well plates for 12 h and washed with PBS three times. Then, the cells were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e). Finally, the cells were stained with calcein-AM and PI, and the images were acquired by an Inverted fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Colony formation assay\u003c/h2\u003e \u003cp\u003eThe HepG2 cells were seeded into 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well for 12 h. The cells were incubated with PBS, MH, MF or MFH ([Zr] 100 \u0026micro;M) for 12 h, and then they were irradiated under US (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e). Then, the cells were replanted into 6-well plates (1000/well, 3 wells in each group) and cultured for 7\u0026ndash;10 days. Finally, the cells were fixed with 4% paraformaldehyde and stained with 0.5% Gentian Violet. Colonies of exceeding 50 cells were counted to calculate survival fractions. Surviving fraction = (surviving colonies)/ (cells seeded \u0026times; plating efficiency).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Apoptosis analysis in vitro\u003c/h2\u003e \u003cp\u003eCellular apoptosis was assessed with an Annexin V-FITC apoptosis detection kit according to the manufacturer\u0026rsquo;s instruction. HepG2 cells were cultured in 12-well plates (2\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells per well) overnight. Afterwards, the cells were treated with PBS, MFH, PBS\u0026thinsp;+\u0026thinsp;US, MH\u0026thinsp;+\u0026thinsp;US, MF\u0026thinsp;+\u0026thinsp;US and MFH\u0026thinsp;+\u0026thinsp;US ([Zr] 100 \u0026micro;M) for 12 h. The cells in the US groups were irradiated with US (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) and cultured for another 12 h. Finally, the cells were incubated with Annexin/PI reagent in the dark for 20 min and immediately measured with FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 ICD induced by MFH\u0026thinsp;+\u0026thinsp;US in vitro\u003c/h2\u003e \u003cp\u003eTo determine different formulations-induced immunogenic cell death (ICD) of the tumor cells, the secretion of adenosine triphosphate (ATP), calreticulin (CRT) exposure, and extracellular release of high mobility group box 1 (HMGB1) were examined in vitro. In order to study the exposed CRT on the cell surface, the Hepa1-6 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) in 1 mL media were added to the confocal dish and incubated at 37\u0026deg;C overnight, and then the cells were treated with PBS, MFH, PBS\u0026thinsp;+\u0026thinsp;US, MH\u0026thinsp;+\u0026thinsp;US, MF\u0026thinsp;+\u0026thinsp;US and MFH\u0026thinsp;+\u0026thinsp;US for 12 h (at the Zr concentration of 100 \u0026micro;M). Further incubated with primary CRT antibody for overnight at 4 ℃ and then incubated with the 488-conjugated secondary antibody. Finally, the cells were stained with DAPI and observed by CLSM. For FCM analysis, cells were seeded in the 12-wells plate at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well overnight. Then the cell treatment was the same as CLSM analysis. Next, the cells were washed with PBS and incubated with CRT antibody. After that, the cells were incubated with secondary antibody for 30 min and then analyzed by FCM.\u003c/p\u003e \u003cp\u003eFor evaluating intracellular HMGB1, Hepa1-6 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) in 1 mL media were added to the confocal dish. After 12 h incubation, the cells were treated with different formulations like the CRT analysis for 24 h. Next, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, incubated with 5% bovine serum albumin for 30 min. After that, the cells were incubated with primary HMGB1 antibody overnight at 4 ℃ and then incubated with secondary antibody. Finally, the cells were examined by CLSM.\u003c/p\u003e \u003cp\u003eFor ATP level assay, the cells were seeded in 12-well plate at a density of 15\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. Subsequently, the cells were treated with different formulations like the CRT analysis for 24 h. To measure the release of ATP from treated cancer cells, the supernatant from each group was collected. ATP assay kit (Beyotime, S0026) was used to determine the RLU values for each set of samples using a chemiluminescence meter (luminometer) according to the manufacturer's protocol, and the concentration was defined by the developed calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 BMDC mature in vitro\u003c/h2\u003e \u003cp\u003eTo evaluate dendritic cells (DCs) activation in vitro, bone-marrow derived dendritic cells (BMDCs) were obtained from C57BL/6 mice. B16F10 cells were pretreated with PBS, MFH, PBS\u0026thinsp;+\u0026thinsp;US, MH\u0026thinsp;+\u0026thinsp;US, MF\u0026thinsp;+\u0026thinsp;US and MFH\u0026thinsp;+\u0026thinsp;US ([Zr] 100 \u0026micro;M) and then co-incubated with BMDCs for 24 h. After that, DCs were stained with anti-CD11-650, anti-CD80-PE and anti-CD86-421 for DCs mature analysis by FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Animal welfare and protocols\u003c/h2\u003e \u003cp\u003e Female BALB/c nude mice and C57BL/6 mice (5\u0026ndash;6 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China), and all animal experiments were performed according to the protocol with the Animal Care and Use Committee (CC/ACUCC) of Xiamen University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.16 In vivo biosafety evaluation\u003c/h2\u003e \u003cp\u003eFemale BALB/c nude mice were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;5 mice per group). On day 1, 3, 5, each group mice were injected with PBS, MH, MFH (5 mg/kg) through tail vein, respectively. Then the mice were sacrificed at 15th days after treatments, and the blood samples of the mice were collected for hematological and serum biochemical analysis. Meanwhile, the main organs including heart, liver, spleen, lung and kidney were also obtained for further analysis. All the tissues were paraffin-embedded and tissue sections were prepared. Then H\u0026amp;E staining were performed to observe pathological features, and images were captured using an Microscopic Digital Section Scanning System Motic VM1 (McAuldie, Hong Kong).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Biodistribution of MFH in vivo\u003c/h2\u003e \u003cp\u003eHepG2 cells were injected subcutaneously into the right flank of the BALB/c mice. MF or MFH (5 mg/kg) was intravenously injected into the mice when the tumor volume reached 150\u0026ndash;200 mm\u003csup\u003e3\u003c/sup\u003e. Afterwards, the MR imaging were acquired at 0, 1, 4, 8, 12 and 24 h post-injection and quantified using the imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Antitumor efficacy evaluation in the HepG2 tumor model\u003c/h2\u003e \u003cp\u003eTo evaluate the antitumor effects of MFH in a human-derived tumor model, the female BALB/c nude mice subcutaneous tumor model were constructed by injecting HepG2 cells into the right flank of the mice, respectively. When the tumor volume reached approximately 60\u0026ndash;80 mm3, the mice were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;5/group) and PBS, PBS\u0026thinsp;+\u0026thinsp;US, MH\u0026thinsp;+\u0026thinsp;US(5 mg/kg), MFH (5 mg/kg), and MFH\u0026thinsp;+\u0026thinsp;US (5 mg/kg) were injected intravenously viathe tail veins. The mice of US groups were irradiated with US at intensity of 1 W/cm\u003csup\u003e2\u003c/sup\u003e for 5 min after 12 h injection. All the mice were treated every two days for 3 cycles, and the tumor volume and body weight were monitored every 2 days. The tumor volumes were calculated by a formula: volume\u0026thinsp;=\u0026thinsp;length\u0026times;width\u003csup\u003e2\u003c/sup\u003e /2. The mice were sacrificed after 15 or 16 days of treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Immunohistochemical analyses\u003c/h2\u003e \u003cp\u003eAfter completion of treatments, the mice were sacrificed and then the major organs and tumors were excised for histological observation by H\u0026amp;E staining and Immunohistochemical (IHC) staining. For H\u0026amp;E staining, excised tumors and organs were fixed in 4% paraformaldehyde solution, embedded in paraffin sections, and stained with hematoxylin and eosin. TUNEL and Ki67 staining were used to assess apoptosis and proliferation of tumor tissues, respectively. The procedures were consistent with the manufacturer's protocol, and finally images were captured using an Microscopic Digital Section Scanning System Motic VM1 (McAuldie, Hong Kong).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.20 Ultrasound-activated immune effects and metastatic tumor models\u003c/h2\u003e \u003cp\u003eHepa 1\u0026ndash;6 cells were injected into the right flank of the C57BL/6J female mice. The mice were randomly grouped (n\u0026thinsp;=\u0026thinsp;5 /group) including PBS, PBS\u0026thinsp;+\u0026thinsp;US, MFH (5 mg/kg), and MFH\u0026thinsp;+\u0026thinsp;US (5 mg/kg) when the tumor volume reached 60\u0026ndash;80 mm\u003csup\u003e3\u003c/sup\u003e. The mice of US groups were irradiated with US (1.0 MHz, 50% duty cycle, 1 W/cm\u003csup\u003e2\u003c/sup\u003e). All the mice were treated every two days for 3 cycles, and the tumor volume and body weight were monitored every 2 days. The mice were sacrificed after 15 or 16 days of treatments. The primary tumors were excised by H\u0026amp;E staining and TUNEL staining and finally images were captured using a Microscopic Digital Section Scanning System Motic VM1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.21 Flow cytometry analysis of the animal tissues\u003c/h2\u003e \u003cp\u003eThe mice were sacrificed after treatments. The obtained fresh tumors and tumor-draining lymph modes were used to prepare single-cell suspension. Then the suspensions were further incubated with different antibody against the immune cells. To detect the DCs maturation in tumor and lymph modes, the cell suspensions were stained with antibodies of anti-CD11-650, anti-CD80-PE and anti-CD86-421, respectively. The matured DCs were marked as CD11\u003csup\u003e+\u003c/sup\u003eCD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003ecells. To detect the infiltration of antitumor T cells and the suspensions of tumor were incubated with anti-CD3-APC, anti-CD8a-FITC and anti-CD4-PE and the CD8\u003csup\u003e+\u003c/sup\u003e T cells were denoted as CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003ecells. Finally, these cells were for FCM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.22 Statistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed at least three times and results were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical significances were analyzed using the one-way ANOVA test or two-way ANOVA test. The difference was regarded as significant when the p value was less than or equal to 0.05. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ns, not significant\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Preparation and characterization of MFH\u003c/h2\u003e \u003cp\u003eBuilding upon prior methodologies, the [Ti₈Zr₂O₁₂(COO)₁₆] cluster was integrated with Bpy-COOH and Ru-COOH via hydrothermal coordination, yielding TiZrRu-MON (M). Transmission electron microscopy (TEM) characterization revealed that M exhibited lamellar structures with surface folding, displaying an average particle size of approximately 200 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u0026amp;S1), and formed a homogeneously dispersed ochre solution in aqueous media. To enhance the photo/sono-catalytic properties of M, Fe\u0026sup3;⁺ ions were immobilized within its lattice through chelation with Bpy-COOH groups, generating TiZrRuFe-MON (MF). MF exhibited a maroon coloration in aqueous solution, its structural integrity remained unaltered compared to M as confirmed by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;S1). Finally, to improve biosafety and prolong systemic circulation, MF was encapsulated with hyaluronic acid (HA), producing TiZrRuFe-MON@HA (MFH). MFH displayed a lighter hue compared to MF and featured distinct 'nano-coatings' in TEM imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026amp;S1).\u003c/p\u003e \u003cp\u003eThe prepared MFH exhibited an increased particle size (\u0026asymp;\u0026thinsp;310 nm) compared to M (\u0026asymp;\u0026thinsp;230 nm) or MF (\u0026asymp;\u0026thinsp;270 nm), likely attributed to HA surface coating (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE \u0026amp; S2). Notably, MFH displayed a higher zeta potential (+\u0026thinsp;25.6 mV), suggesting enhanced nanoparticle stability through HA encapsulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). UV-Vis spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) revealed that M maintained a similar absorption peak to Ru-COOH, indicating new chemical interactions between Ru-COOH and the clusters. Importantly, neither Fe\u0026sup3;⁺ nor HA modified the characteristic Ru absorption peaks.\u003c/p\u003e \u003cp\u003eThe material\u0026rsquo;s elemental composition and valence band structure was analyzed by X-ray photoelectron spectra (XPS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026amp;S3), revealing significant peak variations at Fe and Ru positions indicative of strong coordination effects. MF nanosheets exhibited Ru characteristic peaks at 458.50 and 459.30 eV in the Ru 3p region, contrasted with Ru-COOH's distinct peak at 280.0 eV in the Ru 3d region, demonstrating higher binding energy in MF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The Fe 2p orbital displayed split peaks at 726.41 and 728.33 eV, deviating significantly from conventional Fe\u0026sup3;⁺ binding energies (~\u0026thinsp;710 eV), likely due to interfacial Fe-O-Ru charge transfer-induced binding energy elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These findings suggest successful Fe\u0026sup3;⁺ doping into the nanosheet lattice and Ru local electron structure reorganization, potentially enhancing catalytic activity. HAADF imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ) and EDS mapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK) confirmed homogeneous spatial distribution of Ti, Zr, Ru, and Fe throughout the nanostructure. ICP-OES analysis revealed substantially increased Fe content in MF and MFH compared with M (Fig. S4), verifying precise iron chelation within the nanosheets.\u003c/p\u003e \u003cp\u003eFinally, the T1-weighted magnetic resonance imaging (MRI) of Fe\u003csup\u003e3+\u003c/sup\u003e, M, and MF solutions was investigated using a 1.5T MRI scanner. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, the MF solution exhibited a longitudinal relaxation (r1) of 12.5 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, significantly higher than that of the M solution and Fe\u003csup\u003e3+\u003c/sup\u003e solution, which demonstrated r1 values of merely 1.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3.26 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. These findings suggest that the lattice structure of M enhances the intrinsic magnetic resonance intensity of Fe\u003csup\u003e3+\u003c/sup\u003e, thereby conferring MRI capability to the MF complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Enhanced ROS Generation Efficiency through CDT-SDT Synergy\u003c/h2\u003e \u003cp\u003eUS-activated sonosensitizers facilitate e⁻/h⁺ separation, with the generated electrons not only enhancing Fenton reactions by reducing Fe\u003csup\u003e3+\u003c/sup\u003e but also suppressing e⁻/h⁺ recombination, thereby improving SDT efficiency. Specifically, Fe\u003csup\u003e2+\u003c/sup\u003e participates in the Fenton reaction to convert into Fe\u003csup\u003e3+\u003c/sup\u003e, which is subsequently reduced back to Fe\u003csup\u003e2+\u003c/sup\u003e by US-generated electrons, establishing a \u0026ldquo;Fe\u0026sup3;⁺ \u0026rarr; Fe\u0026sup2;⁺ \u0026rarr; Fe\u0026sup3;⁺\u0026rdquo; catalytic cycle (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To validate this mechanism, the generation of singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) and hydroxyl radicals (\u0026middot;OH) was assessed using electron spin resonance (ESR) spectroscopy and relevant chemical probes, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, the production of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was validated in terms of the sonodynamic effect. When MF was not subjected to US irradiation or with pure US irradiation alone, the ESR spectra showed no significant \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e production. However, upon US irradiation, the ESR spectra of MF nanosheets exhibited a substantially higher intensity of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation compared to M nanosheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, 1,3-diphenylisobenzofuran (DPBF) was used as a \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e probe to detect ROS generated under US irradiation. Briefly, DPBF was mixed with different solutions before US exposure. Comparative results revealed that pure US irradiation alone produced minimal ROS. Notably, the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation efficiency of MF was significantly higher than that of M, confirming that the incorporation of Fe\u003csup\u003e3+\u003c/sup\u003e markedly enhanced the sonodynamic effect of M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, as the US irradiation time increased, the characteristic absorption peak of DPBF at 420 nm gradually decreased, and the solution\u0026rsquo;s yellow color faded, indicating the continuous production of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eNext, the generation of \u0026middot;OH was validated in the context of the chemical fenton reaction. The ESR spectra of MF nanosheets exhibited a significantly higher intensity of \u0026middot;OH production compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Additionally, 3,3',5,5'-tetramethylbenzidine (TMB) was employed as an \u0026middot;OH probe to detect ROS generated via the chemical Fenton reaction. DI water, M, or MF was mixed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;M), and the characteristic absorption peak at 600 nm was monitored to evaluate \u0026middot;OH generation. The results showed that neither pure water nor the MF solution produced any detectable \u0026middot;OH, as evidenced by the absence of color change in the solutions. In contrast, MF generated a substantial amount of \u0026middot;OH, turning the solution into a deep blue color, thereby confirming the potent catalytic activity of MF in CDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Furthermore, as the concentration of the MF system increased, the characteristic absorption peak of TMB at 600 nm gradually intensified, and the solution's color deepened, indicating that higher MF concentrations lead to greater \u0026middot;OH production efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). To verify that US accelerates the catalytic cycle based on the Fenton reaction, \u0026middot;OH generation was quantitatively measured in systems with the same MF concentration. The results demonstrated that \u0026middot;OH production gradually increased with prolonged reaction time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Notably, the nanosheets subjected to US irradiation exhibited a more rapid and efficient \u0026middot;OH generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). These compelling results confirm that the meticulously designed sonosensitizer MF can more effectively produce ROS under US irradiation through a dual ROS generation mechanism.\u003c/p\u003e \u003cp\u003eIn addition, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed as a probe to assess the total ROS production. DCFH-DA itself is non-fluorescent but can be hydrolyzed by low concentrations of NaOH or intracellular esterases to form DCFH, which is further oxidized by ROS to generate highly fluorescent DCF. This fluorescence can be detected using a microplate reader. As shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, in the absence of US irradiation, no significant ROS production by MF was observed. However, under US irradiation, MF demonstrated concentration-dependent ROS generation. These findings indicate that MF possesses remarkable ROS generation efficiency, making it highly suitable for application in combined SDT-CDT cancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.3 In vitro anticancer activity\u003c/h2\u003e \u003cp\u003eThe first step for MFH to exert its anticancer effects is intracellular internalization. Transmission electron microscopy (TEM) observations revealed that HepG2 cells effectively internalized nanoparticles predominantly through an endocytosis mechanism. This process can be divided into four stages: (Ⅰ) recognition and binding, (Ⅱ) formation of endocytic vesicles, (Ⅲ) maturation of endosomes, and (Ⅳ) lysosomal degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). 1.5T magnetic resonance (MR) scanning demonstrated that cells exposed to varying concentrations of MF or MFH, MFH exhibited superior relaxation performance and enhanced signal intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In summary, MFH is more efficiently internalized by HepG2 cells and exhibits superior capabilities for MR imaging.\u003c/p\u003e \u003cp\u003eTo further elucidate the uptake efficiency of MFH, HepG2 cells were incubated with rhodamine b-labeled MFH or MF for varying durations, and the intracellular uptake was analyzed via confocal laser scanning microscopy (CLSM). The results demonstrated a time-dependent increase in fluorescence intensity of the rhodamine-labeled nanoparticles, indicating progressive cellular uptake. Notably, MFH exhibited significantly higher uptake efficiency compared to MF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, cells were incubated with MF or MFH using the same protocol, followed by digestion and centrifugation to collect the cells. After aqua regia digestion, Zr concentrations in each group were measured using ICP-OES. The results demonstrated cellular uptake efficiencies consistent with those observed via CLSM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eROS production was assessed using DCFH-DA as a probe, where stronger green fluorescence correlates with higher ROS levels. CLSM observations of HepG2 cells under different treatments revealed that the pure US group displayed minimal fluorescence, indicating extremely low ROS generation efficiency in the absence of a sonosensitizer. In contrast, the MFH\u0026thinsp;+\u0026thinsp;US group exhibited significantly stronger green fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Notably, groups without ultrasound irradiation showed no ROS production, indicating that MH, MF, or MFH require external US irradiation to generate ROS. Among the ultrasound-treated groups, the MH\u0026thinsp;+\u0026thinsp;US group exhibited weaker green fluorescence compared to the MFH\u0026thinsp;+\u0026thinsp;US group due to the absence of Fe(III) in MH, which are essential for the synergistic SDT-CDT enhancement effect. Similarly, the MF\u0026thinsp;+\u0026thinsp;US group showed lower fluorescence intensity than the MFH\u0026thinsp;+\u0026thinsp;US group, likely because the latter achieved higher cellular uptake, resulting in increased fluorescence intensity. Quantitative analysis of ROS production using flow cytometry (FCM) further confirmed that cells treated with MFH\u0026thinsp;+\u0026thinsp;US generated significantly higher ROS levels than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026amp;S5). These results collectively demonstrate that the MFH\u0026thinsp;+\u0026thinsp;US group possesses superior ROS generation efficiency, attributed to enhanced cellular uptake and the synergistic effect of combined CDT-SDT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eROS can induce cytotoxic effects by damaging biomolecules such as nucleic acids, proteins, and lipids. The cytotoxicity of MH, MF, and MFH against HepG2 cells was assessed using the CCK-8 assay. The results demonstrated that even after 24 hours of incubation with HepG2 cells, the prepared nanostructures did not exhibit significant cytotoxicity at high concentrations ([Zr], 200 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). On the other hand, following US irradiation, MFH group showed significant cytotoxic effects on HepG2 cells, with a 63% cell inhibition rate, which was 2.3 times higher than that of MH group (27%) at a concentration of [Zr] 200 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Further, colony formation assays were performed to evaluate the proliferative ability of the treated cells, with results showing that the colony formation of the MFH\u0026thinsp;+\u0026thinsp;US group was significantly suppressed (Fig. S6). Based on this, the effect of different treatments on HepG2 cell apoptosis was investigated. The results revealed that the apoptosis rate of HepG2 cells treated with MFH\u0026thinsp;+\u0026thinsp;US (59.7%) was 9.5 times higher than that of cells treated with PBS (6.28%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Finally, live/dead cell assays were performed on HepG2 cancer cells, showing that the percentage of dead cells (red) induced by MFH\u0026thinsp;+\u0026thinsp;US was higher compared to MH\u0026thinsp;+\u0026thinsp;US and MF\u0026thinsp;+\u0026thinsp;US groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Taken together, the above results demonstrated that MFH\u0026thinsp;+\u0026thinsp;US was effective in generating ROS, which subsequently triggered cancer cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Excessive ROS Triggering ICD Effect and DCs Maturation\u003c/h2\u003e \u003cp\u003eHigh levels of ROS are critical and indispensable for ICD, which is characterized by the release of DAMPs, such as calreticulin (CRT) exposure, high mobility group box 1 (HMGB1) secretion, and ATP release (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). These DAMPs act as \"danger signals\" that facilitate the recruitment and activation of DCs, thereby bridging innate and adaptive immune responses. Specifically, CRT is exposed on the cell surface which acts as an \u0026lsquo;eat-me\u0026rsquo; signal and ATP serves as a \u0026lsquo;find-me\u0026rsquo; signal, which together trigger the phagocytosis of dying tumor cells by DCs. Moreover, HMGB1 promotes DCs maturation and antigen presentation. These signals together induce immune responses.\u003c/p\u003e \u003cp\u003eTo investigate whether the designed nanosheets can induce ICD in cancer cells under ultrasonic irradiation, we conducted a series of experiments. First, immunofluorescence staining was performed to evaluate HMGB1 (red) in Hepa1-6 cells. The results revealed that the red fluorescence intensity in MFH\u0026thinsp;+\u0026thinsp;US-treated cells was significantly higher than in other treatment groups, indicating a greater capacity of MFH\u0026thinsp;+\u0026thinsp;US to promote HMGB1 release (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026amp;S7). Next, ATP levels were measured across different treatment groups. Hepa1-6 cells treated with MFH\u0026thinsp;+\u0026thinsp;US exhibited the highest ATP release (1.55 \u0026micro;M), which was 7.8-fold and 2.5-fold greater than those treated with PBS (0.2 \u0026micro;M) and MH\u0026thinsp;+\u0026thinsp;US (0.62 \u0026micro;M), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Third, CLSM analysis showed that CRT green fluorescence was markedly elevated in Hepa1-6 cells treated with MFH\u0026thinsp;+\u0026thinsp;US compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026amp;S8). This observation was further quantified using FCM, revealing that cells treated with MFH\u0026thinsp;+\u0026thinsp;US (3163.7) exhibited fluorescence intensities 4.7-fold and 1.2-fold higher than those treated with PBS (678.7) and MH\u0026thinsp;+\u0026thinsp;US (2717.7), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and S9). Collectively, these findings confirm that MFH\u0026thinsp;+\u0026thinsp;US generates substantial ROS, thereby triggering oxidative stress and inducing a potent ICD response in cancer cells.\u003c/p\u003e \u003cp\u003eTo further evaluate the potential of promoting the maturation of bone marrow-derived dendritic cells (BMDCs), we co-cultured treated cancer cells with BMDCs extracted from C57BL/6J mice. Flow cytometric analysis of DC maturation revealed that MFH\u0026thinsp;+\u0026thinsp;US induced the highest proportion of DC maturation (77%), which was 3.8-fold and 1.1-fold greater than that induced by MH\u0026thinsp;+\u0026thinsp;US (20%) and MF\u0026thinsp;+\u0026thinsp;US (69%), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results demonstrate that MFH\u0026thinsp;+\u0026thinsp;US not only induces a robust ICD effect but also significantly enhances DC maturation in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Biodistribution and Antitumor Effects in HepG2 Tumor-Bearing Nude Mice\u003c/h2\u003e \u003cp\u003eA subcutaneous tumor model of HepG2 liver cancer was established in BALB/c nude mice to evaluate the in vivo accumulation and biodistribution of the nanosheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Due to the ability of the nanosheets to enhance magnetic resonance signals, their biodistribution was monitored using a 9.4T magnetic resonance imaging system (Bruke 9.4T MicroMRI) following tail vein injection of MF or MFH. In vivo imaging revealed that the MR signal intensity at the tumor site progressively increased over time, reaching its peak at 12 hours post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Compared to MF group, MFH group demonstrated a higher accumulation at the tumor site, resulting in a significantly stronger magnetic resonance signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This enhanced accumulation can be attributed to the HA coating, which improved tumor uptake of the nanosheets.\u003c/p\u003e \u003cp\u003eTo evaluate the antitumor efficacy of the treatment regimens, nude mice with subcutaneous HepG2 tumors were treated and monitored. To achieve satisfactory tumor elimination, treatments were administered every two days for a total of three cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The body weights and tumor volumes for all groups were recorded every two days. No significant changes in body weight were observed in any group, indicating the low systemic toxicity of the nanomedicine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In addition, vigorous tumor growth inhibition was observed in mice treated with MFH\u0026thinsp;+\u0026thinsp;US group, with a mean tumor volume of 22.3 mm\u003csup\u003e3\u003c/sup\u003e at day 15, compared to 325.4 mm\u0026sup3; in MH\u0026thinsp;+\u0026thinsp;US group and 861.2 mm\u0026sup3; in PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F\u0026amp;S10). Additionally, the average tumor weight in PBS group up to 520 mg, which was 21 times higher than that of mice treated with MFH\u0026thinsp;+\u0026thinsp;US group (25mg) (Fig. S11). Histological analyses were conducted to assess tumor necrosis, apoptosis, and proliferative activity in tumor tissues using H\u0026amp;E staining, TUNEL staining, and Ki-67 staining, respectively. The H\u0026amp;E staining and TUNEL staining results showed extensive tumor necrosis and apoptosis in the MFH\u0026thinsp;+\u0026thinsp;US group. Ki-67 staining revealed that the MFH\u0026thinsp;+\u0026thinsp;US group had the lowest proportion of proliferating cells compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These results demonstrate that MFH\u0026thinsp;+\u0026thinsp;US group effectively suppresses tumor growth by inducing significant necrosis and reducing cellular proliferation in tumors.\u003c/p\u003e \u003cp\u003eTo evaluate the biosafety of all treatments, blood routine tests, biochemical analyses, and organ histology were conducted for all mice. As shown in Fig. S12, the hemolysis rate of MFH at various concentrations was below 4%, indicating that the nanosheets did not cause hemolysis. Blood routine and biochemical parameter analyses further confirmed that all groups exhibited blood values within the normal range (Fig. S13\u0026amp;S14). Additionally, H\u0026amp;E staining of the major organs (heart, liver, spleen, lung and kidney) showed no noticeable pathological changes or toxicity associated with the prepared nanostructures (Fig. S15).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that MFH\u0026thinsp;+\u0026thinsp;US group not only exhibits excellent biosafety but also provides strong magnetic resonance imaging signals and exceptional tumor growth inhibition capabilities, making it a promising candidate for effective and safe cancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Augmentation of SDT-induced anti-tumor immunity through ICD mechanism\u003c/h2\u003e \u003cp\u003eTo gain deeper insights into the mechanism by which MFH-enhanced SDT combats tumors through immunogenic cell death (ICD) induction by amplifying immunogenicity and alleviating the immunosuppressive microenvironment (ISM), relevant immune markers were further monitored. A subcutaneous tumor model of Hepa1-6 was established to evaluate tumor growth dynamics and immune profiles. When tumor volumes reached 60\u0026ndash;80 mm\u0026sup3;, mice were randomized into four groups (n\u0026thinsp;=\u0026thinsp;3 per group) and treated with PBS, MFH, PBS\u0026thinsp;+\u0026thinsp;US, or MFH\u0026thinsp;+\u0026thinsp;US, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe results demonstrated rapid tumor growth in PBS and MFH treated groups. Strikingly, tumors in the MFH\u0026thinsp;+\u0026thinsp;US group exhibited nearly complete suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C\u0026amp;S16). No significant body weight differences were observed across groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). To analyze DC maturation in tumors and lymph nodes, mice were euthanized on day 16 for tumor immune profiling via flow cytometry. The results demonstrated that compared to the PBS group, the MFH\u0026thinsp;+\u0026thinsp;US group exhibited increased proportions of mature DCs in tumors and lymph nodes from 3.57\u0026ndash;10.3% and 14.5\u0026ndash;31.6%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F\u0026amp;S17), indicating that MFH\u0026thinsp;+\u0026thinsp;US possessed the strongest capacity to activate T cells and enhance CD8\u0026thinsp;+\u0026thinsp;T cell infiltration. As shown in the figure G, H, MFH\u0026thinsp;+\u0026thinsp;US-treated mice displayed the highest proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumors (15%), representing a 1.82-fold increase over PBS\u0026thinsp;+\u0026thinsp;US-treated mice (8.23%). Notably, the observed clonal expansion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumor-draining lymph nodes (TDLNs) suggests systemic immune activation, potentially driving the transition from immunologically 'cold' to 'hot' tumor microenvironment (Fig. S18). This lymphoid compartment priming may facilitate subsequent T cell trafficking to tumor sites, as evidenced by enhanced intratumoral CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration - a hallmark feature of hot tumors.\u003c/p\u003e \u003cp\u003eIn summary, MFH induces immunogenic cell death (ICD) effects via ultrasound-induced high-level ROS generation, thereby potentiating the sonodynamic immunotherapy efficacy. This process encompasses CRT exposure, HMGB1 and ATP release, promotion of DC maturation, activation of CD8⁺ T cells, thereby facilitating antitumor immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, we have developed a metal-organic nanostructure (MFH) based on Ti\u003csub\u003ex\u003c/sub\u003eO\u003csub\u003ey\u003c/sub\u003e/Ru catalytic units. The synthesized MFH exhibits the following distinctive features: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) TiZr-O clusters and Ru-COOH form a uniform and stable metal-organic nanosheet (TiZrRu-MON, M) through metal ion coordination. We demonstrate that the clusters transfer ultrasound-triggered energy to Ru-COOH, subsequently generating ROS; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Fe\u003csup\u003e3+\u003c/sup\u003e is incorporated into the M framework (denoted as MF) via chelation. Experimental results confirm that MF significantly enhances ROS production under US, indicating that Fe\u003csup\u003e3+\u003c/sup\u003e doping promotes SDT efficiency. Furthermore, MF generates Fenton-based \u0026middot;OH via H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activation, and US irradiation accelerates the CDT process. This synergy between SDT and CDT induces potent oxidative stress, establishing a critical foundation for efficient cancer therapy; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) A HA-based \"nanocoating\" enables tumor-targeted accumulation of nanosheets by binding to CD44 receptors overexpressed on cancer cells, thereby enhancing in vivo retention and improving MR imaging performance; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) MFH combined with US irradiation triggers ICD in tumor cells, promotes DCs maturation, and recruits cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells into tumors and lymph nodes through antigen-presenting mechanisms, thereby activating robust antitumor immunity. Collectively, our findings validate MFH as a promising sonosensitizer capable of addressing current clinical limitations of SDT. However, further studies are required to optimize its therapeutic window for deep-seated tumor treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTao Jiang, Zixiang Tang and Shumiao Tian contributed to this work equally. Tao Jiang, Zixiang Tang and Shumiao Tian designed experiments and wrote the manuscript. Tao Jiang, Zixiang Tang, Shumiao Tian, Haitian Tang, Zhekun Jia, Fangjian Li, Chenyue Qiu, Lin Deng, Lang Ke, Pan He conducted all experiments and related analysis. Yongfu Xiong, Chengchao Chu and Gang Liu revised the manuscript and supervised this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Sichuan Provincial Health Commission special research project (2024HR03), Sichuan Science and Technology Department Science and Technology Ability Improvement Project (2024JDKP0022), Nanchong Science and Technology Bureau Basic Research Platform Project (23JCYJPT0043), National Natural Science Foundation of China (82302403).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll the animals were raised at Animal Care and Use Committee of Xiamen University (Ethics number XMULAC20190146).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePublisher\u0026rsquo;s note\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGang Liu, Email: [email protected]\u003c/p\u003e\n\u003cp\u003eChengchao Chu: [email protected]\u003c/p\u003e\n\u003cp\u003eYongfu Xiong: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. 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Small (Weinheim an der Bergstrasse, Germany). 2023;19(48):2304032.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1-2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Metal-organic nanostructure, Sonodynamic therapy, Chemodynamic therapy, Magnetic resonance imaging, cancer therapy","lastPublishedDoi":"10.21203/rs.3.rs-6273421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6273421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSonodynamic therapy (SDT) has demonstrated significant clinical potential in malignant tumor treatment due to its deep tissue penetration and spatiotemporal controllability. Its core mechanism relies on ultrasound-activated sonosensitizers to generate reactive oxygen species (ROS), thereby inducing tumor cell apoptosis. However, conventional sonosensitizers face limitations in ROS yield and tumor-targeting efficiency. In this study, we innovatively designed a multifunctional metal-organic nanosheet (TiZrRu-MON) by hydrothermal coordination of [Ru(bpy)\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e2+\u003c/sup\u003e photosensitizing units with TiZr-O clusters, while incorporating Fe\u003csup\u003e3+\u003c/sup\u003e to construct a cascade catalytic system. Experimental results demonstrated that: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Fe\u003csup\u003e3+\u003c/sup\u003e lattice doping significantly enhanced charge carrier mobility and ultrasound-triggered \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e quantum yield via the formation charge transfer channels; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The acidic tumor microenvironment activated Fe\u003csup\u003e3+\u003c/sup\u003e-mediated Fenton reactions, establishing a positive feedback loop with SDT to synergistically amplify ROS generation; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Hyaluronic acid functionalization improved nanosheet internalization in HepG2 tumor cells through CD44 receptor-mediated endocytosis. Remarkably, ultrasound irradiation induced substantial oxidative stress and immunogenic cell death, promoting the release of damage-associated molecular patterns (DAMPs), which elevated the maturation rate of tumor-infiltrating dendritic cells (DCs) and significantly increased the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells. In a mouse subcutaneous tumor model, the system achieved effective tumor suppression with manageable systemic toxicity. This work proposes a metal-ligand coordination strategy to advance the development of high-performance sonosensitizers and immunomodulatory antitumor technologies.\u003c/p\u003e","manuscriptTitle":"Metal-organic nanostructures based on sono/chemo-nanodynamic synergy of TixOy/Ru reaction units: for ultrasound-induced dynamic cancer therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 11:12:12","doi":"10.21203/rs.3.rs-6273421/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-16T16:04:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-16T13:31:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T15:55:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-13T02:34:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T18:53:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49204516312164306564787000691041172214","date":"2025-04-07T09:14:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8351456359477670172650967161263986696","date":"2025-04-05T02:06:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21293874901210329536725843135607739497","date":"2025-04-04T01:46:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272958785378026338865867580048735282375","date":"2025-04-03T17:37:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T16:33:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-31T09:31:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T13:04:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-03-21T02:38:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bdbb3e21-2baa-426b-bc0c-3b3cc6fd3431","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:02:07+00:00","versionOfRecord":{"articleIdentity":"rs-6273421","link":"https://doi.org/10.1186/s12951-025-03599-1","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-07-21 15:57:48","publishedOnDateReadable":"July 21st, 2025"},"versionCreatedAt":"2025-04-25 11:12:12","video":"","vorDoi":"10.1186/s12951-025-03599-1","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03599-1","workflowStages":[]},"version":"v1","identity":"rs-6273421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6273421","identity":"rs-6273421","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}