A Molybdenum Disulfide Nanozyme and Its Preparation Method and Application

Abstract

The present invention provides a molybdenum disulfide nanozyme, a preparation method thereof and an application thereof, relating to the technical field of nanomaterials; the preparation method of the molybdenum disulfide nanozyme includes using a hydrothermal method to uniformly mix a molybdenum source, a sulfur source and a structure-directing agent and then performing a hydrothermal reaction to obtain the molybdenum disulfide nanozyme, and the molybdenum source is a MoO2(OH)(OOH) solution. The MoS2 nanozyme provided by the technical solution of the present invention has good stability within a relatively wide pH range (3 to 11). In particular, within the experimental dose range, it not only has no toxic effect on cells, but also can protect cells, has a good antioxidant stress effect, can significantly reduce the ROS level in cells, increase the activity of SOD in cells, and reduce the MDA content in cells, and can safely and efficiently scavenge oxygen free radicals.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and specifically, relates to a molybdenum disulfide nanozyme and its preparation method and application. Background Art

[0002] Oxidative damage results from an increase in free radicals and ROS. Abnormally elevated ROS can cause cellular oxidative stress. Therefore, living organisms have a series of ROS-scavenging enzymes. To regulate the dynamic balance of intracellular ROS, organisms are equipped with a complete enzymatic antioxidant system. However, this natural antioxidant enzyme is vulnerable to various factors. For example, under strong acid or strong base conditions, the enzyme activity will decrease or disappear. Therefore, constructing artificial enzyme composite materials that can mimic antioxidant enzymes has become a research hotspot today. Nanozymes are a class of nanomaterials with biocatalytic activity that can mimic the catalytic processes of a variety of natural redox enzymes.

[0003] Molybdenum disulfide nanomaterials can mimic the activities of a variety of antioxidant enzymes, and have advantages such as strong stability, high catalytic activity, low cost, and wide application range. In the prior art, molybdenum disulfide nanomaterials (MoS 2 nanozymes) are mainly widely used in various fields as lubricating additives, catalysts, electronic device materials, lubricants, etc.

[0004] X. Zhang et al. (Self-cascade MoS 2 nanozymes for efficient intracellular antioxidation and hepatic fibrosis therapy, Nanoscale 13(29), 2021, 12613-12622) disclosed that MoS 2 nanozymes exhibit the activities of four major cellular cascade antioxidant enzymes, including superoxide dismutase, catalase, peroxidase, and glutathione peroxidase. MoS 2 nanozymes attenuate the electron transfer in cytochrome c / H 2 O 2 to improve the inherent antioxidant defense system under stress conditions. MoS 2 nanozymes, as a self-cascade platform, inhibit the production of intracellular reactive oxygen species (ROS) by regulating mitochondrial function and scavenging a large amount of ROS through its intrinsic antioxidant ability. However, MoS 2Nanozymes also have certain disadvantages and limitations, including synthesis methods and performance stability issues, and the nanozymes themselves may have certain toxicity that can damage normal cells. Chinese invention patent CN106986387A discloses a three-dimensional molybdenum disulfide flower ball and its preparation method, which uses zinc salt to assist sodium molybdate and thiourea to prepare a three-dimensional molybdenum disulfide flower ball by a hydrothermal method. The molar ratio of thiourea to molybdate ions is 6-12:1, and the molar ratio of zinc ions to molybdate ions is 1.0-2.0:1. The hydrothermal reaction conditions are hydrothermal reaction at 200-240°C for 20-24 hours. The obtained molybdenum disulfide is mainly used as the active material of the capacitor electrode sheet, and it does not involve the research on its antioxidant ability and cytotoxic effect on cells. Chinese invention patent CN108680566A provides a new method for detecting hydrogen peroxide based on a MoS 2 nanozyme-like enzyme luminescence system. This MoS 2 nanozyme-like enzyme combined with luminol solution can be used for the detection of hydrogen peroxide, proving that the MoS 2 nanozyme-like enzyme combined with luminol solution has antioxidant effects. Chinese invention patent CN115192606A discloses a single-atom nanozyme Pt@MoS 2 and its preparation method and application. This atomic nanozyme Pt@MoS 2 uses potassium thiocyanate and ammonium molybdate tetrahydrate as sulfur source and molybdenum source respectively to synthesize MoS 2 . Then it is dropped on a conductive carrier, and Pt metal plasma is injected to obtain a single-atom nanozyme Pt@MoS 2 , realizing the effective loading of single atoms and inhibiting the proliferation of 4T1 cells. This invention shows that the single-atom nanozyme Pt@MoS 2 has cytotoxic effects.

[0005] Based on the technical problems existing in the prior art, the present invention provides a novel molybdenum disulfide nanomaterial with a cell-protecting effect and high activity. Summary of the Invention

[0006] The purpose of the present invention is to provide a molybdenum disulfide nanozyme and its preparation method and application. It is prepared by a hydrothermal reaction, which can inhibit the generation of ROS, utilize its antioxidant ability to scavenge abundant reactive oxygen species (ROS), and improve the activity of intracellular SOD and reduce the content of MDA in cells; the MoS 2 nanozyme has a significant therapeutic effect in improving liver fibrosis in a mouse model.

[0007] As one of the invention purposes, the present invention provides a preparation method of a molybdenum disulfide nanozyme, including using a hydrothermal method to mix a molybdenum source, a sulfur source and a zinc salt evenly and then performing a hydrothermal reaction to obtain the molybdenum disulfide nanozyme.

[0008] As a preferred embodiment, the molybdenum source is MoO 2 (OH)(OOH) solution.

[0009] As a preferred embodiment, the sulfur source includes, but is not limited to, one or a combination of several of potassium thiocyanate, sodium thiocyanate, thiourea, etc.

[0010] As a preferred embodiment, the zinc salt includes, but is not limited to, one or a combination of several of zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, etc.

[0011] In the present invention, MoO 2 (OH)(OOH) solution is used as a precursor, and at the same time, the zinc salt plays the roles of a structure-directing agent and a template. Zn 2+ can interact with MoO 2 (OH)(OOH) and the sulfur source (such as KSCN, NaSCN, etc.) in the solution to affect the nucleation and growth process of MoS 2 nanoparticles. Zinc ions can combine with sulfur ions to form tiny nuclei of zinc sulfide (ZnS), and these nuclei can serve as the starting points for the growth of MoS 2 , affecting the orientation and growth direction of the MoS 2 crystal, and promoting the aggregation and cross-linking of nanoparticles to form a cluster-like structure of C-MoS 2 , obtaining C-MoS with different microstructures and morphologies from those in the prior art, such as nano-sheets, nano-particles, coral-like, flower-like, etc. 2 macroscopic properties of nanozymes.

[0012] As a preferred embodiment, the preparation of the precursor solution includes: adding hydrogen peroxide to molybdenum powder slowly under ice-water bath conditions for an oxidation reaction to obtain a molybdic acid peroxide solution.

[0013] As a preferred embodiment, the molar ratio of the sulfur source to the molybdenum source is (3-5):1; the molar ratio of the zinc salt to the molybdenum source is (2-5):1.

[0014] As a preferred embodiment, the conditions of the hydrothermal reaction include heating at 160-180 °C for 20-28 hours.

[0015] As a preferred embodiment, the method for preparing the molybdenum disulfide nanozyme includes the following steps:

[0016] S1. Provide a precursor solution

[0017] Under ice-water bath conditions, add hydrogen peroxide to molybdenum powder slowly for an oxidation reaction to obtain a molybdic acid peroxide solution, which is the precursor solution;

[0018] S2. Hydrothermal reaction

[0019] After uniformly mixing the precursor solution, the sulfur source and the zinc salt, heat it for hydrothermal reaction to obtain a black solid;

[0020] S3. Post-treatment

[0021] Centrifuge and wash the black solid to obtain molybdenum disulfide nanozyme.

[0022] As a preferred embodiment, the concentration of hydrogen peroxide is 30% (volume percentage).

[0023] As a preferred embodiment, the concentration of the precursor solution is 0.01 - 0.1 mol / L.

[0024] Most preferably, the concentration of the precursor solution is 0.05 mol / L.

[0025] As one of the objects of the invention, the present invention also provides a molybdenum disulfide nanozyme prepared by the foregoing preparation method.

[0026] As a preferred embodiment, the structure of the molybdenum disulfide nanozyme is a cluster-like structure formed by aggregation and cross-linking of nanoparticles.

[0027] As a preferred embodiment, the molybdenum disulfide nanozyme has good stability in the range of pH = 3 - 11.

[0028] As a preferred embodiment, the molybdenum disulfide nanozyme has a cell protection effect, and C-MoS 2 The nanozyme has no toxic effect on LO2 hepatocytes in the range of 5 - 50 μg / mL.

[0029] As one of the objects of the invention, the present invention also provides an application of the foregoing molybdenum disulfide nanozyme in H 2 O 2 induced oxidative stress; in cells after H 2 O 2 induced oxidative stress, the molybdenum disulfide nanozyme can restore the activity of the cells.

[0030] As a specific embodiment, when the concentration of the molybdenum disulfide nanozyme is 40 - 50 μg / mL, after co-incubating with LO2 hepatocytes for 24 h, H 2 O 2 induced oxidative stress of LO2 hepatocytes has a cell activity recovery of more than 80%.

[0031] As one of the objects of the invention, the present invention also provides an application of the foregoing molybdenum disulfide nanozyme in scavenging cellular oxygen free radicals.

[0032] As a preferred embodiment, after the molybdenum disulfide nanozyme is co-incubated with cells for 24 h, the cell damage of LO2 hepatocytes after oxidative stress induced by H 2 O 2 can be restored.

[0033] As a preferred embodiment, when the concentration of the molybdenum disulfide nanozyme reaches 40 μg / mL and is co-incubated with LO2 hepatocytes under oxidative stress induced by H 2 O 2 for 24 h, the cell viability of LO2 hepatocytes can be restored to more than 80%.

[0034] As a preferred embodiment, when the concentration of the molybdenum disulfide nanozyme reaches 50 μg / mL and is co-incubated with LO2 hepatocytes under oxidative stress induced by H 2 O 2 for 24 h, the cell viability of LO2 hepatocytes can be restored to the level before induction.

[0035] The MoS 2 nanozyme provided by the technical solution of the present invention can inhibit the production of ROS, utilize its antioxidant ability to scavenge abundant reactive oxygen species (ROS), and increase the activity of intracellular SOD and reduce the content of MDA in cells; the MoS 2 nanozyme has a significant effect on improving the treatment effect of LO2 hepatocytes in a mouse model of liver fibrosis. Based on this, the MoS 2 nanozyme can be used to prepare nano-drugs for oxidative stress-related diseases, and to prepare related drugs for treating liver injury caused by oxidative stress induced by H 2 O 2 .

[0036] The beneficial technical effects obtained by the present invention are as follows:

[0037] 1. The present invention uses a MoO 2 (OH)(OOH) solution as a precursor and synthesizes a cluster-structured MoS 2 nanozyme by a one-step hydrothermal reaction. During the reaction process, the reaction temperature is further reduced, making the reaction conditions milder and the process simpler; the obtained MoS 2 nanozyme has good stability in a wide pH range (3-11). In particular, within the experimental dose range, it not only has no toxic effect on cells, but also has a cell protection effect.

[0038] 2. The synthesis of the MoS 2 nanozyme can be achieved by a one-pot method, which is suitable for large-scale popularization and application; moreover, the whole process of the present invention avoids the use of organic solvents, reduces environmental pollution caused by the volatilization of organic solvents, and is an environmentally friendly synthesis method.

[0039] 3. The molybdenum disulfide nanozyme provided by the technical solution of the present invention has a protective effect on cells. In particular, it can restore the cell damage of LO2 cells against H 2 O 2 -induced oxidative stress. When the concentration of the molybdenum disulfide nanozyme is 40 μg / mL, the cell viability of LO2 cells after H 2 O 2 -induced oxidative stress can be restored to more than 80%. When the concentration reaches 50 μg / mL, the LO2 cells after H 2 O 2 -induction can be restored to the level before induction.

[0040] 4. The MoS 2 nanozyme provided by the technical solution of the present invention can inhibit the production of ROS, utilize its antioxidant ability to scavenge abundant reactive oxygen species (ROS), and increase the activity of intracellular SOD and reduce the content of MDA in cells; the MoS 2 nanozyme has a significant effect on improving the treatment effect of LO2 hepatocytes in a liver fibrosis mouse model. Therefore, the MoS 2 nanozyme can be used in the preparation of nano-drugs for oxidative stress-related diseases. In particular, it can be used in the preparation of related drugs for treating hepatocyte damage caused by H 2 O 2 -induced oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 and Figure 2 are FESEM photos of the molybdenum disulfide nanozyme provided in Example 1 of the present invention at different magnifications.

[0042] Figure 3 is the XRD pattern of the molybdenum disulfide nanozyme provided in Example 1 of the present invention.

[0043] Figure 4 is the EDS pattern of the molybdenum disulfide nanozyme provided in Example 1 of the present invention.

[0044] Figure 5 and Figure 6 are the element distribution maps of the molybdenum disulfide nanozyme provided in Example 1 of the present invention, respectively.

[0045] Figures 7 - 10 are the ultraviolet absorption spectra of the molybdenum disulfide nanozyme provided in Example 1 of the present invention under different pH conditions, respectively.

[0046] Figure 11 is the bar chart showing the effect of different concentrations of the molybdenum disulfide nanozyme provided in Example 1 of the present invention on the viability of LO2 cells.

[0047] Figure 12 Bar graph showing the effect of H at different concentrations 2 O 2 on the viability of LO2 cells.

[0048] Figure 13 Bar comparison graph showing the effect on cell viability of the test groups with different concentrations of C-MoS provided in Example 1 of the present invention, the oxidative stress inducer H 2 O 2 O 2 control group, and the blank control group.

[0049] Figure 14 Bar comparison graph showing the effect on MDA content of the test groups with different concentrations of C-MoS provided in Example 1 of the present invention, the oxidative stress inducer H 2 O 2 O 2 control group, and the blank control group.

[0050] Figure 15 Bar comparison graph showing the effect on SOD activity of the test groups with different concentrations of C-MoS provided in Example 1 of the present invention, the oxidative stress inducer H 2 O 2 O 2 control group, and the blank control group.

[0051] Figure 16 Bar comparison graph showing the effect on ROS level of the test groups with different concentrations of C-MoS provided in Example 1 of the present invention, the oxidative stress inducer H 2 O 2 O 2 control group, and the blank control group.

[0052] Figure 17 Bar comparison graph showing the fluorescence intensity of the effect on the level of reactive oxygen species in cells of the test groups with different concentrations of C-MoS provided in Example 1 of the present invention, the oxidative stress inducer H 2 O 2 O 2 control group, and the blank control group.

[0053] Figure 18 and Figure 19 SEM photos of molybdenum disulfide nanomaterials at different magnifications provided in Comparative Example 1 of the present invention.

[0054] Figure 20 and Figure 21 SEM photos of molybdenum disulfide nanomaterials at different magnifications provided in Comparative Example 4 of the present invention.

[0055] Figure 22 SEM photo of molybdenum disulfide nanomaterials provided in Comparative Example 6 of the present invention. Detailed implementation manners

[0056] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0057] The present invention provides a preparation method of molybdenum disulfide nanozyme, which includes using a hydrothermal method to uniformly mix a molybdenum source, a sulfur source and a structure-directing agent and then performing a hydrothermal reaction to obtain the molybdenum disulfide nanozyme.

[0058] In some specific embodiments, the molybdenum source is MoO 2 (OH)(OOH) solution.

[0059] In some specific embodiments, the sulfur source is a combination of one or more of potassium thiocyanate, sodium thiocyanate, thiourea, etc.

[0060] In some specific embodiments, the zinc salt includes a combination of one or more of zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, etc.

[0061] In some specific embodiments, the preparation method of the molybdenum disulfide nanozyme includes the following steps:

[0062] S1. Provide a precursor solution

[0063] Under the condition of an ice-water bath, hydrogen peroxide is slowly added to molybdenum powder for an oxidation reaction to obtain a molybdic acid peroxide solution, which is the precursor solution;

[0064] S2. Hydrothermal reaction

[0065] The precursor solution, the sulfur source and the zinc salt are uniformly mixed and then heated to perform a hydrothermal reaction to obtain a black solid;

[0066] S3. Post-treatment

[0067] The black solid is centrifuged and washed to obtain the molybdenum disulfide nanozyme.

[0068] Adopting the above technical solutions, no organic reagent needs to be added during the whole preparation process, which can avoid the residue of organic reagents. It can not only avoid the environmental pollution caused by organic reagents, but also improve the operation safety and the use safety of the molybdenum disulfide nanozyme. In particular, when performing cell experiments, the toxic effect of organic reagents on cells can be further avoided.

[0069] In some specific embodiments, the preparation of the precursor solution includes: adding a hydrogen peroxide solution dropwise to molybdenum powder for an oxidation reaction to obtain a molybdic acid peroxide solution, which is the precursor solution.

[0070] In some specific embodiments, the molar ratio of the sulfur source to the molybdenum source is (3 - 5):1; the molar ratio of the zinc salt to the molybdenum source is (2 - 5):1.

[0071] In some specific embodiments, the conditions of the hydrothermal reaction include heating at 160 - 180 °C for 20 - 28 hours.

[0072] In some specific embodiments, the structure of the molybdenum disulfide nanozyme is a cluster - like structure formed by stacking and cross - linking of nanoparticles.

[0073] In some specific embodiments, the molybdenum disulfide nanozyme has good acid - base resistance and good stability in a wide pH range of pH = 3 - 11.

[0074] In some specific embodiments, the molybdenum disulfide nanozyme not only has no toxic effect on cells, but also has the function of protecting cells.

[0075] The molybdenum disulfide nanozyme provided by the technical solution of the present invention has good stability in a relatively wide pH range (3 - 11), enabling it to adapt to changes in environmental pH without affecting its performance in practical applications, especially in cell experiments and bioactivity experiments; in particular, within the experimental dose range, it has no toxic effect on cells, can significantly reduce the ROS level in cells, increase the activity of SOD in cells, and reduce the MDA content in cells, and can play a protective role on cells against H 2 O 2 induced oxidative stress, restore cell activity, and thus can not only be applied to the scavenging of oxygen free radicals in cells or organisms, but also be applied to the preparation of nano - drugs for oxidative stress - related diseases.

[0076] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0077] Sources of experimental drugs and reagents used in the examples:

[0078] MoO 2 (OH)(OOH), KSCN, and zinc nitrate were purchased from Shanghai Macklin Biochemical Co., Ltd.

[0079] The MDA, SOD, and ROS kits were all purchased from Nanjing Jiancheng Bioengineering Institute; LO2 human hepatocytes (referred to as LO2 cells or LO2 hepatocytes in this invention) were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; DMEM high-glucose medium (product number: C11995500BT) was purchased from Gibco, USA; DMEM complete medium was prepared by mixing DMEM high-glucose medium: fetal bovine serum: penicillin-streptomycin mixture in a ratio of 90:10:1.

[0080] Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; penicillin-streptomycin mixture was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0081] The experimental instruments used in the examples and comparative examples: The scanning electron microscope is from Carl Zeiss Optics (China) Co., Ltd.; the transmission electron microscope (TEM) is from Carl Zeiss AG, Germany; the ultraviolet-visible (UV-Vis) spectrophotometer is from Shanghai M-PET Instrument Co., Ltd.; the microplate reader is from Molecular Devices, USA; the fluorescence inverted microscope is from Olympus, Japan.

[0082] For those not specifying specific techniques or conditions in the examples, they can all be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions.

[0083] Performance test:

[0084] This invention conducted performance tests on the cytotoxicity, cell viability, etc. of C-MoS provided in the foregoing examples 2 on LO2 normal human hepatocytes, including measuring the cell viability of LO2 hepatocytes using the MTT method. The specific steps are as follows:

[0085] First, 2 H 2 O

[0086] Using a cell counting chamber, inoculate LO2 hepatocytes at 5.0×10 3 cells / well in a 96-well plate. Add 100 µL of medium to each well and incubate overnight for 12 hours to allow them to adhere. The next day, discard the old medium and add 100 µL of hydrogen peroxide with set concentration gradients of 0, 800, 900, 1000, 1100, 1200, 1300 µmol / L to each well. After culturing in a cell incubator for 3 hours, add 10 µL of MTT solution to each well and continue culturing for 4 hours. After discarding the medium, add 100 µL of DMSO to each well, mix gently on a shaker at low speed for 10 min, and measure the absorbance value using a microplate reader at a wavelength of 490 nm. To calculate the cell survival rate.

[0087] H 2 O 2 is a method commonly used to trigger cellular oxidative stress. Oxidative damage to cells is mainly caused by oxygen utilization reactions within the cells: Cellular respiration accounts for most of the oxygen consumption by cells, so mitochondria are considered to be the generators of oxidative stress. Set the concentration gradients of hydrogen peroxide to 0, 800, 900, 1000, 1100, 1200, 1300 μmol / L, and conduct MTT experiments.

[0088] Second, determination of the cytotoxic effect of MoS 2 nanozyme on LO2 hepatocytes

[0089] In a 96-well plate, inoculate LO2 hepatocytes at a density of 5000 cells / well, add 100 μL of complete medium, and incubate overnight in a cell culture incubator. After completely aspirating the medium, add 100 μL of DMEM complete medium containing C-MoS 2 nanozyme at concentrations of 0, 5, 10, 15, 20, 30, 40, 50 μg / mL (DMEM high-glucose medium: fetal bovine serum: penicillin-streptomycin mixture = 90:10:1). After co-incubating in the incubator for 24 hours, measure the absorbance values of each well at a wavelength of 490 nm by the MTT method.

[0090] Third, determination of the cell protection effect of MoS 2 nanozyme

[0091] Inoculate LO2 hepatocytes at a density of 5000 cells / well in the same manner as the above steps. After seeding into a 96-well plate, expose the cells to a medium containing 1000 μmol / L H 2 O 2 and co-incubate for 3 h. Then, co-incubate with C-MoS 2 nanozyme at concentrations of 0, 5 μg / mL, 20 μg / mL, 40 μg / mL in the medium at 37 °C for 24 hours. Then add MTT solution, and after incubating for 4 h, discard the medium and replace it with 100 μL of DMSO. Measure the absorbance values of each well at a wavelength of 490 nm using a microplate reader.

[0092] Fourth, effect of MoS 2 nanozyme on the MDA content in cells

[0093] The present invention also uses the TBA method to detect the MDA content in LO2 hepatocytes and studies the effect of MoS 2 nanozyme on the MDA content in cells.

[0094] This step uses an MDA kit for detection. The MDA kit is based on the reaction between MDA and TBA under high temperature and acidic conditions to generate a red MDA-TBA adduct, which has a maximum absorption at 535 nm and can be detected by colorimetry.

[0095] The specific steps include:

[0096] 1. Sample pretreatment

[0097] After counting the LO2 hepatocytes, inoculate them at 5.0×10 4 cells / well on a six-well plate, and set up blank control groups, H 2 O 2 control groups, and C-MoS 2 experimental groups, which are divided into C-MoS 2 -L (5 µg / mL), C-MoS 2 -M (20 µg / mL), and C-MoS 2 -H (40 µg / mL). When the density of each well reaches 70%-80% under the microscope, except for the control groups, after 3 h in the medium containing 1000 µmol / L H 2 O 2 for the C-MoS 2 experimental groups, treat them with MoS 2 nanozyme for 24 h. Scrape the cells that have been washed three times with PBS, collect them into 1.5 mL Eppendorf tubes, and make marks. After centrifugation at 1000 rpm for 10 min using a centrifuge, discard the supernatant, add 1 mL of PBS to resuspend, and centrifuge again to discard the supernatant. Under ice-water bath conditions, add 200 µL of PBS buffer to each tube of cells, and ultrasonically disrupt them three times, 3-5 s each time, and then set aside.

[0098] 2. MDA reagent pretreatment

[0099] The composition of the kit and the reagent preparation are shown in Table 1.

[0100] Table 1 Composition of the kit and reagent preparation method

[0101] ;

[0102] Note: If there is no hemolysis or lipemia in the sample, the blank tube can be used instead of the control tube.

[0103] 3. TBA method experiment

[0104] Set up blank tubes, standard tubes, and measurement tubes, which are divided into control groups, H 2 O 2 , C-MoS 2 -L, C-MoS 2 -M, C-MoS 2-H. Add samples according to the ratios in Table 2.

[0105] Table 2 Ratios of Reagents and Samples for TBA Method Testing

[0106] ;

[0107] After adding samples, mix well, incubate in a water bath at 95 °C for 40 min, cool, centrifuge at 4000 rpm for 10 min. Take the supernatant and measure the absorbance of each group at 532 nm, and then calculate the MDA content in LO2 hepatocytes for each group.

[0108] 4. BCA Protein Concentration Determination

[0109] Add 20 μL of each group of samples to a 96-well plate, then add 200 μL of BCA working solution, and incubate at 37 °C for 15 - 30 min. Measure A562nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration according to the standard curve and express the results as mean ± standard deviation (SD).

[0110] Calculation formula (unit: nmol / mgprot):

[0111]

[0112] Fifth, MoS 2 Effect of Nanozyme on Cellular SOD Activity

[0113] The present invention also provides an experiment for detecting the SOD content in LO2 hepatocytes using the WST-1 method, and simultaneously examines the effect of C-MoS2 nanozyme on the cellular SOD content. A SOD activity detection kit is used, which adopts the WST-8 method to determine SOD activity by colorimetry. Add the reaction solution and staining solution to a 96-well plate, and then add the catalytic solution and dilution solution. After gently shaking and mixing the samples, place them in an incubator at 37 °C for 20 minutes, avoiding light. Measure the absorbance using an ELISA reader at a wavelength of 450 nm, construct a SOD standard curve, with the abscissa being the SOD unit concentration per milliliter and the ordinate being the reading of the standard sample (absorbance unit OD value). Calculate the SOD content in the samples according to the standard curve and express the results as mean ± standard deviation (SD).

[0114] The specific steps include:

[0115] 1. Sample Pretreatment

[0116] This step is the same as the sample pretreatment method for detecting the MDA content in LO2 hepatocytes by the TBA method.

[0117] 2. SOD Reagent Pretreatment

[0118] The composition of the kit and the reagent preparation are shown in Table 3.

[0119] Table 3 Composition and Reagent Ratio of the Kit for SOD Test

[0120] ;

[0121] Note: Substrate application solution = substrate stock solution : buffer = 1 : 200; enzyme working solution = enzyme stock solution : enzyme diluent = 1:10; both are prepared freshly before use.

[0122] 3. WST-1 Method Experiment

[0123] Set up control wells, control blank wells, measurement wells (the same as above), and measurement blank wells. Add samples according to the ratio in Table 4.

[0124] Table 4 Ratio of Samples for WST-1 Method Test

[0125] ;

[0126] Note: Before the experiment, select normal LO2 hepatocytes for preliminary experiment. Dilute the tissue homogenate of this sample into different concentrations, measure the OD value according to the above steps, calculate the SOD inhibition rate, and select the concentration with an inhibition rate of 40% - 60% for the formal experiment.

[0127] 4. BCA Protein Concentration Determination

[0128] The method for determining the BCA protein concentration is the same as that for detecting the BCA protein concentration by the TBA method. The calculation formula is (unit: U / mgprot):

[0129] ;

[0130] ;

[0131] Sixth, MoS 2 Effect of Nanozyme on Cellular ROS Content

[0132] The present invention also provides an experiment for detecting the ROS content in LO2 hepatocytes by the chemiluminescence method. It is detected using a ROS activity detection kit. This ROS kit uses DCFH-DA as a fluorescent probe (provided by the kit itself). DCFH-DA itself has no fluorescence and can freely penetrate the cell membrane. Inside the cell, it is hydrolyzed by esterase to generate DCFH. DCFH is oxidized by intracellular ROS to generate the fluorescent substance DCF. According to the positive correlation between the fluorescence intensity and the intracellular ROS level, the content of ROS in the sample is calculated, and the result is expressed as the mean ± standard deviation (SD).

[0133] The specific steps are as follows: Dilute the DCFH-DA probe (provided in the kit) with serum-free medium at a ratio of 1:1000 to make the final concentration 10 µm. The sample pretreatment step is the same as that for detecting the MDA content in LO2 hepatocytes by the TBA method. After washing the cells in the plate three times with PBS, add 1 ml of the diluted probe to each well, incubate at 37 °C for 20 min. After incubation, wash three times with PBS, then add 1 ml of PBS to each well, and observe and take grouped photos using an inverted fluorescence microscope.

[0134] The data of the above experiments were analyzed by variance using Graphpad Prism 9.0 software. All experimental data were the average values from 3 repeated experiments and were presented as the mean ± standard deviation (SD).

[0135] The MDA kit, SOD kit, and ROS kit used in the experiments were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0136] The technical solutions of the present invention will be described in detail below through specific embodiments.

[0137] Example 1

[0138] This example provides a preparation method of molybdenum disulfide nanozyme, and the specific steps include:

[0139] S1. Provide a precursor solution

[0140] Weigh 1.195 g of molybdenum powder into a round-bottom flask. Under an ice-water bath condition, slowly add 10 mL of hydrogen peroxide solution with a concentration of 30%. After the addition is complete, continue stirring the reaction until the two react completely to obtain a precursor solution of molybdic acid peroxide. Dilute it with deionized water to 250 mL to prepare a precursor solution of molybdic acid (MoO 2 (OH)(OOH)) with a concentration of 0.0498 mol / L for standby.

[0141] S2. Provide molybdenum disulfide nanozyme

[0142] First, add 200 mg of KSCN and 300 mg of zinc nitrate to 10 mL of the MoO 2 (OH)(OOH) precursor solution, stir for 20 minutes to obtain a homogeneous solution. Then, transfer the homogeneous solution to a polytetrafluoroethylene-lined autoclave and keep it heated at 180 °C in an oven for 24 hours, and naturally cool to room temperature.

[0143] S3. Post-treatment

[0144] The obtained black solid was centrifuged and washed several times with ethanol and water respectively. Finally, the collected product was dried under vacuum at 80 °C for 12 hours to obtain molybdenum disulfide nanozyme (MoS 2 nanozyme or C-MoS 2 ).

[0145] Structural characterization:

[0146] See Figure 1 and Figure 2 , the morphology and microstructure of the nanomaterial were studied by scanning electron microscopy (SEM) at different resolutions. As can be seen from the figures, Figure 1 The panoramic picture of molybdenum disulfide nanomaterial at low magnification resolution, C-MoS 2 has a uniform cluster-like structure, and there are obvious stacks of nanoparticles. Figure 2 This is the SEM photograph under high-resolution conditions. As can be seen from the figure, the nanoparticles of the molybdenum disulfide nanomaterial are stacked and there is an obvious cross-linking phenomenon between the particles, indicating that the obtained nanoclusters have a rough surface composed of nanoparticles. The particle size of the nanoparticles on the surface of the cluster structure is about 50-80 nm, and the size is uniform.

[0147] See Figure 3 , which is the X-ray powder diffraction (XRD) of C-MoS 2 provided in this example. In this spectrum, according to the angles of the diffraction peaks of the analyte, it can be determined that the obtained MoS 2 is a hexagonal crystal phase structure (JCPDS No. 37-1492). Among them, the diffraction peaks at diffraction angles 2θ of 14.38, 32.68, 35.87, 44.15 and 58.33 can respectively correspond to different crystal planes (002), (100), (102), (006) and (110). At the same time, there are no other obvious impurity peaks, indicating that the material has good crystallinity.

[0148] Refer to Figure 4 , which is the EDS spectrum of the molybdenum disulfide nanomaterial provided in this example. As can be seen from the figure, this material is mainly composed of two elements, Mo and S (Si is the conductive substrate), and the molar ratio of the two elements is 1:2, which is consistent with the molar ratio of Mo and S atoms in MoS 2 .

[0149] Figure 5 and Figure 6 are the S / Mo element distribution maps in the MoS 2 nanoribbons, which confirm the uniform distribution of Mo and S on the surface of the MoS 2 nanoribbons. The above results indicate that the MoS 2 nanomaterials are successfully prepared.

[0150] Figure 7 The molybdenum disulfide nanozyme provided in this example was used to measure C-MoS by ultraviolet spectrophotometer (UV). 2 Absorbance comparison diagrams at pH = 3, pH = 7, and pH = 11 were used to evaluate the stability of this material in acidic and alkaline environments. Figures 8 - 10 They are ultraviolet absorption spectra at 0 h, 4 h, and 24 h at pH = 3, pH = 7, and pH = 11 respectively. As can be seen from the figure, in the range of pH = 3 - 11, C-MoS 2 has good stability, indicating that the C-MoS provided by the present invention 2 has good acid and alkali resistance in a wide range of pH = 3 - 11.

[0151] Example 2

[0152] The difference between this example and Example 1 is only the different addition amounts of zinc nitrate.

[0153] Specifically, it includes:

[0154] First, 200 mg of KSCN and 460 mg of zinc nitrate were added to 10 mL of MoO 2 (OH)(OOH) precursor solution. After stirring for 20 minutes, a homogeneous solution was obtained. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 180 °C for 24 hours, and then naturally cooled to room temperature.

[0155] Finally, molybdenum disulfide nanozyme was obtained.

[0156] Example 3

[0157] The difference between this example and Example 1 is only the different addition amounts of zinc nitrate.

[0158] Specifically, it includes:

[0159] First, 240 mg of KSCN and 300 mg of zinc nitrate were added to 10 mL of MoO 2 (OH)(OOH) precursor solution. After stirring for 20 minutes, a homogeneous solution was obtained. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 180 °C for 24 hours, and then naturally cooled to room temperature.

[0160] Finally, molybdenum disulfide nanozyme was obtained.

[0161] Example 4

[0162] The difference between this example and Example 1 is only the different addition amounts of zinc nitrate.

[0163] Specifically, it includes:

[0164] First, 10 mL of MoO 2150 mg of KSCN and 300 mg of zinc nitrate were added to the (OH)(OOH) solution, and a homogeneous solution was obtained after stirring for 20 minutes. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 180 °C for 24 hours, and naturally cooled to room temperature.

[0165] Finally, molybdenum disulfide nanozyme was obtained.

[0166] Example 5

[0167] The difference between this example and Example 1 is only the hydrothermal reaction conditions, specifically including:

[0168] S2. Provide molybdenum disulfide nanozyme

[0169] First, 200 mg of KSCN and 300 mg of zinc nitrate were added to 10 mL of MoO 2 (OH)(OOH) precursor solution, and a homogeneous solution was obtained after stirring for 20 minutes. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 160 °C for 20 hours, and naturally cooled to room temperature.

[0170] Example 6

[0171] The difference between this example and Example 1 is only the amount of zinc nitrate added, specifically including:

[0172] S2. Provide molybdenum disulfide nanozyme

[0173] First, 200 mg of KSCN and 400 mg of zinc nitrate were added to 10 mL of MoO 2 (OH)(OOH) precursor solution, and a homogeneous solution was obtained after stirring for 20 minutes. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 160 °C for 20 hours, and naturally cooled to room temperature.

[0174] Comparative Example 1

[0175] The difference between this comparative example and Example 1 is only that: 2.5 mmol of MoO 3The powder sample was dispersed in 30 mL of deionized water, then 2 mL of 4 M HCl solution was added, and stirring was continued for 10 min. After stirring was completed, 6.25 mmol of KSCN was weighed and added to the above reaction solution, and stirring was continued for 10 min. Then the above reaction solution was transferred to a 50 mL Teflon liner and placed in a hydrothermal reactor. The reactor was placed in an oven, heated to 180 °C and reacted for 24 h. After the hydrothermal reaction was completed, the reactor was taken out and allowed to cool naturally. Finally, the reaction product was filtered and collected, washed three times with water and ethanol respectively, and dried in a vacuum oven at 80 °C for 10 h to obtain a black powder sample.

[0176] The method of this comparative example can also be referred to Xianzhong Zeng et al., Solid-contact K + -selective electrode based on three-dimensional molybdenum sulfide nanoflowers as ion-to-electron transducer + , Sensors and Actuators B , 234 (2016), 80~83.

[0177] Refer to Figure 18 and Figure 19 , the morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of nanosheets arranged in an interlaced manner.

[0178] Comparative Example 2

[0179] The difference between this comparative example and Example 1 is only that: KSCN and zinc nitrate were directly added to the sodium molybdate solution, and a homogeneous solution was obtained after stirring for 20 minutes, wherein the molar ratio of sodium molybdate, KSCN and zinc nitrate was 1:4:1. Then, the homogeneous solution was transferred to a Teflon-lined autoclave and heated in an oven at 180 °C for 24 hours, and naturally cooled to room temperature. Other steps were the same.

[0180] The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of nanosheets arranged in an interlaced manner, similar to that of Comparative Example 1.

[0181] Comparative Example 3

[0182] The difference between this comparative example and Example 1 is only that: KSCN and zinc nitrate were directly added to the sodium molybdate solution, and a homogeneous solution was obtained after stirring for 20 minutes. Among them, the molar ratio of sodium molybdate, KSCN and zinc nitrate was 1:6:1. Then, the homogeneous solution was transferred to a high-pressure reactor lined with polytetrafluoroethylene and heated in an oven at 180 °C for 24 hours, and then naturally cooled to room temperature. Other steps were the same.

[0183] The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of nanosheets arranged in a staggered manner, which is similar to Comparative Example 1.

[0184] Comparative Example 4

[0185] The difference between this comparative example and Example 1 is only that: zinc nitrate was not added.

[0186] Specifically, it includes:

[0187] First, 200 mg of KSCN was added to 10 mL of the MoO 2 (OH)(OOH) precursor solution, and a homogeneous solution was obtained after stirring for 20 minutes. Then, the homogeneous solution was transferred to a high-pressure reactor lined with polytetrafluoroethylene and heated in an oven at 200 °C for 24 hours, and then naturally cooled to room temperature.

[0188] Finally, a molybdenum disulfide nanomaterial was obtained.

[0189] See Figure 20 and Figure 21 , which are SEM photos of the molybdenum disulfide nanomaterial synthesized in this comparative example at different magnifications. It can be seen from the figure that the synthesized molybdenum disulfide nanomaterial is formed by the aggregation and fusion of nanoparticles and has an overall porous coral structure.

[0190] Comparative Example 5

[0191] The difference between this comparative example and Example 1 is only that zinc nitrate was not added.

[0192] Specifically, it includes:

[0193] First, 200 mg of KSCN was added to 10 mL of the MoO 2 (OH)(OOH) precursor solution, and a homogeneous solution was obtained after stirring for 20 minutes. Then, the homogeneous solution was transferred to a high-pressure reactor lined with polytetrafluoroethylene and heated in an oven at 180 °C for 24 hours, and then naturally cooled to room temperature.

[0194] Finally, a molybdenum disulfide nanomaterial was obtained.

[0195] The molybdenum disulfide nanomaterial synthesized in this comparative example has a structure similar to that of Comparative Example 4.

[0196] Comparative Example 6

[0197] This comparative example provides a molybdenum disulfide nanomaterial. The specific steps include:

[0198] Weigh 25 mg of MoO 3 sample and disperse it in 30 mL of deionized water. Then weigh and add 25 mg of PVP, and continue stirring for 10 min. After the stirring is completed, weigh 50.67 mg of NaSCN and add it to the above reaction solution, and continue stirring for 10 min. After the stirring is completed, transfer the above reaction solution to a polytetrafluoroethylene liner with a volume of 50 mL and place it in a hydrothermal reactor. Then place the reactor in an oven and carry out a hydrothermal reaction at 180 °C for 24 h. After the reaction is completed, let the reactor cool naturally, then filter and collect the product, wash it three times with water and ethanol respectively, and dry it in a vacuum oven at 80 °C for 10 h to obtain a black powder, which is the molybdenum disulfide nanomaterial.

[0199] The method of this comparative example can also be referred to Juan Li et al., A facile synthesis of porous MoS 2 coralloids for efficient application in water treatment, Materials Letters , 182(2016), 347~350.

[0200] Refer to Figure 22 , the nanomaterial obtained in this comparative example is molybdenum disulfide particles. The boundary contours between the particles are obvious. The surface of the particles is rough, the shape is irregular, and the particle sizes are also uneven, with an average particle size of 50~100 nm.

[0201] Comparative Example 7

[0202] The difference between this comparative example and Example 1 is only the different hydrothermal reaction conditions in step S2. Specifically, the steps of S2 include:

[0203] S2. Provide molybdenum disulfide nanozyme (C-MoS 2 )

[0204] First, add 200 mg of KSCN and 100 mg of zinc nitrate to 10 mL of MoO 2 (OH)(OOH) precursor solution, stir for 20 minutes to obtain a homogeneous solution. Then, transfer the homogeneous solution to a high-pressure reactor lined with polytetrafluoroethylene and heat it in an oven at 200 °C for 24 hours, and cool it naturally to room temperature.

[0205] The molybdenum disulfide nanomaterial obtained in this comparative example is similar in morphology to that in Example 1, but the surface of the cluster structure is rougher, and the particle size of the surface particles is 100-150 nm, indicating that increasing the temperature of the hydrothermal reaction speeds up the reaction rate and the particles become larger.

[0206] Regarding the molybdenum disulfide nanomaterials synthesized in the above examples and comparative examples, in terms of morphology, the molybdenum disulfide nanomaterial obtained by reacting with MoO 2 (OH)(OOH) as the precursor material is a cluster-like structure formed by cross-linking and overlapping of nanoparticles. By controlling the ratio between the sulfur source, zinc salt and molybdenum source and the hydrothermal reaction conditions, a cluster-like structure with uniform particle size and stacked and cross-linked together can be obtained. For Comparative Examples 1-3, the molybdenum disulfide obtained using molybdenum oxide or sodium molybdate as the molybdenum source is in a flaky structure, and the nanosheets can agglomerate to form a flower ball structure under the action of zinc salt. In Comparative Examples 4 and 5, zinc nitrate was not added, and the obtained molybdenum disulfide nanomaterial agglomerated into a porous coral structure. The molybdenum disulfide nanomaterial obtained in Comparative Example 6 is in a dispersed granular structure. After increasing the temperature of the hydrothermal reaction in Comparative Example 7, the surface of the obtained cluster structure is rougher due to the larger particles; obviously, when the molar ratio of the molybdenum source to the zinc salt is 1:1, due to different precursors of the molybdenum source, the structures of the obtained molybdenum disulfide are also different.

[0207] Based on the different morphologies of molybdenum disulfide nanomaterials obtained under different hydrothermal reaction conditions and precursors, etc., the present invention tested the related properties of the cluster-like molybdenum disulfide nanomaterial provided by the present invention as a nanozyme-like, and compared the properties with those of other morphologies.

[0208] Performance test:

[0209] 1. Construction of hydrogen peroxide-induced oxidative stress model

[0210] Using the oxidative stress inducer H 2 O 2 to test the cytotoxicity of LO2 human normal hepatocytes.

[0211] By using the IC 50 concentration of H 2 O 2 to induce cell damage, the antioxidant defense mechanism of the cells themselves and the protective effect of exogenous antioxidants can be studied. Selecting the median lethal dose (IC 50 ) as the concentration of the hydrogen peroxide-induced oxidative stress model can effectively balance the degree of cell damage and the survival rate, simulate the real pathological process, facilitate experimental operation and result analysis, and has a high degree of standardization.

[0212] Referring to Figure 12 , when different concentrations of the oxidative stress inducer H2 O 2 When co-incubated with cells for 3 h at concentrations of 0, 800 μmol / L, 900 μmol / L, 1000 μmol / L, 1100 μmol / L, 1200 μmol / L, and 1300 μmol / L respectively, the cell viability of LO2 hepatocytes showed a significant decrease, and the degree of decrease was positively correlated with the concentration of H 2 O 2 . Compared with the H 2 O 2 control group, after the cells were treated with H 2 O 2 , with the increase of concentration, the cell survival rate showed a dose-dependent decrease. Experiments showed that when the concentration of H 2 O 2 was 1000 mmol / L, the cell survival rate was about 50%. The hydrogen peroxide-induced oxidative stress model selected a concentration of 1000 μmol / L of H 2 O 2 as the H 2 O 2 control group for cell activity, oxidative damage and other tests.

[0213] 2. Determination of the toxicity of molybdenum disulfide nanozyme to LO2 hepatocytes

[0214] To explore the effect of molybdenum disulfide nanozyme on LO2 hepatocytes, different concentrations of C-MoS 2 at 0, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL were used to act on LO2 hepatocytes. After continuous culture for 24 h, MTT experiments were carried out.

[0215] See Figure 11 , using different concentrations of C-MoS 2 provided in Example 1 as the test groups, and co-incubated with LO2 hepatocytes for 24 h respectively. The results showed that there was no obvious change in the cell viability of the control group (the concentration of C-MoS 2 was 0) and the test groups, and the biological activity of LO2 hepatocytes remained above 80%, indicating that in the range of 0-50 μg / mL, the effect on the cell survival rate was not obvious, indicating that the C-MoS 2 provided by the present invention has no toxic effect on cells.

[0216] 3. Test of the restoration of cell activity induced by C-MoS 2 nanozyme against H 2 O 2 oxidative stress

[0217] Using the C-MoS 2The concentration of 5 μg / mL is designated as C-MoS 2 -L group, C-MoS 2 The concentration of 20 μg / mL is designated as C-MoS 2 -M group and C-MoS 2 The concentration of 40 μg / mL is designated as C-MoS 2 -H group, the oxidative stress inducer H 2 O 2 The concentration of 1000 μmol / L is designated as H 2 O 2 Control group; the blank control group is C-MoS 2 and the oxidative stress inducer H 2 O 2 The concentrations are both 0, that is, the control group not induced by H 2 O 2 oxidative stress.

[0218] See Figure 13 For the C-MoS provided in Example 1 2 Set C-MoS at different concentrations 2 The experimental groups and the oxidative stress inducer H 2 O 2 The control group and the blank control group were compared for cell viability. As the concentration of C-MoS 2 nanozyme (C-MoS 2 -L group, C-MoS 2 -M group and C-MoS 2 -H group) increased, the cell viability of LO2 hepatocytes induced by H 2 O 2 was 67.50%, 75.42% and 82.37% respectively, and the cell viability gradually recovered. Further, the present invention also adjusted the concentration of C-MoS 2 to 50 μg / mL, and the results showed that the recovery of the viability of LO2 hepatocytes was close to that of the blank control group, indicating that the recovery of the cell viability of LO2 hepatocytes induced by H 2 O 2 was proportional to the concentration of C-MoS 2 . In the figure, compared with the blank control group, ; compared with H 2 O 2 , ##P < 0.01, P < 0.001; compared with C-MoS 2 -L, ^P < 0.05, ^^P < 0.01, ^^^P < 0.001. The data are expressed as mean ± standard deviation (SD). It shows that C-MoS 2 has an effect on H 2 O 2The activity of cells induced by oxidative stress has a restorative effect, and in C-MoS 2 at a concentration of 50 µg / mL on H 2 O 2 LO2 hepatocytes induced by oxidative stress can recover to an activity close to that before induction.

[0219] Furthermore, the molybdenum disulfide nanozyme provided in Examples 2-6 was used as the experimental group. When the concentration of C-MoS 2 was 40 µg / mL, the cell viability of LO2 hepatocytes could also reach more than 80%, indicating that the molybdenum disulfide nanozyme provided by the present invention can effectively inhibit H 2 O 2 induced oxidative damage. The specific experimental results are the same as those in Example 1 and will not be elaborated here.

[0220] In Comparative Examples 1-3, when the concentration of C-MoS 2 was 40 µg / mL, the cell viability of LO2 hepatocytes was about 60%. Among them, the highest cell viability in Comparative Examples 1-3 recovered to 62.14%, 61.06%, and 60.89%; compared with Example 1, it shows that molybdenum disulfide nanomaterials synthesized from different precursors as nanozyme mimetics all have a certain restorative effect on the cell viability of LO2 hepatocytes induced by H 2 O 2 induced oxidative stress, and the molybdenum disulfide nanozyme synthesized using MoO 2 (OH)(OOH) as the precursor in this application has significantly better ability to restore cell viability than other precursors.

[0221] In Comparative Examples 4, 5, and 7, when the concentration of C-MoS 2 was 40 µg / mL, the highest cell viability of LO2 hepatocytes recovered to 70.52%, 72.06%, and 72.94%. Compared with Example 1, the molybdenum disulfide nanozyme provided in Example 1 is more significant in inhibiting the oxidative damage induced by H 2 O 2 indicating that the cluster-like structure provided by the present invention is superior to the flower ball structure and the porous coral structure; in Comparative Example 6, when the concentration of C-MoS 2 was 40 µg / mL, the cell viability of LO2 hepatocytes was all below 40%. On the one hand, it may be related to its structure, and on the other hand, the organic solvents used in its synthesis process or the recovery of cell damage also have a certain impact.

[0222] In the porous coral structures generated in Comparative Example 4 and Comparative Example 5, although the porous structure can provide more active sites, its ability to restore cell activity is relatively poorer than that of the present invention. The possible reason is that the addition of zinc nitrate can not only crosslink and overlap the nanoparticles to obtain a cluster-like structure, but also regulate the particle size of the nanoparticles. The nanoparticles with smaller particle sizes form a cluster-like structure with a rough surface, and the molybdenum disulfide nanozyme with a small particle size can provide more active sites, thus showing more excellent biological activity. Although the porous coral structure has a higher specific surface area, the particle size of the molybdenum disulfide particles is larger.

[0223] 4. Effect on the MDA content in cells

[0224] The MDA kit was used for detection. MDA reacts with TBA under high temperature and acidic conditions to generate a red MDA-TBA adduct, which has the maximum absorption at 535 nm and can be detected by colorimetry.

[0225] See Figure 14 , which shows the effect of molybdenum disulfide nanozyme on the MDA content. The MDA content of the blank control group was 1.06 ± 0.09 nmol / mgprot; H 2 O 2 The cell MDA content of the control group was 5.81 nmol / mgprot.

[0226] The content of MDA in the sample was calculated and the results were expressed as the mean ± standard deviation (SD). The results showed that the MDA content in the C-MoS 2 experimental group was significantly higher than that in the blank control group, indicating that the cell MDA content increased in the presence of H 2 O 2 . After intervention with molybdenum disulfide nanozyme, compared with the H 2 O 2 group, the MDA contents in the C-MoS 2 -L group, C-MoS 2 -M group and C-MoS 2 -H group were 4.82 nmol / mgprot, 3.95 nmol / mgprot, and 1.98 nmol / mgprot, respectively. The MDA contents all decreased significantly, indicating that molybdenum disulfide nanozyme can reduce the MDA content in cells and shows a dose-dependent relationship. When the concentration of C-MoS 2 reached 50 μg / mL, the MDA content could be reduced to the level of the blank control group.

[0227] 5. Effect on the SOD content in cells

[0228] Use an SOD activity detection kit, which adopts the WST-8 method to determine SOD activity by colorimetry. Add the reaction solution and staining solution into a 96-well plate, and then add the catalytic solution and dilution solution. After gently shaking and mixing the sample, place it in an incubator at 37 °C for 20 minutes, avoiding light. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 450 nm, construct an SOD standard curve, with the abscissa being the SOD unit concentration per milliliter and the ordinate being the standard sample reading (absorbance unit OD value). Calculate the SOD content in the sample according to the standard curve, and express the results as the mean ± standard deviation (SD). See Figure 15 , for C-MoS 2 The effect of the nanozyme on SOD activity. The SOD content in the blank control group was 9.14 ± 0.01 U / mgprot, H 2 O 2 The SOD activity of the cells in the control group was 4.52 ± 0.26 U / mgprot. The significant decrease in SOD activity indicated that hydrogen peroxide inhibited the SOD activity in LO2 hepatocytes; compared with the H 2 O 2 group, the SOD activities of the C-MoS 2 -L group, C-MoS 2 -M group and C-MoS 2 -H group were 6.34 ± 0.25 U / mgprot, 7.26 ± 0.10 U / mgprot and 8.17 ± 0.18 U / mgprot respectively. When the C-MoS 2 concentration reached 50 µg / mL, the SOD activity could be restored to the level of the blank control group. Obviously, it increased with the increase of the C-MoS 2 dose, indicating that the C-MoS 2 nanozyme could increase the activity of intracellular SOD.

[0229] 6. Effect on the intracellular ROS content

[0230] Use a ROS activity detection kit, which uses DCFH-DA as a fluorescent probe. DCFH-DA itself has no fluorescence and can freely cross the cell membrane. It is hydrolyzed by esterase in the cell to generate DCFH, and DCFH is oxidized by intracellular ROS to generate the fluorescent substance DCF. According to the positive correlation between the fluorescence intensity and the intracellular ROS level, calculate the ROS content in the sample, and express the results as the mean ± standard deviation (SD). See Figure 16 , for C-MoS 2 The effect of the nanozyme on the ROS level. The ROS fluorescence intensity in the blank control group was 56.13 ± 5.87 AU, H 2 O 2The fluorescence intensity of the control group was 189.34 AU, which was significantly increased compared with the blank control group. After intervention with C-MoS2 nanozyme, compared with the H 2 O 2 control group, the fluorescence intensities of the C-MoS 2 -L group, C-MoS 2 -M group and C-MoS2-H group were 38.43 AU, 57.69 AU and 102.38 AU respectively, all of which showed significant decreases, indicating that C-MoS 2 significantly reduced the ROS level in cells. Further referring to Figure 17 , for the blank control group, H 2 O 2 control group, C-MoS 2 -L group, C-MoS 2 -M group and C-MoS 2 -H group, the fluorescence intensity comparison chart of intracellular reactive oxygen species in cells, which shows that under the stimulation of H 2 O 2 , the intracellular reactive oxygen species level is the highest, while after adding different concentrations of C-MoS 2 nanozyme, the reactive oxygen species level gradually decreases. When there is reactive oxygen species in cells, DCFH is oxidized to a strong green fluorescent substance DCF, and its fluorescence intensity is proportional to the intracellular reactive oxygen species level. The increase in intracellular reactive oxygen species is one of the markers of cell apoptosis. Obviously, the C-MoS 2 nanozyme provided by the present invention has a protective and antioxidant effect on cells and can significantly reduce the intracellular reactive oxygen species level induced by H 2 O 2 .

[0231] Obviously, according to the foregoing test results, MoS 2 nanozyme has a significant effect on improving the treatment effect of LO2 hepatocytes in a mouse model of liver fibrosis. MoS 2 nanozyme can be used to prepare nano-drugs for oxidative stress-related diseases, and, for preparing related drugs for treating liver injury induced by oxidative stress caused by H 2 O 2 .

[0232] Based on the above analysis, the molybdenum disulfide nanozyme synthesized from MoO 2 (OH)(OOH) as a precursor provided by the present invention is different from the molybdenum disulfide nanomaterials with nano-sheet, granular and coral-like structures in the prior art. In particular, it not only has no toxic effect on cells, but also has a protective effect and can more effectively inhibit the oxidative damage induced by H 2 O 2 . When C-MoS 2When the concentration is 40 µg / mL, the cell viability of LO2 hepatocytes can be restored by more than 80%, while for C-MoS 2 When the concentration is 50 µg / mL, the cell viability of LO2 hepatocytes can be restored to the level before injury.

[0233] The description of the above 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 method for preparing a molybdenum disulfide nanozyme, characterized in that: The method comprises using a MoO2(OH)(OOH) solution as a molybdenum source; The molybdenum source, the sulfur source and the zinc salt are uniformly mixed and then subjected to a hydrothermal reaction to obtain the molybdenum disulfide nanozyme; The following steps are involved: S1. Provide precursor solution In an ice-water bath, hydrogen peroxide is slowly added to molybdenum powder for oxidation reaction to obtain a peroxymolybdic acid solution, which is a precursor solution; S2. Hydrothermal reaction After uniformly mixing the precursor solution, the sulfur source and the zinc salt, heating and performing the hydrothermal reaction to obtain a black solid; S3. Post-processing The black solid is centrifuged and washed to obtain the molybdenum disulfide nanozyme; The molar ratio of the zinc salt to the molybdenum source is (2-5):

1.

2. The method for preparing the molybdenum disulfide nanozyme according to claim 1, characterized in that: The conditions of the hydrothermal reaction include heating at 160-180° C. for 20-28 hours.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the sulfur source to the molybdenum source is (3-5):

1.

4. The method for preparing the molybdenum disulfide nanozyme according to claim 1, characterized in that: The sulfur source is any one of potassium thiocyanate, sodium thiocyanate, and thiourea; The zinc salt is one or a combination of zinc nitrate, zinc sulfate, zinc acetate and zinc chloride.

5. The method for preparing the molybdenum disulfide nanozyme according to claim 1, characterized in that: The volume percentage concentration of hydrogen peroxide is 30%; the concentration of the precursor solution is 0.01-0.1 mol / L.

6. A molybdenum disulfide nanozyme prepared by the preparation method according to any one of claims 1 to 5.

7. The molybdenum disulfide nanozyme according to claim 6, characterized in that The structure of the molybdenum disulfide nanozyme is a cluster structure formed by cross-linking of nanoparticles; And / or, the molybdenum disulfide nanozyme has no toxic effect on cells and has the function of protecting cells and scavenging oxygen free radicals; And / or, the molybdenum disulfide nanozyme can restore cell damage after H2O2-induced oxidative stress.

8. A use of the molybdenum disulfide nanozyme as described in any one of claims 6-7 for removing cellular oxygen free radicals or H2O2-induced oxidative stress for non-therapeutic purposes.

9. Use of the molybdenum disulfide nanozyme as described in any one of claims 6 to 7 in the preparation of nanomedicines for oxidative stress-related diseases.

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