A molybdenum disulfide nanozyme, its preparation method and application

Abstract

The present invention provides a molybdenum disulfide nanozyme and a preparation method and application thereof, and relates to the technical field of nanomaterials; the preparation method of the molybdenum disulfide nanozyme comprises using a hydrothermal method to evenly mix a molybdenum source, a sulfur source and a structure directing agent, and then performing a hydrothermal reaction to obtain the molybdenum disulfide nanozyme, wherein the molybdenum source is a MoO2(OH)(OOH) solution. The MoS2 nanozyme provided by the technical solution of the present invention has good stability in a wide pH range (3-11), and in particular, it not only has no toxic effect on cells within the experimental dosage range, but also can protect cells, has a good anti-oxidative stress effect, can significantly reduce the ROS level in cells, increase the activity of SOD in cells, reduce the MDA content in cells, and can safely and efficiently remove 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 a preparation method and application thereof. Background Art

[0002] Oxidative damage comes from the increase of free radicals and ROS. Abnormally elevated ROS can cause oxidative stress in cells, so living organisms have a series of ROS scavenging enzymes. In order to regulate the dynamic balance of ROS in cells, organisms are equipped with a complete enzyme antioxidant system. However, this natural antioxidant enzyme is easily affected by a variety of factors. For example, under strong acid and alkaline conditions, the enzyme activity will decrease or disappear. Therefore, the construction of artificial enzyme composite materials that can simulate antioxidant enzymes has become a hot topic of research today. Nanozymes are a class of nanomaterials with biocatalytic activity that can simulate the catalytic process of a variety of natural oxidoreductases.

[0003] Molybdenum disulfide nanomaterials can mimic the activities of a variety of antioxidant enzymes and have the advantages of strong stability, high catalytic activity, low cost and a wide range of applications. In the existing technology, molybdenum disulfide nanomaterials (MoS2 nanozymes) are mainly used as lubricating additives, catalysts, electronic device materials, lubricants, etc. and are widely used in various fields.

[0004] X. Zhang et al. (Self-cascade MoS2nanozymes for efficient intracellularantioxidation and hepatic fibrosis therapy, Nanoscale,13(29), 2021, 12613-12622) disclosed that MoS2 nanozymes exhibit the activities of four major cellular cascade antioxidant enzymes, including superoxide dismutase, catalase, peroxidase and glutathione peroxidase. MoS2 nanozymes weaken electron transfer in cytochrome c / H2O2 to improve the inherent antioxidant defense system under stress conditions. MoS2 nanozymes, as a self-cascading platform, inhibit the production of intracellular reactive oxygen species (ROS) by regulating mitochondrial function and clearing a large amount of ROS through its intrinsic antioxidant capacity. However, MoS2 nanozymes also have certain disadvantages and limitations, including problems with synthesis methods and performance stability, and the fact that the nanozymes themselves have certain toxicity that may damage normal cells. Chinese invention patent CN106986387A discloses a three-dimensional molybdenum disulfide flower ball and its preparation method, using zinc salt to assist sodium molybdate and thiourea to prepare a three-dimensional molybdenum disulfide flower ball through a hydrothermal method, the molar ratio of thiourea to molybdate ions is 6-12:1, the molar ratio of zinc ions to molybdate ions is 1.0-2.0:1, the hydrothermal reaction conditions are 200-240°C for 20-24 hours, and the molybdenum disulfide obtained is mainly used as an active substance for capacitor electrode sheets, and it does not involve research on its antioxidant capacity and toxic effects on cells. Chinese invention patent CN108680566A provides a new method for detecting hydrogen peroxide based on a MoS2 nano-enzyme luminescence system, and the MoS2 nano-enzyme cooperates with luminol solution to detect hydrogen peroxide, proving that the MoS2 nano-enzyme cooperates with luminol solution to have an antioxidant effect. Chinese invention patent CN115192606A discloses a single-atom nanozyme Pt@MoS2, its preparation method and application. The atomic nanozyme Pt@MoS2 uses potassium thiocyanate and ammonium molybdate tetrahydrate as sulfur source and molybdenum source respectively to synthesize MoS2, which is then dropped on a conductive carrier and injected with Pt metal plasma to obtain the single-atom nanozyme Pt@MoS2, achieving the effective load of single atoms and inhibiting the proliferation of 4T1 cells. The invention shows that the single-atom nanozyme Pt@MoS2 has cytotoxic effects.

[0005] Based on the technical problems existing in the prior art, the present invention provides a novel molybdenum disulfide nanomaterial which has the function of protecting cells and has high activity. Summary of the invention

[0006] The purpose of the present invention is to provide a molybdenum disulfide nanozyme and a preparation method and application thereof. It is prepared by hydrothermal reaction, can inhibit the production of ROS, use its antioxidant ability to remove abundant reactive oxygen (ROS), and increase the activity of intracellular SOD and reduce the MDA content in cells; MoS2 nanozyme has the effect of significantly improving the therapeutic effect in a mouse model of liver fibrosis.

[0007] As one of the objects of the invention, the present invention provides a method for preparing a molybdenum disulfide nanozyme, comprising using a hydrothermal method to evenly mix a molybdenum source, a sulfur source and a zinc salt and then conducting a hydrothermal reaction to obtain the molybdenum disulfide nanozyme.

[0008] As a preferred embodiment, the molybdenum source is a MoO2(OH)(OOH) solution.

[0009] As a preferred embodiment, the sulfur source includes but is not limited to one or a combination 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 zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, etc.

[0011] The present invention uses MoO2(OH)(OOH) solution as a precursor, and zinc salt plays the role of structure directing agent and template. 2+ It can interact with MoO2(OH)(OOH) and sulfur sources (such as KSCN, NaSCN, etc.) in solution to affect the nucleation and growth process of MoS2 nanoparticles. Zinc ions can combine with sulfur ions to form tiny crystal nuclei of zinc sulfide (ZnS). These crystal nuclei can serve as the starting point for the growth of MoS2, affecting the orientation and growth direction of MoS2 crystals, and promoting the agglomeration and cross-linking of nanoparticles to form a clustered structure of C-MoS2, thereby obtaining the macroscopic performance of C-MoS2 nanozymes with different microstructures and morphologies such as nanosheets, particles, corals, and flower balls in the prior art.

[0012] As a preferred embodiment, the preparation of the precursor solution includes: slowly adding hydrogen peroxide to molybdenum powder in an ice-water bath to carry out an oxidation reaction to obtain a molybdenum 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 comprises the following steps: S1. Provide precursor solution In an ice-water bath, slowly adding hydrogen peroxide to molybdenum powder for oxidation reaction to obtain a molybdenum peroxide solution, i.e., the precursor solution; S2. Hydrothermal reaction After uniformly mixing the precursor solution, the sulfur source and the zinc salt, heating and performing a hydrothermal reaction to obtain a black solid; S3. Post-processing The black solid is centrifuged and washed to obtain molybdenum disulfide nanozyme.

[0016] As a preferred embodiment, the concentration of hydrogen peroxide is 30% (volume percentage).

[0017] As a preferred embodiment, the concentration of the precursor solution is 0.01-0.1 mol / L.

[0018] Most preferably, the concentration of the precursor solution is 0.05 mol / L.

[0019] As one of the objects of the invention, the present invention also provides a molybdenum disulfide nanozyme prepared by the aforementioned preparation method.

[0020] As a preferred embodiment, the structure of the molybdenum disulfide nanozyme is a cluster structure formed by agglomeration and cross-linking of nanoparticles.

[0021] As a preferred embodiment, the molybdenum disulfide nanozyme has good stability in the pH range of 3 to 11.

[0022] As a preferred embodiment, the molybdenum disulfide nanozyme has a cell-protective effect, and the C-MoS2 nanozyme has no toxic effect on LO2 hepatocytes in the range of 5-50 µg / mL.

[0023] As one of the purposes of the invention, the present invention also provides an application of the aforementioned molybdenum disulfide nanozyme in H2O2-induced oxidative stress; in cells after H2O2-induced oxidative stress, the molybdenum disulfide nanozyme can restore the activity of the cells.

[0024] As a specific embodiment, when the concentration of molybdenum disulfide nanozyme was 40~50µg / mL, after incubation with LO2 hepatocytes for 24 hours, the cell activity of LO2 hepatocytes induced by H2O2 oxidative stress was restored by more than 80%.

[0025] As one of the purposes of the invention, the present invention also provides an application of the aforementioned molybdenum disulfide nanozyme in removing cellular oxygen free radicals.

[0026] As a preferred embodiment, the molybdenum disulfide nanozyme can restore cell damage of LO2 hepatocytes after oxidative stress induced by H2O2 after being co-incubated with cells for 24 hours.

[0027] As a preferred embodiment, when the concentration of the molybdenum disulfide nanozyme reaches 40 µg / mL, after incubation with H2O2-induced oxidative stress LO2 hepatocytes for 24 hours, the cell activity of LO2 hepatocytes can be restored to more than 80%.

[0028] As a preferred embodiment, when the concentration of the molybdenum disulfide nanozyme reaches 50 µg / mL, after incubation with H2O2-induced oxidative stress LO2 hepatocytes for 24 hours, the cell activity of LO2 hepatocytes can be restored to the level before induction.

[0029] The MoS2 nanozyme provided by the technical solution of the present invention can inhibit the production of ROS, utilize its antioxidant ability to remove abundant reactive oxygen species (ROS), and increase the activity of intracellular SOD and reduce the MDA content in the cells; MoS2 nanozyme has a significant improvement in the therapeutic effect on LO2 hepatocytes in a liver fibrosis mouse model. Based on this, MoS2 nanozyme can be used to prepare nanomedicines for oxidative stress-related diseases, as well as for preparing related drugs for treating H2O2-induced oxidative stress liver damage.

[0030] Beneficial technical effects obtained by the present invention: 1. The present invention uses MoO2(OH)(OOH) solution as a precursor and adopts a hydrothermal reaction to synthesize a clustered MoS2 nanozyme in one step. During the reaction, the reaction temperature is further reduced, making the reaction conditions milder and the process simpler. The obtained MoS2 nanozyme has good stability in a wide pH range (3-11). In particular, it is not only non-toxic to cells within the experimental dosage range, but also has a cell-protective effect.

[0031] 2. The one-pot method can be used to synthesize MoS2 nanozymes, which is suitable for large-scale promotion and application. Moreover, the whole process of the present invention avoids the use of organic solvents, reduces the pollution of the environment caused by the volatilization of organic solvents, and is an environmentally friendly synthesis method.

[0032] 3. The molybdenum disulfide nanozyme provided by the technical solution of the present invention has a protective effect on cells, especially can restore the cell damage of LO2 cells induced by H2O2 oxidative stress. When the concentration of molybdenum disulfide nanozyme is 40µg / mL, the cell activity of LO2 cells after H2O2-induced oxidative stress can be restored to more than 80%, and when the concentration reaches 50µg / mL, the LO2 cells after H2O2 induction can be restored to the level before induction.

[0033] 4. The MoS2 nanozyme provided by the technical solution of the present invention can inhibit the production of ROS, utilize its antioxidant ability to remove abundant reactive oxygen species (ROS), and increase the activity of intracellular SOD and reduce the MDA content in the cells; MoS2 nanozyme has a significant improvement in the therapeutic effect on LO2 hepatocytes in a liver fibrosis mouse model. Therefore, MoS2 nanozyme can be used to prepare nanomedicines for oxidative stress-related diseases, especially, it can be used to prepare related drugs for treating H2O2-induced oxidative stress hepatocyte damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 and Figure 2 They are FESEM photos of molybdenum disulfide nanozyme at different magnifications provided in Example 1 of the present invention.

[0035] Figure 3 This is the XRD diagram of the molybdenum disulfide nanozyme provided in Example 1 of the present invention.

[0036] Figure 4 This is the EDS image of the molybdenum disulfide nanozyme provided in Example 1 of the present invention.

[0037] Figure 5 and Figure 6 They are respectively the element distribution diagrams of the molybdenum disulfide nanozyme provided in Example 1 of the present invention.

[0038] Figure 7-10 They are respectively the ultraviolet absorption spectra of the molybdenum disulfide nanozyme provided in Example 1 of the present invention under different pH conditions.

[0039] Fig.11 This is a bar graph showing the effects of different concentrations of molybdenum disulfide nanozymes provided in Example 1 of the present invention on the viability of LO2 cells.

[0040] Fig.12 It is a bar graph showing the effect of different concentrations of H2O2 on the viability of LO2 cells.

[0041] Fig.13 This is a bar graph comparing the effects of different concentrations of C-MoS2 test groups, oxidative stress inducer H2O2 control groups and blank control groups on cell activity provided in Example 1 of the present invention.

[0042] Fig.14 This is a bar graph comparing the effects of different concentrations of C-MoS2 test groups, oxidative stress inducer H2O2 control groups and blank control groups on MDA content provided in Example 1 of the present invention.

[0043] Fig.15This is a bar graph comparing the effects of different concentrations of C-MoS2 test groups, oxidative stress inducer H2O2 control groups and blank control groups on SOD activity provided in Example 1 of the present invention.

[0044] Fig.16 This is a bar graph comparing the effects of different concentrations of C-MoS2 test groups, oxidative stress inducer H2O2 control groups and blank control groups on ROS levels provided in Example 1 of the present invention.

[0045] Fig.17 This is a comparison chart of the fluorescence intensity of the effects of different concentrations of C-MoS2 test groups, oxidative stress inducer H2O2 control groups and blank control groups on the level of reactive oxygen in cells provided in Example 1 of the present invention.

[0046] Fig.18 and Fig.19 SEM photos of molybdenum disulfide nanomaterials at different magnifications provided in Comparative Example 1 of the present invention.

[0047] Fig. 20 and Fig.21 SEM photographs of molybdenum disulfide nanomaterials at different magnifications provided for Comparative Example 4 of the present invention.

[0048] Fig. 22 This is a SEM photograph of the molybdenum disulfide nanomaterial provided for Comparative Example 6 of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The present invention provides a method for preparing a molybdenum disulfide nanozyme, comprising adopting 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.

[0051] In some specific embodiments, the molybdenum source is a MoO2(OH)(OOH) solution.

[0052] In some specific embodiments, the sulfur source is one or a combination of, including but not limited to, potassium thiocyanate, sodium thiocyanate, thiourea, and the like.

[0053] In some specific embodiments, the zinc salt includes but is not limited to one or a combination of zinc nitrate, zinc sulfate, zinc acetate, zinc chloride, etc.

[0054] In some specific embodiments, the method for preparing the molybdenum disulfide nanozyme comprises the following steps: S1. Provide precursor solution Slowly adding hydrogen peroxide to the molybdenum powder in an ice-water bath to carry out an oxidation reaction to obtain a molybdenum peroxide solution, which is the precursor solution; S2. Hydrothermal reaction After the precursor solution, the sulfur source and the zinc salt are uniformly mixed, the mixture is heated to perform a hydrothermal reaction to obtain a black solid; S3. Post-processing The black solid is centrifuged and washed to obtain molybdenum disulfide nanozyme.

[0055] By adopting the above-mentioned technical scheme, no organic reagents need to be added during the entire preparation process, and the residue of organic reagents can be avoided. This can not only avoid the pollution of the environment by organic reagents, but also improve the safety of operation and the safety of the use of molybdenum disulfide nanozymes. In particular, when conducting cell experiments, the toxic effects of organic reagents on cells can be further avoided.

[0056] In some specific embodiments, the preparation of the precursor solution includes: adding hydrogen peroxide solution to molybdenum powder for oxidation reaction to obtain molybdenum peroxide acid solution, which is the precursor solution.

[0057] 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.

[0058] In some specific embodiments, the conditions of the hydrothermal reaction include heating at 160-180° C. for 20-28 hours.

[0059] In some specific embodiments, the structure of the molybdenum disulfide nanozyme is a cluster structure formed by stacking and cross-linking nanoparticles.

[0060] In some specific embodiments, the molybdenum disulfide nanozyme has good acid and alkali resistance and good stability in a wide range of pH = 3~11.

[0061] In some specific embodiments, the molybdenum disulfide nanozyme is not only non-toxic to cells, but also has the function of protecting cells.

[0062] The molybdenum disulfide nanozyme provided by the technical solution of the present invention has good stability in a wide pH range (3-11), so that in practical applications, especially cell experiments and biological activity experiments, it can adapt to changes in environmental pH without affecting performance; in particular, it has no toxic effect on cells within the experimental dosage range, can significantly reduce the ROS level in cells, increase the activity of intracellular SOD, and reduce the MDA content in cells, can protect cells against H2O2-induced oxidative stress, and restore cell activity, so that it can not only be used to remove oxygen free radicals in cells or organisms, but also can be used to prepare nanomedicines for oxidative stress-related diseases.

[0063] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0064] The experimental drugs and reagents used in the examples are from: MoO2(OH)(OOH), KSCN, and zinc nitrate were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; MDA, SOD and ROS kits were purchased from Nanjing Jiancheng Bioengineering Institute; LO2 human hepatocytes (referred to as LO2 cells or LO2 hepatocytes in the present invention) were from Shanghai Institute of Cell Biology, Chinese Academy of Sciences; DMEM high glucose medium (Cat. No.: 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.

[0065] Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; penicillin-streptomycin mixture was purchased from Beijing Solaibao Biotechnology Co., Ltd.

[0066] The experimental instruments used in the embodiments and comparative examples are as follows: scanning electron microscope from Carl Zeiss Optics (China) Co., Ltd.; transmission electron microscope (TEM) from Zeiss Instruments AG, Germany; ultraviolet-visible (UV-Vis) spectrophotometer from Shanghai Mepda Instrument Co., Ltd.; microplate reader from Molecular Devices, Inc., USA; and fluorescent inverted microscope from Olympus Corporation, Japan.

[0067] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0068] Performance Test: The present invention tests the cytotoxicity and cell activity of C-MoS2 provided in the above embodiment on LO2 normal human liver cells, including determining the cell viability of LO2 liver cells using the MTT method. The specific steps include: First, the construction of H2O2 oxidative damage model of hepatocytes Using a cell counting agarose, LO2 hepatocytes were counted at 5.0 × 10 3 100µL of culture medium was added to each well and cultured overnight for 12 hours to allow the cells to adhere to the wall. The next day, the old culture medium was discarded and 100µL of hydrogen peroxide with a concentration gradient of 0, 800, 900, 1000, 1100, 1200, and 1300µmol / L was added to each well. After culturing in a cell culture incubator for 3 hours, 10µL of MTT solution was added to each well and cultured for another 4 hours. After discarding the culture medium, 100µL of DMSO was added to each well and mixed at a low speed on a shaker for 10 minutes. The absorbance value was measured using an enzyme reader at a wavelength of 490nm to calculate the cell survival rate.

[0069] H2O2 is a commonly used method to trigger cellular oxidative stress. Oxidative damage to cells is mainly caused by oxygen usage reactions in cells: cellular respiration accounts for most of the cellular oxygen consumption, so mitochondria are considered to be the generators of oxidative stress. The concentration gradient of hydrogen peroxide was set to 0, 800, 900, 1000, 1100, 1200, and 1300 µmol / L for MTT experiments.

[0070] Second, determination of the toxic effect of MoS2 nanozymes on LO2 hepatocytes LO2 hepatocytes were seeded at a density of 5000 cells / well in a 96-well plate, 100 µL of complete medium was added, and the cells were incubated overnight in a cell culture incubator. After the medium was completely aspirated, 100 µL of DMEM complete medium (DMEM high glucose medium: fetal bovine serum: penicillin-streptomycin mixture = 90:10:1) containing C-MoS2 nanozymes at concentrations of 0, 5, 10, 15, 20, 30, 40, and 50 µg / mL was added. After incubation in the incubator for 24 hours, the absorbance of each well at a wavelength of 490 nm was measured by the MTT method.

[0071] Third, determination of the cell protective effect of MoS2 nanozymes The LO2 hepatocytes were seeded at a density of 5000 cells / well in the same manner as above, and then inoculated into a 96-well plate. The cells were exposed to a medium containing 1000µmol / LH2O2 and incubated for 3h. After incubation at 37°C for 24h with 0, 5µg / mL, 20µg / mL, and 40µg / mL C-MoS2 nanozymes, the MTT solution was added, and after incubation for 4h, the medium was discarded and replaced with 100µL DMSO. The absorbance of each well was measured at a wavelength of 490nm using a microplate reader.

[0072] Fourth, the effect of MoS2 nanozymes on cellular MDA content The present invention also uses the TBA method to detect the MDA content of LO2 hepatocytes and studies the effect of MoS2 nanozyme on the cellular MDA content.

[0073] This step uses an MDA kit for detection. The MDA kit is based on the reaction of 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.

[0074] The specific steps include: 1. Sample Pretreatment After LO2 hepatocytes were counted, 5.0×10 4 Each well was inoculated on a six-well plate and set as a blank control group, a H2O2 control group, and a C-MoS2 test group, namely C-MoS2-L (5µg / mL), C-MoS2-M (20µg / mL), and C-MoS2-H (40µg / mL). When the density of each well reached 70%-80% under a microscope, the cells of the control group were treated with MoS2 nanozymes for 24 hours after being in a medium containing 1000µmol / LH2O2 for 3h. The cells washed three times with PBS were scraped off and collected in a 1.5mL EP tube and marked. After centrifugation at 1000rpm for 10min, the supernatant was discarded and 1mL PBS was added to resuspend, centrifuged again, and the supernatant was discarded. Under ice-water bath conditions, 200µL PBS buffer was added to each tube of cells and ultrasonically disrupted three times, each time for 3-5s.

[0075] 2. MDA reagent pretreatment The composition of the kit and the preparation of reagents are shown in Table 1.

[0076] Table 1 Kit composition and reagent configuration method ; Note: If there is no hemolysis or lipemia in the sample, a blank tube can be used instead of the control tube.

[0077] 3. TBA method experiment Set up blank tubes, standard tubes, and test tubes to be divided into control group, H2O2, C-MoS2-L, C-MoS2-M, and C-MoS2-H. Add samples according to the proportions in Table 2.

[0078] Table 2 Ratio of TBA test reagents and samples ; After the addition of samples, mix well, place in a 95℃ water bath for 40 minutes, cool, and centrifuge at 4000 rpm for 10 minutes. Take the supernatant, measure the absorbance of each group at 532 nm, and then calculate the MDA content of each group in LO2 hepatocytes.

[0079] 4. BCA Protein Concentration Determination Add 20 µL of each sample to a 96-well plate, then add 200 µL of BCA working solution and place at 37°C for 15-30 min. Measure A562nm with an ELISA reader, calculate the protein concentration based on the standard curve and express the results as mean ± standard deviation (SD).

[0080] Calculation formula (unit: nmol / mgprot):

[0081] Fifth, the effect of MoS2 nanozymes on cellular SOD activity The present invention also provides an experiment for detecting the SOD content of LO2 hepatocytes by the WST-1 method, and at the same time, the effect of C-MoS2 nanozyme on the SOD content of cells is carried out, and a SOD activity detection kit is used, which adopts the WST-8 method to determine the SOD activity by colorimetry. The reaction solution and the staining solution are added to a 96-well plate, and then the catalytic solution and the diluent are added. After the sample is gently shaken and mixed, it is placed in a 37°C incubator and incubated for 20 minutes to avoid light. The absorbance is detected at a wavelength of 450nm using an ELISA instrument to construct a SOD standard curve, with the horizontal axis being the SOD unit concentration per milliliter and the vertical axis being the standard sample reading (absorbance unit OD value). The SOD content in the sample is calculated according to the standard curve, and the results are expressed as mean ± standard deviation (SD).

[0082] The specific steps include: 1. Sample Pretreatment This step is the same as the sample pretreatment method for detecting the MDA content of LO2 hepatocytes by TBA method.

[0083] 2. SOD reagent pretreatment The composition of the kit and the reagent preparation are shown in Table 3.

[0084] Table 3 Composition and reagent ratio of the SOD test kit ; Note: Substrate application solution = substrate stock solution: buffer solution = 1:200; enzyme working solution = enzyme stock solution: enzyme diluent = 1:10; all should be prepared before use.

[0085] 3. WST-1 method experiment Set up control wells, control blank wells, assay wells (same as above), and assay blank wells. Add samples according to the ratio in Table 4.

[0086] Table 4 Proportions of samples tested by WST-1 method ; Note: Before the experiment, select normal LO2 hepatocytes for preliminary experiment. Dilute the tissue homogenate of the 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.

[0087] 4. BCA Protein Concentration Determination The determination method of BCA protein concentration is the same as that of TBA method, and the calculation formula is (unit: U / mgprot): ; ; 6. Effect of MoS2 nanozymes on cellular ROS content The present invention also provides an experiment for detecting the ROS content of LO2 liver cells by a chemiluminescence method, and a ROS activity detection kit is used for detection. The ROS kit uses DCFH-DA as a fluorescent probe (provided by the kit). DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. It is hydrolyzed by esterase in the cell to generate DCFH. DCFH is oxidized by intracellular ROS to generate a fluorescent substance DCF. The ROS content in the sample is calculated based on the proportional relationship between the fluorescence intensity and the intracellular ROS level, and the result is expressed as mean value ± standard deviation (SD).

[0088] The specific steps include: diluting the DCFH-DA probe (provided in the kit) with serum-free medium at 1:1000 to a final concentration of 10µM. Sample pretreatment This step is the same as the sample pretreatment method for detecting the MDA content of LO2 hepatocytes by TBA method. After washing the cells in the plate three times with PBS, 1 ml of the diluted probe was added to each well and incubated at 37°C for 20 minutes. After the incubation is completed, rinse with PBS three times, add 1 ml of PBS to each well, observe using an inverted fluorescence microscope and take pictures in groups.

[0089] The data from the above experiments were analyzed by variance analysis using Graphpad Prism 9.0 software. All experimental data are the average of three repeated experiments and are presented as mean ± standard error (SD).

[0090] The MDA kit, SOD kit and ROS kit used in the experiment were purchased from Nanjing Jiancheng Bioengineering Institute.

[0091] The technical solution of the present invention is described in detail below through specific embodiments.

[0092] Example 1 This embodiment provides a method for preparing molybdenum disulfide nanozyme, and the specific steps include: S1. Provide precursor solution Weigh 1.195g of molybdenum powder in a round-bottom flask, slowly add 10mL of 30% hydrogen peroxide solution in an ice-water bath, and continue to stir until the two react completely to obtain a precursor solution of molybdenum peroxide. Dilute to 250mL with deionized water to prepare a precursor solution of molybdenum peroxide (MoO2(OH)(OOH)) with a concentration of 0.0498mol / L for later use.

[0093] S2. Providing MoS2 Nanozymes First, 200 mg KSCN and 300 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 180°C in an oven for 24 hours, and then cooled naturally to room temperature.

[0094] S3. Post-processing The resulting black solid was centrifuged and washed several times with ethanol and water. Finally, the collected product was dried under vacuum at 80 °C for 12 hours to obtain molybdenum disulfide nanozyme (MoS2 nanozyme or C-MoS2).

[0095] Structural characterization: See also Figure 1 and Figure 2 The morphology and microstructure of the nanomaterial were studied at different resolutions using a scanning electron microscope (SEM). Figure 1 Panoramic picture of molybdenum disulfide nanomaterials at low resolution. C-MoS2 has a uniform cluster structure and the nanoparticles are clearly stacked. Figure 2This is a SEM photo taken under high-resolution conditions. It can be seen from the figure that the particles of molybdenum disulfide nanomaterials are stacked and the cross-linking phenomenon between the particles is obvious, 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 50~80nm, and the size is uniform.

[0096] See also Figure 3 , which is the X-ray powder diffraction (XRD) of C-MoS2 provided in this embodiment. In this spectrum, according to the angle of the diffraction peak of the test object, it can be determined that the obtained MoS2 is a hexagonal crystal phase structure (JCPDS No.37-1492), wherein the diffraction peaks at diffraction angles 2θ of 14.38, 32.68, 35.87, 44.15 and 58.33 correspond to different crystal planes (002), (100), (102), (006) and (110), respectively, and there are no other obvious miscellaneous peaks, indicating that the material has good crystallinity.

[0097] See also Figure 4 , which is the EDS spectrum of the molybdenum disulfide nanomaterial provided in this embodiment. It can be seen from the figure that the material is mainly composed of two elements, Mo and S (Si is a 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 MoS2.

[0098] Figure 5 and Figure 6 This is the S / Mo element distribution diagram in MoS2 nanobelts, which confirms the uniform distribution of Mo and S on the surface of MoS2 nanobelts. The above results indicate that the MoS2 nanomaterial was successfully prepared.

[0099] Figure 7 The molybdenum disulfide nanozyme provided in this embodiment was measured by ultraviolet spectrophotometer (UV) to compare the absorbance of C-MoS2 at pH = 3, pH = 7, and pH = 11 to evaluate the stability of the material in an acidic and alkaline environment. Figure 8-Figure 10 They are respectively the UV absorption spectra at 0h, 4h and 24h at pH = 3, pH = 7 and pH = 11. As can be seen from the figure, within the range of pH = 3 to 11, the stability of C-MoS2 is good, indicating that the C-MoS2 provided by the present invention has good acid and alkali resistance within a wide range of pH = 3 to 11.

[0100] Example 2 The difference between this embodiment and embodiment 1 is only that the addition amount of zinc nitrate is different.

[0101] Specifically include: First, 200 mg KSCN and 460 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 180°C in an oven for 24 hours, and then cooled naturally to room temperature.

[0102] Finally, molybdenum disulfide nanozyme was obtained.

[0103] Example 3 The difference between this embodiment and embodiment 1 is only that the addition amount of zinc nitrate is different.

[0104] Specifically include: First, 240 mg KSCN and 300 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 180°C in an oven for 24 hours, and then cooled naturally to room temperature.

[0105] Finally, molybdenum disulfide nanozyme was obtained.

[0106] Example 4 The difference between this embodiment and embodiment 1 is only that the addition amount of zinc nitrate is different.

[0107] Specifically include: First, 150 mg KSCN and 300 mg zinc nitrate were added to 10 ml MoO2(OH)(OOH) solution and stirred for 20 minutes to obtain a homogeneous solution. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 180°C in an oven for 24 hours and cooled naturally to room temperature.

[0108] Finally, molybdenum disulfide nanozyme was obtained.

[0109] Example 5 The difference between this embodiment and embodiment 1 is only that the conditions of the hydrothermal reaction are different, specifically including: S2. Providing MoS2 Nanozymes First, 200 mg KSCN and 300 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 160°C in an oven for 20 hours, and then cooled naturally to room temperature.

[0110] Example 6 The difference between this embodiment and embodiment 1 is only that the amount of zinc nitrate added is different, specifically including: S2. Providing MoS2 Nanozymes First, 200 mg KSCN and 400 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 160°C in an oven for 20 hours, and then cooled naturally to room temperature.

[0111] Comparative Example 1 The difference between this comparative example and Example 1 is that 2.5mmol MoO3 powder sample is dispersed in 30mL deionized water, and then 2mL 4M HCl solution is added, and stirring is continued for 10min. After stirring is completed, 6.25mmol KSCN is weighed and added to the above reaction solution, and stirring is continued for 10min. Then the above reaction solution is transferred to a polytetrafluoroethylene liner with a volume of 50mL and placed in a hydrothermal kettle. The reactor is placed in an oven, heated to 180°C and reacted for 24h. After the hydrothermal reaction is completed, the reactor is taken out and allowed to cool naturally. Finally, the reaction product is filtered and collected, and rinsed with water and ethanol three times each, and dried in a vacuum oven at 80°C for 10h to obtain a black powder sample.

[0112] The method of this comparative example can also be found in Xianzhong Zeng et al., Solid-contact K + -selectiveelectrode based on three-dimensional molybdenum sulfide nanoflowers as ion-to-electron transducer + , Sensors and Actuators B , 234 (2016), 80~83.

[0113] See also Fig.18 and Fig.19 The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of staggered nanosheets.

[0114] Comparative Example 2 The difference between this comparative example and Example 1 is that KSCN and zinc nitrate are directly added to the sodium molybdate solution, and a uniform solution is obtained after stirring for 20 minutes, wherein the molar ratio of sodium molybdate, KSCN and zinc nitrate is 1:4:1. Then, the uniform solution is transferred to a polytetrafluoroethylene-lined autoclave, and heated at 180°C in an oven for 24 hours, and naturally cooled to room temperature. The other steps are the same.

[0115] The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of staggered nanosheets, which is similar to that of comparative example 1.

[0116] Comparative Example 3 The difference between this comparative example and Example 1 is that KSCN and zinc nitrate are directly added to the sodium molybdate solution, and a uniform solution is obtained after stirring for 20 minutes, wherein the molar ratio of sodium molybdate, KSCN and zinc nitrate is 1:6:1. Then, the uniform solution is transferred to a polytetrafluoroethylene-lined autoclave, and heated at 180°C in an oven for 24 hours, and naturally cooled to room temperature. The other steps are the same.

[0117] The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is a spherical structure composed of staggered nanosheets, which is similar to that of comparative example 1.

[0118] Comparative Example 4 The only difference between this comparative example and Example 1 is that no zinc nitrate is added.

[0119] Specifically include: First, 200 mg of KSCN was added to 10 ml of MoO2(OH)(OOH) precursor solution and stirred for 20 minutes to obtain a homogeneous solution. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 200 °C in an oven for 24 hours and cooled naturally to room temperature.

[0120] Finally, molybdenum disulfide nanomaterial is obtained.

[0121] See also Fig. 20 and Fig.21 , 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 agglomeration and fusion of nanoparticles, and the whole has a pore-like coral structure.

[0122] Comparative Example 5 The difference between this comparative example and Example 1 is only that zinc nitrate is not added.

[0123] Specifically include: First, 200 mg of KSCN was added to 10 ml of MoO2(OH)(OOH) precursor solution and stirred for 20 minutes to obtain a homogeneous solution. Then, the homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 180°C in an oven for 24 hours and then cooled naturally to room temperature.

[0124] Finally, molybdenum disulfide nanomaterial is obtained.

[0125] The structure of the molybdenum disulfide nanomaterial synthesized in this comparative example is similar to that of comparative example 4.

[0126] Comparative Example 6 This comparative example provides a molybdenum disulfide nanomaterial, and the specific steps include: Weigh 25 mg of MoO3 sample and disperse it in 30 mL of deionized water, then weigh and add 25 mg of PVP and continue stirring for 10 minutes. After stirring, weigh 50.67 mg of NaSCN and add it to the above reaction solution, and continue stirring for 10 minutes. After stirring, transfer the above reaction solution to a polytetrafluoroethylene liner with a volume of 50 mL and place it in a hydrothermal kettle. Then place the reactor in an oven and perform a hydrothermal reaction at 180°C for 24 hours. After the reaction is completed, let the reactor cool naturally, then filter and collect the product, rinse it with water and ethanol three times each, and dry it in a vacuum oven at 80°C for 10 hours to obtain a black powder, which is molybdenum disulfide nanomaterial.

[0127] This comparative method can also be found in Juan Li et al., A facile synthesis of porous MoS2coralloids for efficient application in water treatment, Materials Letters , 182(2016), 347~350.

[0128] See also Fig. 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, the particle size is uneven, and the average particle size is 50~100nm.

[0129] Comparative Example 7 The difference between this comparative example and Example 1 is that the conditions of the hydrothermal reaction in step S2 are different. Specifically, step S2 includes: S2. Provide molybdenum disulfide nanozyme (C-MoS2) First, 200 mg KSCN and 100 mg zinc nitrate were added to 10 ml of MoO2(OH)(OOH) precursor solution, and a uniform solution was obtained after stirring for 20 minutes. Then, the uniform solution was transferred to a polytetrafluoroethylene-lined autoclave and heated at 200 ° C in an oven for 24 hours, and then cooled naturally to room temperature.

[0130] The morphology of the molybdenum disulfide nanomaterial obtained in this comparative example is similar 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 accelerates the reaction rate and increases the particle size.

[0131] The molybdenum disulfide nanomaterial synthesized in the above embodiments and comparative examples, from the morphology, is a cluster structure obtained by cross-linking and overlapping nanoparticles by reacting with MoO2(OH)(OOH) as a precursor material. By controlling the ratio between the sulfur source, zinc salt and molybdenum source and the conditions of the hydrothermal reaction, a cluster structure with uniform particle size and stacked and cross-linked can be obtained. In Comparative Examples 1 to 3, the molybdenum disulfide obtained by using molybdenum oxide or sodium molybdate as the molybdenum source is a sheet structure, and the nanosheets can be agglomerated to form a flower ball structure under the action of zinc salt. No zinc nitrate was added in Comparative Examples 4 and 5, and the obtained molybdenum disulfide nanomaterials agglomerated into a coral structure containing holes. The molybdenum disulfide nanomaterial obtained in Comparative Example 6 is a dispersed granular structure. After the temperature of the hydrothermal reaction was increased in Comparative Example 7, the surface of the cluster structure obtained was rougher due to the larger particles; obviously, when the molar ratio of the molybdenum source to the zinc salt was 1:1, the structure of the obtained molybdenum disulfide was different due to the different precursors of the molybdenum source.

[0132] Based on the different morphologies obtained by different hydrothermal reaction conditions and precursors of molybdenum disulfide nanomaterials, the present invention tests the nanozyme-like related properties of the clustered molybdenum disulfide nanomaterials provided by the present invention, and compares the performance with other morphologies.

[0133] Performance Test: 1. Construction of hydrogen peroxide-induced oxidative stress model Experiments to test the cytotoxicity of LO2 to normal human hepatocytes using the oxidative stress inducer H2O2.

[0134] By using IC 50 The concentration of H2O2 induced cell damage, which can be used to study the cell's own antioxidant defense mechanism and the protective effect of exogenous antioxidants. The median lethal dose (IC 50 ) as the concentration of hydrogen peroxide-induced oxidative stress model, can effectively balance the degree of cell damage and survival rate, simulate the real pathological process, facilitate experimental operation and result analysis, and has a high degree of standardization.

[0135] See also Fig.12When different concentrations of oxidative stress inducer H2O2 (concentrations were 0, 800µmol / L, 900µmol / L, 1000µmol / L, 1100µmol / L, 1200µmol / L and 1300µmol / L) were co-incubated with cells for 3h, the cell viability of LO2 hepatocytes showed a significant decrease, and the degree of decrease was positively correlated with the concentration of H2O2. Compared with the H2O2 control group, after H2O2 acted on cells, the cell survival rate showed a dose-dependent decrease with the increase of concentration. Experiments have shown that when the concentration of H2O2 is 1000mmol / L, the cell survival rate is about 50%. The hydrogen peroxide-induced oxidative stress model is based on the H2O2 concentration of 1000µmol / L selected as the H2O2 control group for cell activity, oxidative damage and other tests.

[0136] 2. Determination of the toxicity of MoS2 nanozymes on LO2 hepatocytes In order to explore the effect of molybdenum disulfide nanozymes on LO2 hepatocytes, different concentrations of C-MoS2 were applied to LO2 hepatocytes at 0, 5µg / mL, 10µg / mL, 15µg / mL, 20µg / mL, 30µg / mL, 40µg / mL, and 50µg / mL. After culturing for 24 hours, the MTT experiment was performed.

[0137] See also Fig.11 The different concentrations of C-MoS2 provided in Example 1 were used as the test group and incubated with LO2 hepatocytes for 24 hours. The results showed that there was no significant change in the cell viability between the control group (C-MoS2 concentration was 0) and the test group, and the biological activity of LO2 hepatocytes was still maintained above 80%, indicating that within the range of 0~50μg / mL, the effect on the survival rate of the cells was not obvious, indicating that the C-MoS2 provided by the present invention has no toxic effect on the cells.

[0138] 3. Test of C-MoS2 nanozyme on cell activity recovery induced by H2O2 oxidative stress The C-MoS2 concentration provided in Example 1 is 5 µg / mL, which is calculated as the C-MoS2-L group, the C-MoS2 concentration is 20 µg / mL, which is calculated as the C-MoS2-M group, and the C-MoS2 concentration is 40 µg / mL, which is calculated as the C-MoS2-H group. The concentration of the oxidative stress inducer H2O2 is 1000 µmol / L, which is calculated as the H2O2 control group; the blank control group is a control group in which the concentrations of C-MoS2 and the oxidative stress inducer H2O2 are both 0, that is, a control group that has not undergone H2O2 oxidative stress induction.

[0139] See also Fig.13, the C-MoS2 test group set at different concentrations of C-MoS2 provided in Example 1 was compared with the oxidative stress inducer H2O2, the control group and the blank control group for cell activity. With the increase in the concentration of C-MoS2 nanozymes (C-MoS2-L group, C-MoS2-M group and C-MoS2-H group), the cell activity of LO2 hepatocytes induced by H2O2 oxidative stress was 67.50%, 75.42% and 82.37%, respectively, and the cell activity gradually recovered. Furthermore, the present invention also adjusted the concentration of C-MoS2 to 50µg / mL. The results showed that the recovery of the activity of LO2 hepatocytes was close to that of the blank control group, indicating that the recovery of the cell activity of LO2 hepatocytes induced by H2O2 oxidative stress is proportional to the concentration of C-MoS2. In the figure, compared with the blank control group, ; Compared with H2O2, ##P<0.01, ###P<0.001; compared with C-MoS2-L, ^P<0.05, ^^P<0.01, ^^^P<0.001. Data are expressed as mean ± standard deviation (SD). This indicates that C-MoS2 has a restorative effect on the activity of cells induced by H2O2 oxidative stress, and at a C-MoS2 concentration of 50 µg / mL, the activity of LO2 hepatocytes induced by H2O2 oxidative stress can be restored to a level close to that before induction.

[0140] Furthermore, the molybdenum disulfide nanozymes provided in Examples 2 to 6 were used as the test group. When the C-MoS2 concentration was 40 µg / mL, the cell activity of LO2 hepatocytes was also able to reach more than 80%, indicating that the molybdenum disulfide nanozymes provided by the present invention can effectively inhibit H2O2-induced oxidative damage. The specific experimental results are the same as those in Example 1 and will not be repeated here.

[0141] For Comparative Examples 1 to 3, when the C-MoS2 concentration was 40 µg / mL, the cell activity of LO2 hepatocytes was approximately 60%, among which the cell activities of Comparative Examples 1 to 3 were recovered to 62.14%, 61.06% and 60.89% at the highest; compared with Example 1, it is shown that the molybdenum disulfide nanomaterials synthesized from different precursors have a certain restoring effect on the cell activity of LO2 hepatocytes induced by H2O2 oxidative stress as nanozymes, and the molybdenum disulfide nanozyme synthesized using MoO2(OH)(OOH) as a precursor in this application has a significantly better ability to restore cell activity than other precursors.

[0142] In Comparative Examples 4, 5 and 7, when the C-MoS2 concentration was 40 µg / mL, the cell activity of LO2 hepatocytes recovered to 70.52%, 72.06% and 72.94% at the highest. Compared with Example 1, the molybdenum disulfide nanozyme provided in Example 1 is more effective in inhibiting H2O2-induced oxidative damage, indicating that the cluster structure provided by the present invention is superior to the flower ball structure and the pore coral structure. In Comparative Example 6, when the C-MoS2 concentration was 40 µg / mL, the cell activity of LO2 hepatocytes was all below 40%, which may be related to its structure on the one hand, and the organic solvent used in its synthesis process may also have a certain effect on the recovery of cell damage on the other hand.

[0143] In the pore-like coral structures generated in Comparative Examples 4 and 5, since the pore-like structures can provide more active sites, their ability to restore cell activity is relatively poorer than that of the present invention. The possible reasons are: due to the addition of zinc nitrate, not only can the clustered structure obtained by cross-linking and overlapping the nanoparticles, but also the particle size of the nanoparticles can be regulated. Nanoparticles with smaller particle sizes form clustered structures with rough surfaces, and small-sized molybdenum disulfide nanozymes can provide more active sites, thereby showing better biological activity; although the pore-like coral structure has a higher specific surface area, the particle size of the molybdenum disulfide particles is larger.

[0144] 4. Effect on cell MDA content The MDA kit is used for detection. MDA reacts with 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.

[0145] See also Fig.14 , is the effect of MoS2 nanozyme on MDA content. The MDA content of the blank control group was 1.06±0.09nmol / mgprot; the cell MDA content of the H2O2 control group was 5.81nmol / mgprot. According to the calculated content of MDA in the samples, the results were expressed as mean ± standard deviation (SD). The results showed that the MDA content in the C-MoS2 experimental group was significantly higher than that in the blank control group, indicating that the cellular MDA content increased with the participation of H2O2; after intervention with molybdenum disulfide nanozymes, compared with the H2O2 group, the MDA contents in the C-MoS2-L group, C-MoS2-M group and C-MoS2-H group were 4.82nmol / mgprot, 3.95nmol / mgprot and 1.98nmol / mgprot, respectively, and the MDA contents were significantly decreased, indicating that molybdenum disulfide nanozymes can reduce the MDA content in cells in a dose-dependent manner. When the concentration of C-MoS2 reaches 50µg / mL, the MDA content can be reduced to the level of the blank control group.

[0146] 5. Effect on Cellular SOD Content Use the SOD activity detection kit, which uses the WST-8 method to measure SOD activity by colorimetry. Add the reaction solution and staining solution to a 96-well plate, and then add the catalyst solution and diluent. After gently shaking the sample to mix, place it in a 37°C incubator and incubate it for 20 minutes, avoiding light. Use an ELISA reader to detect the absorbance at a wavelength of 450nm to construct a SOD standard curve. The horizontal axis is the concentration of SOD units per milliliter, and the vertical axis is the standard sample reading (absorbance unit OD value). Calculate the SOD content in the sample based on the standard curve, and express the results as the mean ± standard deviation (SD). See Fig.15 , is the effect of C-MoS2 nanozyme on SOD activity. The SOD content of the blank control group is 9.14±0.01U / mgprot, and the SOD activity of the cells in the H2O2 control group is 4.52±0.26U / mgprot. The significant decrease in SOD activity indicates that hydrogen peroxide inhibits SOD activity in LO2 hepatocytes. Compared with the H2O2 group, the SOD activities of the C-MoS2-L group, C-MoS2-M group and C-MoS2-H group were 6.34±0.25U / mgprot, 7.26±0.10U / mgprot and 8.17±0.18U / mgprot, respectively. When the C-MoS2 concentration reached 50µg / mL, the SOD activity could be restored to the level of the blank control group. Obviously, it increased with the increase of C-MoS2 dosage, indicating that C-MoS2 nanozyme can increase the activity of intracellular SOD.

[0147] 6. Effect on cellular ROS content A ROS activity detection kit was used. The kit uses DCFH-DA as a fluorescent probe. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. It is hydrolyzed by esterase in the cell to generate DCFH. DCFH is oxidized by intracellular ROS to generate the fluorescent substance DCF. The ROS content in the sample is calculated based on the positive relationship between the fluorescence intensity and the intracellular ROS level, and the results are expressed as mean ± standard deviation (SD). Fig.16 , is the effect of C-MoS2 nanozyme on ROS level. The ROS fluorescence intensity of the blank control group was 56.13±5.87AU, and the fluorescence intensity of the H2O2 control group was 189.34AU, which was significantly higher than that of the blank control group. After intervention with C-MoS2 nanozyme, compared with the H2O2 control group, the fluorescence intensities of the C-MoS2-L group, C-MoS2-M group and C-MoS2-H group were 38.43AU, 57.69AU and 102.38AU, respectively, which were significantly decreased, indicating that C-MoS2 significantly reduced the ROS level in cells. Further reference Fig.17 , is a comparison of the fluorescence intensity of intracellular reactive oxygen species in the blank control group, H2O2 control group C-MoS2-L group, C-MoS2-M group and C-MoS2-H group. The figure shows that under the stimulation of H2O2, the level of reactive oxygen species in the cell is the highest, and after adding different concentrations of C-MoS2 nanozymes, the level of reactive oxygen species gradually decreases. When reactive oxygen species are present in the cell, DCFH is oxidized to a strong green fluorescent substance DCF, and its fluorescence intensity is proportional to the level of intracellular reactive oxygen species. The increase in intracellular reactive oxygen species is one of the signs of apoptosis. Obviously, the C-MoS2 nanozyme provided by the present invention has a protective effect and an antioxidant effect on cells, and can significantly reduce the level of reactive oxygen species in cells induced by H2O2.

[0148] Obviously, according to the aforementioned test results, MoS2 nanozymes have a significant effect on improving the therapeutic effect on LO2 hepatocytes in the liver fibrosis mouse model. MoS2 nanozymes can be used to prepare nanomedicines for oxidative stress-related diseases, as well as for the preparation of related drugs for the treatment of H2O2-induced oxidative stress-induced liver damage.

[0149] Based on the above analysis, the molybdenum disulfide nanozyme synthesized with MoO2(OH)(OOH) as a precursor provided by the present invention is different from the molybdenum disulfide nanomaterials with nanosheet, granular and coral-like structures in the prior art. In particular, it is not only non-toxic to cells, but also has a protective effect, and can more effectively inhibit H2O2-induced oxidative damage. When the concentration of C-MoS2 is 40µg / mL, the cell activity of LO2 hepatocytes can be restored to more than 80%, and when the concentration of C-MoS2 is 50µg / mL, the cell activity of LO2 hepatocytes can be restored to the pre-damage level.

[0150] The description of the above embodiments is to facilitate the understanding and use of the invention by those skilled in the art. 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 work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention 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, sulfur source and zinc salt are uniformly mixed and then subjected to a hydrothermal reaction to obtain the molybdenum disulfide nanozyme.

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; the molar ratio of the zinc salt to the molybdenum source is (2-5):

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 any one of claims 1 to 4, characterized in that: 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 molybdenum disulfide nanozyme.

6. The method for preparing the molybdenum disulfide nanozyme according to claim 5, 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.

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

8. The molybdenum disulfide nanozyme according to claim 7, 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.

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

10. Use of the molybdenum disulfide nanozyme as described in any one of claims 7 to 8 in the preparation of nanomedicines for oxidative stress-related diseases.

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