Monatomic iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment as well as preparation method and application of monatomic iron-doped molybdenum disulfide piezoelectric catalytic material

Abstract

The invention relates to a monatomic iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment and a preparation method and application thereof.The piezoelectric catalytic material is Fe-MoS2 in a two-dimensional form, MoS2 in the two-dimensional form serves as a carrier of the piezoelectric catalytic material, monatomic iron is doped on MoS2, the doping amount of the monatomic iron is 0.35-0.55 at%, the doping amount of the MoS2 is 0.35-0.55 at%, the doping amount of the MoS2 is 0.35-0.55 at%, and the doping amount of the MoS2 is 0.35-0.55 at%. The preparation method comprises the following steps: S1, synthesizing MoS2 through a hydrothermal method; s2, doping monatomic iron on MoS2 through a dip-coating and calcining method, and synthesizing Fe-MoS2. Compared with the prior art, copper death is started through a piezoelectric and multienzyme combined therapy and mediation of a copper-free piezoelectric catalytic material, so that the tumor treatment effect is improved, in addition, when the copper-free piezoelectric catalytic material is used as a tumor treatment drug, the drug distribution is wider, the copper-free piezoelectric catalytic material can adapt to treatment of various tumors, and the application prospect is wide. The selectivity of various medicines is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly to a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, its preparation method and application. Background Art

[0002] Copper (Cu) is an essential trace element in human physiological processes and is crucial for a wide range of biochemical reactions. Imbalance of copper homeostasis can lead to cell damage and trigger copper death. Copper death, a unique form of copper-dependent programmed cell death, was first described in 2022. Different from traditional cell death pathways such as apoptosis, necrosis, and ferroptosis, copper death is characterized by abnormal accumulation of intracellular copper ions and disruption of key metabolic enzymes. In particular, excessive accumulation of copper ions and association with the dihydrolipoamide acetyltransferase (DLAT) subunit in the pyruvate dehydrogenase complex (PDH) lead to aggregation of iron-sulfur (Fe-S) cluster proteins and subsequent loss of function. This disruption causes metabolic disorders and activates stress responses, ultimately resulting in programmed cell death. During this process, ferredoxin 1 (FDX1) promotes DLAT lipoylation by reducing Cu 2+ to Cu + and downregulates the level of Fe-S cluster proteins by directly interacting with lipoic acid synthase (LIAS), ultimately inducing cell death. ATP7B, a key copper transporter responsible for removing copper ions from cells through ATP hydrolysis, is crucial for maintaining intracellular copper homeostasis. Dysfunction of ATP7B leads to copper accumulation, homeostasis disorder, and initiation of copper death. During tumor progression, copper death is usually inhibited by enhanced ATP7B expression, antioxidant defense systems, and reprogrammed metabolic microenvironments. Therefore, activating copper death by increasing intracellular copper accumulation, inhibiting the function of ATP7B, disrupting antioxidant defense systems, and accelerating the metabolic pathways of tumor cells is a promising strategy for inhibiting tumor growth.

[0003] Nanotechnology has developed rapidly and made remarkable breakthroughs in various disciplines, especially in the biomedical field. The unique physicochemical properties of nanomaterials, including size effect, quantum confinement, surface reactivity, and dielectric properties, have made nanomedicine a highly adaptable method for disease diagnosis, treatment, prognosis, monitoring, and prevention. Significant progress has been made in using nanomaterials to trigger copper death for cancer treatment. For example, Zhang et al. developed a copper death-mediated immunotherapy nanoreactor by integrating CaO encapsulated with a copper-based shell in a DSPE-PEG-FA matrix 2 and JQ-1 (a bromodomain protein 4 inhibitor). The resulting Cu + inhibits intracellular glycolysis and ATP production, while blocking Cu +Exportin ATP7B, thereby enhancing the sensitivity of cells to cuproptosis. Similarly, Lin et al. developed ferroelectric Bi 2 CuO 4 nanoparticles with abundant copper vacancies, which induce cuproptosis and trigger a strong immune response by blocking the Cu + efflux pathway. Currently, the nanosystems for inducing cuproptosis mainly use copper-based nanomaterials to deliver exogenous toxic copper ions into cells, but this limits the application of biomaterials in cuproptosis-mediated therapy. In addition, the existing designs of copper-based nanomaterials cannot effectively promote the aggregation of copper ions, which will be rapidly excreted from cells by specific copper ion transporters, resulting in an unsatisfactory copper conversion efficiency.

[0004] Therefore, developing a copper-free nanoplatform to initiate cuproptosis is crucial for enhancing the application of nanotechnology in cuproptosis-mediated disease treatment to improve the therapeutic effect and biosafety. Summary of the Invention

[0005] The purpose of the present invention is to provide a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, its preparation method and application, which initiate cuproptosis by preparing a copper-free piezoelectric catalytic material and improve the tumor treatment effect.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] On the one hand, the present invention provides a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, and the piezoelectric catalytic material is two-dimensional Fe-MoS 2 , and the piezoelectric catalytic material uses two-dimensional MoS 2 as a carrier, and single-atom iron is doped on the MoS 2 .

[0008] Preferably, each single-atom iron is doped into MoS 2 in the form of coordination with 3.2 ± 0.3 sulfur atoms.

[0009] Preferably, the doping amount of the single-atom iron is 0.35 - 0.55 at%.

[0010] More preferably, the doping amount of the single-atom iron is 0.45 at%.

[0011] In the present invention, the doping amount of single-atom iron is relatively low, which can further reduce the biological toxicity of the material in vivo.

[0012] Preferably, the MoS 2 comprises 1 - 7 odd atomic layers, and the layer spacing is 0.65 - 0.68 nm, and the Fe-MoS 2It contains 1 to 7 odd atomic layers with an interlayer spacing of 0.68 to 0.70 nm.

[0013] Preferably, the two-dimensional MoS 2 and Fe-MoS 2 are in the form of nanosheet structures.

[0014] Preferably, the particle size of the MoS 2 is 284.1 ± 8.4 nm, and the particle size of the Fe-MoS 2 is 298.2 ± 11.0 nm.

[0015] Preferably, the surface of the Fe-MoS 2 is further modified with PEG (polyethylene glycol) to improve its water solubility for intravenous drug injection and to enhance its physiological stability and biocompatibility.

[0016] Preferably, the molecular weight of the PEG is 500 to 600.

[0017] More preferably, the molecular weight of the PEG is 544.7.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, comprising the following steps:

[0019] S1: Synthesize MoS 2 by hydrothermal method;

[0020] S2: Dope single-atom iron on MoS 2 by dip coating and calcination methods to synthesize Fe-MoS 2 .

[0021] Preferably, step S1 specifically includes the following steps: Dissolve sodium molybdate and thiourea in ultrapure water, adjust the pH and then stir, followed by hydrothermal reaction. After the reaction is completed, cool, centrifuge, wash, and dry to obtain the MoS 2 .

[0022] Preferably, in step S1, the molar ratio of sodium molybdate to thiourea is 1:(4.5 - 6), more preferably 1:5.

[0023] Preferably, in step S1, the adjustment of pH means adjusting the pH value to below 1 with hydrochloric acid.

[0024] Preferably, in step S1, the stirring time is 90 - 150 minutes, more preferably 120 minutes.

[0025] Preferably, in step S1, the temperature of the hydrothermal reaction is 180 - 220 °C and the time is 20 - 28 hours.

[0026] Further preferably, in step S1, the temperature of the hydrothermal reaction is 200 °C and the time is 24 hours.

[0027] Further preferably, in step S1, the hydrothermal reaction is carried out in a stainless steel autoclave with a polytetrafluoroethylene lining.

[0028] Preferably, in step S1, the drying temperature is 50 - 70 °C and the time is 10 - 14 hours.

[0029] Further preferably, in step S1, the drying temperature is 60 °C and the time is 12 hours.

[0030] Preferably, in step S1, the washing refers to washing with ultrapure water and ethanol.

[0031] Preferably, step S2 specifically includes the following steps: Immerse the MoS prepared in step S1 2 into FeCl 3 solution, adjust the pH and then stir, centrifuge to obtain a precipitate, and calcine the precipitate to obtain the Fe-MoS 2 .

[0032] Preferably, in step S2, the solvent of the FeCl 3 solution is a mixed solution of water and ethanol, and the ratio of water to ethanol is 1:(0.8 - 1.2).

[0033] Preferably, in step S2, in the mixed solution obtained after the MoS 2 is immersed into the FeCl 3 solution, the molar concentration of MoS 2 :FeCl 3 is 1:(1 - 3), and further preferably 1:2.

[0034] Further preferably, in step S2, the ratio of water to ethanol is 1:1.

[0035] Further preferably, in step S2, the water is ultrapure water.

[0036] Preferably, in step S2, the adjusting of the pH refers to adjusting the pH value to 0.8 - 1.2 with hydrochloric acid, and further preferably adjusting the pH value to 1.

[0037] Preferably, in step S2, the stirring is carried out at 50 - 90 °C for 90 - 150 minutes, and further preferably at 70 °C for 120 minutes.

[0038] Preferably, in step S2, the calcination is carried out in an Ar / H 2 atmosphere, and the Ar and H2 The flow volume ratio is (92 - 98)∶(2 - 8), the calcination temperature is 650 - 750 °C, and the time is 90 - 150 minutes.

[0039] Further preferably, in step S2, the flow volume ratio of Ar and H 2 is 95∶5, the calcination temperature is 700 °C, and the time is 120 minutes.

[0040] Further preferably, in step S2, the calcination is carried out in a tube furnace.

[0041] Thirdly, the present invention also provides an application of the above-mentioned single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment in the preparation of tumor treatment drugs.

[0042] Preferably, the single-atom iron-doped molybdenum disulfide piezoelectric catalytic material mediates cuproptosis of a copper-free piezoelectric catalytic material through piezoelectric and multi-enzyme combination therapy for tumor treatment.

[0043] Further preferably, when the single-atom iron-doped molybdenum disulfide piezoelectric catalytic material is used in tumor treatment drugs, the administration method is intravenous administration.

[0044] In the present invention, when used as a tumor treatment drug, the administration method is intravenous administration. Intravenous administration can make the drug distribution more extensive. Intravenous administration transports the drug to all parts of the body through the blood, ensuring that the drug can reach tumor cells throughout the body, especially those that have metastasized. This is particularly important for the treatment of multiple tumors or metastatic tumors; intravenous administration can be adapted to various tumor treatments. Intravenous administration is applicable to the treatment of various types of cancers, including chemotherapy, targeted therapy, immunotherapy, etc. This method can be used for various types of tumors, whether primary tumors or metastatic tumors; intravenous administration has multiple drug selectivities. Many chemotherapy drugs, targeted drugs, and immune checkpoint inhibitors are administered through intravenous injection. This method provides multiple drug options, and doctors can select the most suitable drug and dosage according to the specific situation of the patient.

[0045] In the present invention, a two-dimensional (2D) single-atom iron-doped molybdenum disulfide (Fe-MoS 2 ) piezoelectric catalytic material ( Figure 1 A) is constructed. This piezoelectric catalytic material has multifunctional catalytic activity. In addition to excellent piezoelectric catalytic performance, it also exhibits various enzyme activities, including peroxidase (POD), glutathione oxidase (GSHOx), oxidase (OXD), and catalase (CAT) - like activities. The present invention integrates piezoelectric catalysis and enzyme-like effects and can achieve effective tumor treatment through a copper-free-dependent cuproptosis pathway.

[0046] Piezoelectrocatalytic therapy is an emerging treatment method that utilizes the piezoelectric effect of materials to drive chemical reactions and is increasingly being applied in disease treatment. Piezoelectric materials generate piezopotentials under mechanical stimulation, which helps with efficient charge carrier separation and promotes directional redox reactions. This piezoelectrocatalytic effect improves the precision of drug delivery, enhances antibacterial activity, and improves treatment outcomes. Compared with traditional therapies, piezoelectrocatalytic therapy releases charge carriers through controlled stimuli (such as ultrasound), triggering redox reactions in matrices (such as water and oxygen) to generate a large number of reactive oxygen species (ROS), and is widely used in fields such as tumor elimination, organic pollutant degradation, and sterilization.

[0047] In the present invention, by doping single-atom iron, it is possible to promote charge redistribution and enhance the piezoelectric polarization of MoS 2 ( Figure 1 B). Under ultrasonic (US) irradiation, the periodic vibration continuously separates electron-hole pairs, establishing a strong built-in electric field and surface charges, and catalyzing the generation of ROS. At the same time, single-atom iron doping changes the oxidation states of Fe and Mo, thereby enhancing the multi-enzyme catalytic process similar to enzymes, significantly altering the hypoxic tumor microenvironment and reducing the level of endogenous glutathione (GSH), thus improving the anti-tumor effect. The Fe-MoS 2 nano-catalytic material combines the characteristics of piezoelectrocatalysis and multi-enzyme catalysis similar to enzymes, resulting in a large amount of ROS generation and significant GSH depletion ( Figure 1 C). The increased ROS level leads to mitochondrial damage, reduces the intracellular ATP concentration, and impairs the activity of ATP7B, hindering the excretion of copper ions from tumor cells. In addition, the reduced GSH level promotes the accumulation of intracellular copper ions, leading to the aggregation of DLAT and the downregulation of Fe-S cluster proteins. These biological changes cause metabolic disorders and cellular stress, ultimately resulting in cuproptosis ( Figure 1 D). In addition, the disrupted redox homeostasis can trigger ferroptosis and ferritin autophagy ( Figure 1 E and F), which can act synergistically with cuproptosis to jointly inhibit tumor growth.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The present invention provides a single-atom iron-doped molybdenum disulfide piezoelectrocatalytic material (Fe-MoS 2 ) for tumor treatment, its preparation method and application. Through the combined therapy of piezoelectrocatalysis and multi-enzyme, it mediates the initiation of cuproptosis by a copper-free piezoelectrocatalytic material, thereby improving the tumor treatment effect.

[0050] (2) The present invention synthesizes two-dimensional molybdenum disulfide by a hydrothermal synthesis method and incorporates single-atom iron by a dip-coating and calcination method. The prepared Fe-MoS 2, wherein the doping amount of single-atom iron is only 0.35 - 0.55 at%, which can greatly reduce the biotoxicity of the material in the body, and the surface is modified with PEG, which can greatly improve the water solubility and biocompatibility of the drug, so that intravenous injection of the drug can be adopted.

[0051] (3) The Fe-MoS of the present invention 2 The piezoelectric catalytic material has both piezoelectric catalytic performance and multiple enzyme activities. Fe-MoS 2 Generates a large amount of reactive oxygen species (ROS) and depletes glutathione (GSH) through the synergistic piezoelectric catalysis and enzyme kinetic effects, thereby disrupting the homeostasis of copper ions in tumor cells and triggering cuproptosis, thus inhibiting tumor growth.

[0052] (4) The Fe-MoS of the present invention 2 The piezoelectric catalytic material is a copper-free nanomaterial. Compared with copper-based nanomaterials, the present invention can effectively promote the aggregation of copper ions in cells without providing exogenous copper ions, enhance the application of nanotechnology in the treatment of diseases mediated by cuproptosis, and improve the treatment effect and biosafety.

[0053] (5) When used as a tumor treatment drug, the administration method of the present invention is intravenous administration. Intravenous administration can make the drug distribute more widely, can be adapted to various tumor treatments, and has multiple drug selectivities. Brief Description of the Drawings

[0054] Figure 1 Schematic diagram of the use of single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment by cuproptosis ((A) MoS 2 and Fe-MoS 2 structures. (B) Scheme showing the piezoelectric catalytic mechanism and multi-enzyme-like activity of Fe-MoS 2 ; (C) Schematic diagram of ROS generation and GSH depletion; Fe-MoS 2 Induces tumor cell death synergistically through three different biological mechanisms, including (D) cuproptosis, (E) ferritin autophagy, and (F) ferroptosis).

[0055] Figure 2 For the synthesis and characterization results of Fe-MoS 2 ((A) Schematic diagram of the synthesis process of Fe-MoS 2 ; (B) Chemical structures of MoS 2 and Fe-MoS 2 ; (C) Transmission electron microscope (TEM) image of Fe-MoS 2 ; (D) High-resolution transmission electron microscope (HRTEM) image of Fe-MoS 2 ; (E) Fe-MoS 2Scanning transmission electron microscope (STEM) image; (F) Fe-MoS 2 Energy-dispersive X-ray spectroscopy (EDX) mapping image; (G) MoS 2 and Fe-MoS 2 X-ray diffraction (XRD) pattern; (H) MoS 2 and Fe-MoS 2 Raman spectrum; Fe-MoS 2 and MoS 2 Mo 3d (I) and S 2p (J) X-ray photoelectron spectroscopy (XPS) spectra; (K) Fe-MoS 2 Aberration-corrected transmission electron microscope (TEM) image, red circles indicate obvious defects, and yellow circles indicate spatially isolated Fe atoms; Fe-MoS 2 、Fe 2 O 3 Fe-K edge normalized X-ray absorption near-edge structure (XANES) spectra (L) and Fourier transform amplitude spectra of extended X-ray absorption fine structure (EXAFS) in R space (M) of Fe-MoS 2 EXAFS fitting results of Fe-MoS in k space; Fe-MoS 2 (O), wavelet transform spectra of Fe K-edge EXAFS of iron foil (P), FeS (Q) and Fe2O3 (R).

[0056] Figure 3 For the piezocatalytic performance and multi-enzyme-like activity of Fe-MoS 2 ((A) Time dependence reflected by the decrease in peak absorbance of DPBF at 415 nm 1 O 2 generation; (B) ESR spectrum of 1 O 2 captured by TEMP; (C) Schematic diagram showing multi-enzyme-like ROS generation activity of Fe-MoS 2 ; (D) Influence of Fe-MoS 2 on oxygen generation in the evaluation of CAT-like activity; (E) OXD-like activity at different concentrations of Fe-MoS 2 ; (F) ESR spectrum of O 2 ·- captured by DMPO; POD-like activity of Fe-MoS 2 at different treatments (G) and material concentrations (H); (I) POD-like activity of Fe-MoS 2 at different pH levels; (J) Enhanced POD-like activity of Fe-MoS 2 under ultrasonic irradiation; (K) ESR spectrum of ·OH captured by DMPO; (L) Fe-MoS at different concentrations2 GSHOx-like activity; (M) Fe-MoS under ultrasonic irradiation 2 Enhanced GSHOx-mimicking properties; (N) MoS 2 and Fe-MoS 2 band gap; MoS 2 (O) and Fe-MoS 2 (P) Mott-Schottky plots; (Q) Schematic diagram of band tilting for strain application and accompanying redox reactions under ultrasound-driven pressure; (R) MoS 2 and Fe-MoS 2 EIS plots; (S) MoS detected by time-resolved photoluminescence spectroscopy 2 and Fe-MoS 2 (decay time plots).

[0057] Figure 4 The in vitro antitumor therapeutic effect achieved by the synergistic action of piezocatalysis and enzyme catalysis of Fe-MoS 2 ((A) Bio-TEM images of B16-F10 cells after co-incubation with Fe-MoS 2 (80 μg / mL) for 0, 6, and 12 hours; (B) Relative cell viability of HUVEC and B16-F10 cells after incubation with different concentrations of Fe-MoS 2 for 24 hours (n = 4); (C) Heatmap showing the relative survival rate of B16-F10 cells after treatment with different treatments and different concentrations of Fe-MoS 2 (n = 4); (D) Relative cell viability of B16-F10 cells after different treatments (n = 4), where the data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; (E) Confocal microscopy (CLSM) images of B16-F10 cells stained with calcein acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) after various treatments; (F) Intracellular ROS levels in B16-F10 cells after different treatments; (G) Flow cytometry analysis of intracellular ROS levels in B16-F10 cells after various treatments; (H) Flow cytometry analysis of apoptosis rate of B16-F10 cells stained with Annexin-FITC and PI after different treatments; (I) Microscopic observation of the migration ability of B16-F10 cells after various treatments).

[0058] Figure 5 Copper death induced by copper-free Fe-MoS 2 piezocatalyst ((A) Control group and Fe-MoS 2 +US+H2 O 2 Volcano plot of differentially expressed genes in the group; (B) Principal component analysis of the control group and Fe-MoS 2 +US+H 2 O 2 group; (C) GSEA shows the oxidative stress response pathway related to DEGs; (D) Circular heat map shows DEGs related to the oxidative stress response; (E) Circular diagram of GO enrichment analysis; (F) Heat map shows the differential expression of genes of interest (left) and the pathways related to cuproptosis (right); (G) Heat map shows DEGs related to the cuproptosis pathway; (H) Schematic diagram shows the biological mechanism of cuproptosis induced by copper-free Fe-MoS 2 piezoelectric catalyst).

[0059] Figure 6 For Fe-MoS 2 In vivo anticancer performance of piezoelectric catalyst ((A) Schematic diagram of the in vivo treatment protocol; Tumor-bearing mice were intravenously injected with Fe-MoS 2 After 2 hours (B) and 24 hours (C), the distribution of Fe-MoS 2 in the main organs and tumors (n = 3); (D) Changes in body weight of mice in different groups (n = 5); (E) Tumor volume growth curve of mice receiving the designated treatment (n = 5); (F) Average weight of tumors harvested from tumor-bearing mice in different groups after 12 days of treatment (n = 5); (G) Tumor inhibition rate of different groups after treatment (n = 5); (H to K) Tumor volume growth curve of each mouse during treatment. (L) Copper ion content in tumor tissues of the control group and Fe-MoS 2 +US group; (M and N) Histological images of tumor sections of tumor-bearing mice collected after 12 days stained with H&E, TUNEL, Ki-67, DCFH-DA, DLAT, FDX1, NCOA4, and GPX4. The data in (E, F, G, and L) are expressed as mean ± standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0060] Figure 7 For MoS 2 Characterization ((A) TEM image of MoS 2 ; (B) HRTEM image of MoS 2 ; (C) Energy-dispersive X-ray spectroscopy (EDX) map of MoS 2 ).

[0061] Figure 8 For MoS 2 and Fe-MoS 2 ESR spectra.

[0062] Figure 9 For the amplitude image and phase image ((A) MoS 2 and Fe-MoS 2 magnitude-voltage curve and (B) phase lag loop).

[0063] Figure 10 For the UV-visible spectra of DPBF solutions containing deionized water (A), MoS 2 (B), and Fe-MoS 2 (C) under US irradiation for different durations.

[0064] Figure 11 For the photocurrent curves of MoS 2 and Fe-MoS 2 ;

[0065] Figure 12 For the particle size distribution diagrams of (A) MoS 2 and Fe-MoS 2 ; (B) size distribution diagram of Fe-MoS 2 -PEG; (C) Zeta potential diagrams of MoS 2 , Fe-MoS 2 and Fe-MoS 2 -PEG.

[0066] Figure 13 Cell viability diagram under ultrasonic stimulation (cell viability of B16-F10 co-cultured with Fe-MoS 2 (80 μg / mL) at different ultrasonic frequencies (A) and ultrasonic durations (B)).

[0067] Figure 14 For the heatmap of DEGs obtained for the control group and the Fe-MoS 2 +US+H 2 O 2 group (n = 3).

[0068] Figure 15 For the complete blood cell count and blood biochemical indices of ICR mice collected on the 28th day after treatment with PBS and Fe-MoS 2 at different concentrations (5 mg / mL, 10 mg / mL, 20 mg / mL).

[0069] Figure 16 For the H&E staining images of major organs (heart, liver, spleen, lung, and kidney) after treatment with different concentrations of Fe-MoS 2 .

[0070] Figure 17 For Fe-MoS 2In vivo biodistribution map (biodistribution of Mo in major organs and tumors of tumor-bearing mice 6 hours (A) and 12 hours (B) after intravenous injection of Fe-MoS2).

[0071] Figure 18 H&E staining images of major organs (heart, liver, spleen, lung, and kidney) after different treatments.

[0072] Figure 19 Tumor photos of tumor-bearing nude mice in different groups harvested after 12 days of treatment (n = 5).

[0073] Figure 20 For Fe-MoS 2 Confocal laser scanning microscopy (CLSM) photos. Detailed implementation mode

[0074] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0075] Unless otherwise specified, the reagents, methods, instruments, and equipment used in the present invention are conventional reagents, methods, instruments, and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0076] Example 1

[0077] A single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, as Figure 2 shown in A, and its preparation process is as follows:

[0078] (1) Synthesize MoS 2 (molybdenum disulfide) by hydrothermal method:

[0079] a. Dissolve Na 2 MoO 4 ·2H 2 O (sodium molybdate) and CH 4 N 2 S (thiourea) in ultrapure water at a molar ratio of 1:5.

[0080] b. Adjust the pH value of the solution to below 1 with HCl (hydrochloric acid), and then stir for 120 minutes.

[0081] c. Transfer the resulting mixture to a stainless steel autoclave lined with polytetrafluoroethylene and heat at 200 °C for 24 hours.

[0082] d. After cooling to room temperature, collect the precipitate by centrifugation, wash it several times with deionized water and ethanol, and dry it at 60 °C for 12 hours.

[0083] (2) Synthesize Fe-MoS by dip coating and calcination 2 Composite material:

[0084] a. Immerse the synthesized MoS above 2 into the FeCl 3 solution (the solvent is a water-ethanol mixture with a volume ratio of 1:1). The molar concentration of MoS 2 :FeCl 3 is 1:2. Adjust the pH to 1 with HCI and stir at 70 °C for 2 hours.

[0085] b. Centrifuge to separate the precipitate and calcine it in a tubular furnace at 700 °C for 2 hours under an Ar / H 2 (95:5) atmosphere to produce the Fe-MoS 2 piezoelectrocatalytic material.

[0086] 1. Conduct structural characterization on the Fe-MoS 2 piezoelectrocatalytic material prepared in Example 1 above

[0087] Transmission electron microscope (TEM) images show that both MoS 2 and Fe-MoS 2 exhibit typical two-dimensional (2D) piezoelectrocatalyst morphologies ( Figure 2 C and Figure 7 A). High-resolution TEM (HRTEM) images further confirm that both MoS 2 and Fe-MoS 2 contain one to seven odd atomic layers, and the interlayer spacings are approximately 0.68 nm and 0.70 nm respectively ( Figure 2 D and Figure 7 B). Scanning transmission electron microscope (STEM) combined with energy-dispersive X-ray spectroscopy (EDX) imaging shows that the Mo and S elements in MoS 2 and Fe-MoS 2 are evenly distributed, and Fe is successfully doped into MoS 2 ( Figure 2 E, F and Figure 7 C). Determined by inductively coupled plasma optical emission spectrometry (ICP-OES), the single-atom Fe doping amount is 0.45 at%. The X-ray diffraction (XRD) patterns of MoS 2 and Fe-MoS 2 show very little difference, indicating that the incorporation of single-atom Fe causes negligible structural changes ( Figure 2 G). In the Raman spectra of MoS 2 and Fe-MoS 2 as shown in Figure 2As shown in H, two characteristic peaks are located at 375 and 402 cm -1 , corresponding to the E1 2 g and A1g modes of 2H MoS 2 . In addition, after single-atom Fe doping, both peaks shift by 1 cm -1 , indicating that due to the incorporation of single-atom Fe, the strain at the MoS 2 position increases. The chemical states and elemental compositions of MoS 2 and Fe-MoS 2 were analyzed by X-ray photoelectron spectroscopy (XPS), and it was found that the Mo 3d and S2p peaks of Fe-MoS 2 are significantly redshifted compared to those of pure MoS 2 ( Figure 2 I and J), indicating that single-atom Fe doping makes the Fermi level closer to the bottom of the conduction band (CBM).

[0088] The aberration-corrected transmission electron microscopy (TEM) image confirmed the presence of atomically dispersed Fe (highlighted by yellow circles) and obvious defects (highlighted by red circles) in Fe-MoS 2 , and no nanoparticles or clusters were observed, indicating that Fe atoms are highly isolated ( Figure 2 K). The chemical states and bonding environments of isolated Fe atoms in Fe-MoS 2 were analyzed by synchrotron radiation X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy. Figure 2 L shows the Fe K-edge XANES spectrum of Fe-MoS 2 , with Fe 2 O 3 , FeS, and metallic Fe as reference standards, corresponding to the Fe 3+ , Fe 2+ , and Fe 0 states, respectively. The near-edge absorption energy of Fe-MoS 2 is located between FeS and Fe 2 O 3 , indicating that the oxidation state of Fe is between +2 and +3. In the Fourier transform extended X-ray absorption fine structure (FT-EXAFS) spectrum ( 2 M) of Fe-MoS Figure 2 , a single main peak is observed at , while the Fe-Fe bond of Fe 0 in metallic Fe is expected to be at , and the Fe-Fe bond of Fe 2 O 3 in Fe 3+ is expected to be at At none of these positions did they appear. Quantitative fitting of the EXAFS curves in k-space and R-space further revealed that each Fe atom was coordinated with 3.2 ± 0.3 S atoms in Fe-MoS 2 (Table N and Table 1). In addition, high-resolution wavelet transform (WT) analysis was performed on the Fe K-edge oscillations ( Figure 2 O to R). The Fe-MoS Figure 2 sample exhibited the maximum WT intensity in k-space, attributed to the Fe-S structure. The lack of the maximum WT intensity at positions usually associated with Fe-Fe bonding further confirmed the atomic dispersion of Fe atoms in the synthesized Fe-MoS 2 . Subsequently, electron spin resonance (ESR) spectroscopy was used to confirm the presence of defects, and a signal at g = 2.0 was observed, indicating the presence of sulfur vacancies. This signal was present in the ESR spectra of both Fe-MoS and MoS , although it was significantly stronger in Fe-MoS 2 , indicating that Fe doping promoted the removal of surface unsaturated S atoms, thereby generating a large number of sulfur vacancies ( 2 ). 2 The signal was present in the ESR spectra of both Fe-MoS 2 and MoS Figure 8 ).

[0089] Table 1 Fe K-edge EXAFS fitting parameters of different samples

[0090]

[0091] a CN, number of coordinating atoms; b R, distance between adjacent atoms; c σ 2 , mean square relative displacement; d ΔE 0 , inner potential energy correction; R, goodness of fit; S0 2 (correction factor) was 0.740.

[0092] 2. The piezocatalytic performance of the Fe-MoS 2 piezocatalytic material prepared in Example 1 above was characterized

[0093] The piezoelectric properties of MoS 2 and Fe-MoS 2 were evaluated using piezoresponse force microscopy (PFM). When a bias voltage ranging from -10 V to 10 V was applied, a characteristic butterfly-shaped amplitude loop and a phase change of approximately 180° were observed ( Figure 9 ), indicating that Fe-MoS 2 had significant piezoelectric properties. Notably, Fe-MoS 2The amplitude change exceeded that of MoS 2 , indicating that Fe-MoS 2 has a stronger piezoelectric response. The piezoelectric coefficient (d 33 ) is a key parameter of piezoelectric materials, reflecting the efficiency of converting mechanical energy into electrical energy. Therefore, a higher d 33 value is crucial for practical applications. The d 2 value of Fe-MoS 33 measured from the amplitude-voltage loop is approximately 9.22 pm V -1 , which is 1.5 times higher than that of MoS 2 at 6.16 pm V -1 , confirming the superior piezoelectric properties of Fe-MoS 2 .

[0094] Evaluated the efficiency of generating reactive oxygen species (ROS) in MoS 2 and Fe-MoS 2 under low-frequency ultrasonic (US) irradiation, using 1,3-diphenylisobenzofuran (DPBF) as an indicator for singlet oxygen ( 1 O 2 ). After ultrasonic treatment, the characteristic absorption peak at 416 nm of all samples gradually decreased, verifying the 1 O 2 generation. Notably, under ultrasonic irradiation, compared with MoS 2 , the characteristic peak of Fe-MoS 2 at 416 nm decreased more significantly with time, highlighting the enhanced piezoelectric properties after Fe doping ( Figure 3 A and Figure 10 ). Electron spin resonance (ESR) spectroscopy was employed, using 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TEMP) as a spin trap, to detect the 2 generation of 2 in MoS 1 O 2 under ultrasonic irradiation. ESR analysis showed that under ultrasonic irradiation, a characteristic triplet corresponding to the intensity ratio of 2 / TEMP of 1:1:1 appeared in both MoS 2 and Fe-MoS 1 O 2 ( Figure 3 B). In addition, the Fe-MoS 2 +US group showed a stronger signal, further verifying the enhanced piezoelectric properties of Fe-MoS 2 .

[0095] For the above Fe-MoS 2To explore the piezocatalytic activity mechanism, the present invention studied the energy levels and electronic configurations of MoS 2 and Fe-MoS 2 First, the Tauc plots obtained using the Kubelka-Munk function were used to determine the band gaps of MoS 2 and Fe-MoS 2 The results showed that the band gaps of Fe-MoS 2 and MoS 2 were 1.03 eV and 1.24 eV, respectively, and the band gap of Fe-MoS 2 was narrower, which was conducive to the separation of electron-hole pairs ( Figure 3 N). The flat band potentials calculated from the Mott-Schottky plots indicated that the conduction band (CB) potentials of MoS 2 and Fe-MoS 2 were -0.06 V and -0.11 V, respectively ( Figure 3 O and P). Using the band gap and CB values, the valence band (VB) potentials of MoS 2 and Fe-MoS 2 were determined to be 1.18 V and 0.92 V, respectively. Notably, the electrons generated in the CB of Fe-MoS 2 were not energetically suitable for generating O 2 ·-(the redox potential of O 2 / O 2 ·-is -0.33 V), and the holes in the VB were also not suitable for generating ·OH (the redox potential of H 2 O / ·OH is 1.99 V) or 1 O 2 (the redox potential of O 2 / 1 O 2 is 0.98 V) ( Figure 3 Q). Under ultrasonic (US) irradiation, the local dipole moment of Fe-MoS 2 changed, inducing piezopolarization and generating an internal piezoelectric field inside the catalyst, which further promoted the directional migration of positive and negative charges to the opposite sides of the catalyst. Due to the tilting of the CB and VB caused by the piezoelectric field, the generation of O 2 ·-, ·OH, and 1 O 2 became energetically favorable, which was confirmed by ESR data ( Figure 3 B, F, and K).

[0096] The present invention evaluated the separation efficiency of ultrasound-induced carriers after single-atom Fe doping through ultrasonic transient current and electrochemical impedance spectroscopy (EIS) analysis. Under ultrasonic irradiation, Fe-MoS 2 showed higher separation efficiency of ultrasound-induced carriers than MoS2 Higher and more stable current density ( Figure 11 ). Similarly, the Nyquist plot of Fe-MoS 2 shows a much smaller semicircle ([[]] 2 R) than that of MoS Figure 3 , indicating a significant improvement in the interfacial charge transfer efficiency. Then, the present invention analyzed the time-resolved photoluminescence (PL) spectrum ( Figure 3 S), and the results showed that the PL intensity of Fe-MoS 2 was lower than that of MoS 2 , which confirmed that the single-atom Fe doping effectively inhibited charge recombination under ultrasonic irradiation. Therefore, Fe doping enhanced charge migration and reduced recombination, thereby improving the piezocatalytic performance of MoS 2 and leading to an increase in the generation of ROS (O 2 ·−, ·OH, and 1 O 2 ).

[0097] 3. Characterize the peroxidase-like catalytic performance of the Fe-MoS 2 piezocatalytic material prepared in Example 1 above

[0098] Considering the elevated concentration of hydrogen peroxide (H 2 O 2 ) at the tumor site, the CAT-like activity was evaluated by measuring the O 2 O 2 generated from the decomposition of H 2 . The oxygen level increased proportionally with the increase in H 2 O 2 and Fe-MoS 2 concentrations, confirming that Fe-MoS 2 has CAT-like catalytic activity ( Figure 3 D). Evaluate its OXD-like activity, in which substrate oxidation occurs simultaneously with O 2 reduction to generate superoxide anion (O 2 ·−). Using 3,3′,5,5′-tetramethylbenzidine (TMB) as the chromogenic substrate, the absorption intensity at 652 nm increased significantly with the increase in Fe-MoS 2 concentration, indicating that its OXD-like activity is concentration-dependent ( Figure 3 E). Applying electron spin resonance (ESR) spectroscopy technology and using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap, the generation of O 2 ·− was detected. Compared with MoS 2 alone, the Fe-MoS 2 sample showed an obvious sextet signal, confirming the generation of O2 ·- generation ( Figure 3 F). In addition, clear sextet signals were detected in both the MoS 2 +US and Fe-MoS 2 +US groups, indicating that O 2 ·- was generated in these two samples under ultrasonic irradiation due to their piezoelectric properties. The sextet signal of the Fe-MoS 2 +US group was significantly stronger than that of the individual Fe-MoS 2 , indicating that the piezocatalytic effect synergistically increased the generation of O 2 ·- with the OXD-like catalytic activity.

[0099] Since tumor tissues usually exhibit higher hydrogen peroxide (H 2 O 2 ) concentrations (50 - 100 μM) than normal tissues (0.1 - 10 μM), the peroxidase (POD)-like activity of Fe-MoS 2 was further evaluated by monitoring the degradation of methylene blue (MB). Similar to the Fenton-like reaction that generates hydroxyl radicals (·OH) through the decomposition of H 2 O 2 , POD uses H 2 O 2 as an electron acceptor to promote substrate oxidation. In the Fe-MoS 2 +MB+H 2 O 2 group, the absorption at 664 nm decreased significantly over time ( Figure 3 G). In addition, the degradation rate of MB was strongly affected by the concentrations of Fe-MoS 2 and H 2 O 2 ( Figure 3 H). Compared with neutral pH, the weakly acidic conditions of the tumor microenvironment enhanced the POD-like activity of Fe-MoS 2 ( Figure 3 I). Notably, compared with the Fe-MoS 2 +H 2 O 2 group, the Fe-MoS 2 +H 2 O 2 +US group exhibited significantly higher ·OH generation ( Figure 3 J). Electron spin resonance (ESR) spectroscopy, using DMPO as a spin trap, was used to quantify the generation of ·OH. In Fe-MoS 2 +US, Fe-MoS 2 +H 2 O 2 and Fe-MoS 2+H 2 O 2 A significant ·OH / DMPO adduct with a relative intensity of 1:2:2:1 was detected in both the Fe-MoS 2 +US and Fe-MoS 2 +H 2 O 2 groups, confirming the generation of ·OH. ·OH was present in both the Fe-MoS 2 demonstrating the ability of Fe-MoS 2 +H 2 O 2 +US group showed the strongest signal intensity, indicating that the combination of piezocatalysis and POD-like activity led to abundant ·OH generation under ultrasonic irradiation ( Figure 3 K). GSH is a key regulator of tumor progression and acts by scavenging excessive ROS. Therefore, its depletion is beneficial for ROS-mediated anticancer therapies. GSHOx plays a key role in catalyzing the oxidation of GSH, thus promoting the depletion of GSH and increasing the oxidative stress of tumor cells. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) reacts with GSH to form yellow 5-thiobis-2-nitrobenzoic acid, which shows a characteristic absorption peak at a wavelength of 412 nm. As Figure 3 shown in 2 L, Fe-MoS 2 effectively depleted GSH in a concentration-dependent manner, confirming its GSHOx-like activity. In addition, under US irradiation, compared with the use of Fe-MoS 2 alone, the Fe-MoS 2 +US group showed a greater consumption of GSH, indicating that US irradiation enhanced the GSH consumption ability of Fe-MoS Figure 3 M).

[0100] Application Example 1

[0101] The surface of Fe-MoS 2 was PEGylated (polyethyleneglycolated) with PEG having a molecular weight of 544.7 to improve its physiological stability and biocompatibility. The results of dynamic light scattering (DLS) measurements showed that the particle sizes of MoS 2 , Fe-MoS 2 and Fe-MoS 2 -PEG were approximately 284.1 ± 8.4 nm, 298.2 ± 11.0 nm and 322.5 ± 13.1 nm, respectively ( Figure 12 A and 12B). In addition, Fe-MoS 2-The surface potential of PEG is closer to neutral. Compared with MoS 2 and Fe-MoS 2 , it confirms the success of the surface modification of PEG-NH 2 ( Figure 12 C).

[0102] 1. Under ultrasonic irradiation, the Fe-MoS2 prepared in Example 1 was applied to in vitro treatment

[0103] Based on the excellent piezoelectric properties and multi-enzyme activities of Fe-MoS 2 , the anti-tumor efficacy of this material under ultrasonic stimulation was evaluated in cell experiments. Bio-transmission electron microscopy (Bio-TEM) showed that mouse skin melanoma cells (B16-F10) ingested Fe-MoS 2 within 6 hours, and the intake increased after 12 hours, indicating that the cells ingested and retained Fe-MoS 2 ( Figure 4 A). Confocal laser scanning microscopy (CLSM) further confirmed the cell uptake ( Figure 20 ), which is a prerequisite for Fe-MoS 2 to produce cytotoxic effects.

[0104] Evaluate the in vitro biocompatibility of the Fe-MoS 2 of the present invention on B16-F10 cells and human umbilical vein endothelial cells (HUVECs). Co-culture Fe-MoS 2 at concentrations (0 - 80 μg / mL) with B16-F10 cells and HUVECs for 24 hours. Even at the highest concentration of 80 μg / mL, Fe-MoS 2 had no significant effect on the viability of both cell types, indicating its good in vitro biosafety ( Figure 4 B). Evaluate the therapeutic effect of Fe-MoS 2 ultrasonic irradiation on B16-F10 cells by the standard CCK-8 assay (a method for measuring cell proliferation and cytotoxicity), and the results showed that the therapeutic effect gradually enhanced with the increase of ultrasonic irradiation time and power ( Figure 13 ). Given that the concentration of H 2 O 2 in tumor tissues (50 - 100 μM) is higher than that in normal tissues, 100 μM of H 2 O 2 was introduced in subsequent experiments. In all groups, as the concentration of Fe-MoS 2 increased, the viability of B16-F10 cells decreased, while Fe-MoS 2 +US+H 2 O 2The group showed the lowest survival rate. This highlights the enhanced anti-cancer effect of the synergistic action of piezocatalysis and enzyme catalysis( Figure 4 C).

[0105] To further confirm the significant anti-cancer effect mediated by Fe-MoS 2 B16-F10 cancer cells were divided into 6 groups: control group (treated with complete medium), US group, Fe-MoS 2 group, Fe-MoS 2 +US group, Fe-MoS 2 +H 2 O 2 group, and Fe-MoS 2 +US+H 2 O 2 group. At the same Fe-MoS 2 concentration (80 μg / mL), the cytotoxic effects of the Fe-MoS 2 +US group and the Fe-MoS 2 +H 2 O 2 group were significantly stronger than those of the Fe-MoS 2 group. Meanwhile, the anti-cancer function of the Fe-MoS 2 +US+H 2 O 2 group was significantly stronger than those of the Fe-MoS 2 +US group and the Fe-MoS 2 +H 2 O 2 group, further confirming the synergistic therapeutic effect of piezocatalysis and enzyme catalysis of Fe-MoS 2 . The live-dead staining images also showed that higher green fluorescence intensity was observed in other groups compared with the Fe-MoS Figure 4 +US+H 2 O 2 group( 2 E), indicating that Fe-MoS Figure 4 exhibited a powerful anti-tumor therapeutic effect under ultrasound irradiation. The ROS concentration in B16-F10 cells was measured using the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe. As 2 shown in Figure 4 F, weak green fluorescence of oxidized DCFH was detected in the groups treated with only ultrasound or Fe-MoS 2 . In contrast, both the Fe-MoS 2 +US group and the Fe-MoS 2 +H 2 O 2 group showed bright green fluorescence, indicating an increase in ROS levels. Fe-MoS 2+US + H 2 O 2 The group exhibited the highest fluorescence intensity compared to other groups, confirming the substantial ROS generation driven by the synergistic effect of the piezocatalytic and enzyme catalytic activities of Fe-MoS 2 . Flow cytometry analysis of DCFH-DA further supported these findings ( Figure 4 G).

[0106] The apoptotic response of B16-F10 cells under different treatments was quantitatively evaluated by flow cytometry ( Figure 4 H). The apoptosis rates of the Fe-MoS 2 +US group and the Fe-MoS 2 +H 2 O 2 group were 37.01% and 41.23%, respectively. After treatment with Fe-MoS 2 +US + H 2 O 2 , the apoptosis rate increased significantly to approximately 60.03%, indicating that Fe-MoS 2 -mediated ROS generation effectively induced tumor cell apoptosis. Melanoma is characterized by strong invasiveness, manifested as the destructive invasion of tumor cells into host tissues and distant metastasis. Therefore, next, it was investigated whether Fe-MoS 2 could inhibit tumor migration. As Figure 4 I shows, the migration experiment clearly demonstrated that Fe-MoS 2 +US + H 2 O 2 treatment significantly inhibited the migration of tumor cells, and the effect was significantly better than that of Fe-MoS 2 +US or Fe-MoS 2 +H 2 O 2 treatment alone. This effect may be attributed to the enhanced ROS generation when Fe-MoS 2 is combined with US and H 2 O 2 , which disrupts the biological mechanism of tumor metastasis, thereby limiting the invasiveness of tumor cells.

[0107] 2. Copper-free Fe-MoS 2 -induced cuproptosis mechanism

[0108] For the antitumor mechanism of the synergistic piezocatalytic and enzyme catalytic therapy mediated by Fe-MoS 2 , transcriptome analysis was performed. Cells were collected using complete medium (control group) or Fe-MoS 2 +US + H 2 O 2RNA sequencing analysis was performed on the treated B16-F10 cells. In the Fe-MoS 2 +US+H 2 O 2 group, a total of 2335 genes showed differential expression, among which 1144 genes were up-regulated and 1191 genes were down-regulated ( Figure 5 A). Principal component analysis (PCA) showed significant differences in the gene expression profiles between the control group and the Fe-MoS 2 +US+H 2 O 2 group, and the heatmap results also confirmed this ( Figure 5 B and Figure 14 ). Gene set enrichment analysis (GSEA) showed that the pathways related to the oxidative stress response were significantly positively enriched in the Fe-MoS 2 +US+H 2 O 2 group ( Figure 5 C). Further analysis of the differentially expressed genes (DEGs) related to oxidative stress showed that genes such as G6pdx, Gclc, and Prdx1 were significantly up-regulated in the Fe-MoS 2 +US+H 2 O 2 group, highlighting a significant oxidative stress response in tumor cells ( Figure 5 , D and F).

[0109] Imbalances in oxidative stress and redox homeostasis, especially those triggered by ROS, often trigger cell death pathways including apoptosis, necrosis, pyroptosis, ferroptosis, and cuproptosis. Cuproptosis is a newly recognized form of programmed cell death triggered by excessive copper influx and its reduction to the more toxic Cu+ state. Disruption of intracellular copper homeostasis leads to the direct binding of copper ions to aconitase proteins in the tricarboxylic acid (TCA) cycle, causing the aggregation of sulfur-containing enzymes, impairing the function of iron-sulfur cluster proteins, and disrupting TCA cycle metabolism. This chain reaction triggers cellular proteotoxic stress and ultimately leads to cell death. During ROS-induced cuproptosis, GSH relieves oxidative stress by scavenging ROS and binds to copper ions to form an inactive copper-GSH complex, which helps remove excessive copper from the cell. Therefore, reducing intracellular GSH levels may be used to enhance cuproptosis in tumor cells. To confirm whether the Fe-MoS prepared by the present invention 2 can initiate cuproptosis, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. GO enrichment analysis showed that in Fe-MoS 2 +US+H2 O 2 After treatment, metabolic pathways including fatty acid synthesis, modified amino acid metabolism, GSH metabolism, and NADP metabolism were significantly altered, and these pathways are all crucial for the TCA cycle ( Figure 5 E). Similarly, post-treatment analysis showed significant changes in genes related to the TCA cycle, glycolysis / gluconeogenesis, amino acid metabolism, purine metabolism, GSH metabolism, and copper ion response pathways ( Figure 5 F). Differentially expressed genes (DEGs) related to cuproptosis also showed significant alterations, including upregulation of Pdhb, Pgls, Cdkn2aip, Lipt1, and Nfe2l2, and downregulation of Atp7b and Atp7a, further supporting the involvement of cuproptosis ( Figure 5 G).

[0110] Based on the above results, the Fe-MoS of the present invention 2 can simultaneously generate a large amount of ROS and deplete GSH through piezoelectric catalysis and enzyme-catalyzed therapy, thereby inducing cuproptosis ( Figure 5 H). On the one hand, the elevated ROS level leads to mitochondrial dysfunction, which in turn reduces the synthesis of intracellular ATP. This decline affects the function of ATP7B, thereby hindering the effective efflux of copper ions from the cytoplasm. On the other hand, the decrease in GSH level promotes the accumulation of intracellular copper ions, which bind to DLAT, resulting in its functional inactivation and damage to iron-sulfur cluster proteins. This leads to metabolic disorders and cellular stress. In addition, FDX1 plays a key role in this process, by reducing Cu 2+ to the more toxic Cu + , promoting the acetylation of DLAT, and contributing to the depletion of iron-sulfur cluster proteins. These mechanisms work together to trigger cuproptosis.

[0111] These results strongly verify that Fe-MoS 2 enables piezoelectric catalysis and enzyme-catalyzed therapy to synergistically enhance the ROS level in tumor cells and consume GSH, inhibit copper efflux and reduce intracellular copper consumption, ultimately leading to a large accumulation of mitochondrial copper and inducing cuproptosis, even in the absence of exogenous copper ions.

[0112] 3. Application of the Fe-MoS 2 piezoelectric catalytic material prepared in Example 1 to in vivo anti-tumor therapy

[0113] After observing the powerful anti-cancer effect of Fe-MoS 2 at the cellular level, the present invention further studied its anti-cancer efficacy in vivo using a subcutaneous B16-F10 tumor model ( Figure 6 A). First, for Fe-MoS 2In vivo biosafety assessment was performed. Blood biochemical analysis showed no significant changes in liver and kidney functions, and histopathological examination showed no tissue damage or inflammation in major organs, confirming the biocompatibility and safety of Fe-MoS 2 at all tested doses ( Figure 15 and 16 ). Next, the biodistribution of Fe-MoS 2 in major organs and tumors ( Figure 6 B and C, as well as Figure 17 ) was evaluated, and the results showed that Fe-MoS 2 accumulated in tumors at 2.58% ID / g within 2 h, 1.09% at 6 h, and 1.50% at both 12 and 24 h, indicating its high tumor retention ability.

[0114] When the tumor volume approached 50 mm 3 , BALB / c nude mice bearing B16-F10 tumors were randomly divided into four groups (n = 5 per group): control group (saline), ultrasound (US) group, Fe-MoS 2 group, and Fe-MoS 2 +US group (10 mg / kg) to evaluate its in vivo therapeutic effect. No significant weight changes or evidence of organ toxicity (heart, liver, spleen, lung, and kidney) were observed in the entire treatment group, verifying the safety of Fe-MoS 2 -mediated therapy ( Figure 6 D and Figure 18 ). The tumor volume growth curves ( Figure 6 E and Figures H to K) showed that compared with the control group and the ultrasound-alone group, the tumor growth in the Fe-MoS 2 group was moderately inhibited, while the Fe-MoS 2 +US group showed the strongest tumor growth inhibitory effect. At the treatment endpoint, the tumor weight in the Fe-MoS 2 +US group was significantly smaller than that in other groups ( Figure 6 F), with the highest inhibition rate (78.5%), compared with 16.9% in the ultrasound-alone group and 46.9% in the Fe-MoS 2 group ( Figure 6 G and Figure 19 ), demonstrating the superior anti-cancer effect of Fe-MoS 2 under ultrasound irradiation.

[0115] Subsequently, a comprehensive histological assessment was performed to explore Fe-MoS 2Therapeutic effects and potential mechanisms under ultrasonic irradiation. First, hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, Ki-67 immunostaining, and dihydroethidium (DHE) fluorescence staining were performed. Compared with other groups, the Fe-MoS 2 +US group showed the most extensive tumor necrosis area in H&E staining. In addition, this group showed the lowest Ki-67 positive rate and the highest TUNEL positive rate, indicating that Fe-MoS 2 +US significantly inhibited tumor cell proliferation and induced apoptosis ( Figure 6 M). DCFH-DA staining showed that compared with the Fe-MoS 2 alone group, the Fe-MoS 2 +US group produced more ROS.

[0116] Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to measure the copper concentration in the control group and the Fe-MoS 2 +US group to confirm cuproptosis ( Figure 6 L). The copper ion level in the tumor tissue of the Fe-MoS 2 +US group was significantly higher than that in the control group, which was consistent with the previous results. Through immunofluorescence detection of DLAT and FDX1, it was confirmed that the Fe-MoS 2 +US group had the highest DLAT fluorescence and the lowest FDX1 fluorescence, confirming cuproptosis ( Figure 6 N). Figure 6 In N, immunofluorescence detection of NCOA4 and GPX4 showed the trends of ferritinophagy and ferroptosis, which were consistent with the previous results. Based on these data, it can be concluded that under ultrasonic irradiation, the Fe-MoS of the present invention 2 synergistically induces piezoelectric catalytic effect and enzyme catalytic effect, generates a large amount of ROS, depletes GSH, and induces cuproptosis, ferritinophagy and ferroptosis, thereby jointly enhancing tumor cell death and achieving ideal therapeutic effects.

[0117] In summary, the present invention significantly promotes the application of biomaterials in the treatment of cuproptosis-related diseases by introducing copper-free piezoelectric catalytic materials to initiate cuproptosis. Fe-MoS 2Piezoelectric catalysis mediated by [specific mediator] and enzyme-catalyzed therapy synergistically increase the ROS level in tumor cells and deplete GSH. On the one hand, the elevated ROS level triggers mitochondrial damage, reduces intracellular ATP supply, and impairs the function of ATP7B, thus hindering the efflux of copper ions from the cytoplasm. On the other hand, the reduced GSH level promotes the binding of copper ions to dihydrolipoamide S-acetyltransferase (DLAT), leading to DLAT dysfunction and damage to iron-sulfur (Fe-S) cluster proteins, thereby triggering metabolic disorders and cellular stress, and ultimately inducing cuproptosis.

[0118] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment, characterized in that: The piezoelectric catalytic material is Fe-MoS2 in a two-dimensional form. The piezoelectric catalytic material uses MoS2 in a two-dimensional form as a carrier, and the MoS2 is doped with single-atom iron.

2. The single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 1, characterized in that: The doping amount of the single-atom iron is 0.35-0.55 at %.

3. The single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 1, characterized in that: The MoS2 contains 1 to 7 odd-numbered atomic layers with an interlayer spacing of 0.65 to 0.68 nm; the Fe-MoS2 contains 1 to 7 odd-numbered atomic layers with an interlayer spacing of 0.68 to 0.70 nm; the particle size of the MoS2 is 284.1±8.4 nm, and the particle size of the Fe-MoS2 is 298.2±11.0 nm.

4. The single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 1, characterized in that: The Fe-MoS2 surface is also modified with PEG.

5. A method for preparing a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Synthesis of MoS2 by hydrothermal method; S2: Fe-MoS2 was synthesized by doping single-atom iron on MoS2 via dip coating and calcination.

6. The method for preparing a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 5, characterized in that: Step S1 specifically includes the following steps: dissolving sodium molybdate and thiourea in ultrapure water, adjusting the pH and stirring, and then performing a hydrothermal reaction. After the reaction is completed, cooling, centrifuging, washing, and drying to obtain the MoS2.

7. The method for preparing a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 6, characterized in that: In step S1, The molar ratio of sodium molybdate to thiourea is 1:(4.5-6); The pH adjustment refers to adjusting the pH value to below 1 with hydrochloric acid; The stirring time is 90 to 150 minutes; The temperature of the hydrothermal reaction is 180-220°C and the time is 20-28 hours; The drying temperature is 50-70° C. and the drying time is 10-14 hours.

8. The method for preparing a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 5, characterized in that: Step S2 specifically includes the following steps: immersing the MoS2 prepared in step S1 in a FeCl3 solution, stirring after adjusting the pH, centrifuging to obtain a precipitate, and calcining the precipitate to obtain the Fe-MoS2.

9. The method for preparing a single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment according to claim 8, characterized in that: In step S2, The solvent of the FeCl3 solution is a mixed solution of water and ethanol, and the ratio of water to ethanol is 1: (0.8-1.2); In the mixed solution obtained after the MoS2 is immersed in the FeCl3 solution, the molar concentration ratio of MoS2:FeCl3 is 1:(1-3); The pH adjustment refers to adjusting the pH value to 0.8-1.2 with hydrochloric acid; The stirring is stirring at 50-90° C. for 90-150 minutes; The calcination is carried out in an Ar / H2 atmosphere, the flow volume ratio of Ar to H2 is 95:5, the calcination temperature is 650-750°C, and the calcination time is 90-150 minutes.

10. Use of the single-atom iron-doped molybdenum disulfide piezoelectric catalytic material for tumor treatment as claimed in any one of claims 1 to 4 in the preparation of tumor treatment drugs.

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