Preparation method of Mn-based multifunctional nano-enzyme, Mn-based multifunctional nano-enzyme and application of Mn-based multifunctional nano-enzyme

Abstract

According to the preparation method of the Mn-based multifunctional nano-enzyme, the Mn-based multifunctional nano-enzyme and the application, the metal frame ZIF-8 is used as a template, Mn is uniformly dispersed in a metal organic frame, and then calcination is performed in an inert atmosphere to successfully realize uniform dispersion of an Mn catalyst on a ZIF-8-derived nitrogen-doped dodecahedron carbon material to form the Mn-based multifunctional nano-enzyme. The optimal valence state proportion of Mn and the optimal manganese coordination structure are obtained by regulating and controlling the Mn doping amount, the carbonization temperature and the like, so that the Mn-based multifunctional nano-enzyme with good UOD, CAT and SOD performance is obtained, and the purposes of rapidly reducing uric acid and hydrogen peroxide and eliminating inflammation are achieved.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a preparation method of a Mn-based multifunctional nanozyme, a Mn-based multifunctional nanozyme and applications. Background Art

[0002] Gout is a recurrent inflammatory disease caused by excessive uric acid production or poor uric acid excretion, which leads to increased uric acid in the blood, and the deposition of monosodium urate crystals (MSU) in the synovium, bursa, cartilage and other tissues of the joints. Current treatments for gout include pain relief during acute attacks (nonsteroidal anti-inflammatory drugs, colchicine and corticosteroids) and long-term treatment to reduce uric acid (uricase preparations and non-enzymatic drugs). More severe urate deposition forms tophi (MSU) and causes joint deformities, which requires surgical treatment. However, these treatments have side effects, and there are few drugs that directly act on the degradation of uric acid in clinical practice. Natural uricase (UOx) can convert uric acid into more water-soluble allantoin and H2O2, thereby reducing plasma uric acid levels. However, natural enzymes are expensive and unstable, and the degradation of uric acid will lead to the accumulation of excessive hydrogen peroxide (H2O2) byproducts in the joint cavity, which will not only cause oxidative stress in cells, but also cause irreversible damage to surrounding joints.

[0003] Most of the existing technologies only focus on the reduction of uric acid levels during the stable period of gout (curing the root cause), or anti-inflammatory and analgesic treatment during the acute gout attack period (curing the symptoms), and fail to organically combine "curing the root cause" and "curing the symptoms". In addition to causing an increase in the byproduct H2O2 of uric acid degradation, the introduction of natural enzymes may also potentially cause an immune response in the human body. The precious metal nanozymes reported in the literature currently perform well in catalytic reactions by introducing expensive precious metals such as platinum (Pt), ruthenium (Ru), and palladium (Pd) as precious metal catalysts, but their potential side effects on the human body cannot be ignored. For example, Pt as a cisplatin anticancer drug can damage DNA and proteins, and Ru and Pd can bind to hemoglobin and affect blood oxygen transport. Summary of the invention

[0004] In view of the limitations of current gout treatments that focus only on lowering uric acid during the stable period and only on anti-inflammatory and analgesic measures during the acute attack period, it is necessary to provide a preparation method, a Mn-based multifunctional nanozyme and its application that can simultaneously degrade uric acid and inhibit inflammatory response.

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] One of the purposes of the present application is to provide a method for preparing a Mn-based multifunctional nanozyme, comprising the following steps:

[0007] Using a metal framework ZIF-8 as a template and uniformly dispersing Mn in the metal framework ZIF-8 to obtain a nanomaterial precursor;

[0008] The nanomaterial precursor is calcined in an inert atmosphere to obtain the Mn-based multifunctional nanozyme.

[0009] In some embodiments, the step of using the metal framework ZIF-8 as a template and uniformly dispersing Mn ions in the metal framework ZIF-8 to obtain a nanomaterial precursor specifically includes the following steps:

[0010] A manganese chloride solution, a zinc nitrate solution and a dimethyl imidazole solution are mixed and sealed and stirred for reaction, wherein the molar ratio of manganese chloride in the manganese chloride solution to zinc nitrate in the zinc nitrate solution is 5% to 50%;

[0011] The stirred mixed solution is centrifuged to obtain a precipitate;

[0012] The precipitate is washed and dried to obtain the nano material precursor.

[0013] In some embodiments, the step of mixing the manganese chloride solution, the zinc nitrate solution and the dimethylimidazole solution and then sealing and stirring the mixture for reaction specifically includes the following steps:

[0014] After mixing the manganese chloride solution and the zinc nitrate solution, seal the mixture and stir at room temperature for 30 minutes. Then add the dimethylimidazole solution to the mixed solution and keep stirring during the process. After the addition of the dimethylimidazole solution is completed, seal the mixture again and stir the mixture for 3 hours at room temperature.

[0015] In some embodiments, the step of centrifuging the stirred mixed solution to obtain a precipitate specifically includes the following steps: centrifuging the stirred mixed solution at 10000 rpm for 7 minutes at room temperature to obtain a precipitate.

[0016] In some embodiments, the step of washing and drying the precipitate to obtain the nanomaterial precursor specifically includes the following steps:

[0017] Cleaning and drying of nanoparticles: The precipitate is cleaned with anhydrous methanol, centrifuged at 10,000 rpm for 7 min at room temperature, and the supernatant is removed; then centrifuged once at 1,000 rpm for 2-3 min, retaining the milky white suspension and removing the brown-black precipitate; then centrifuged 3-5 times at 10,000 rpm for 7 min. After each centrifugation, place it on an oscillator to loosen the precipitate, and ultrasonicate to disperse the material, then add the original volume of the anhydrous methanol to resuspend it, and finally place the washed material in a 40°C oven to dry the precipitate to obtain the nanomaterial precursor.

[0018] In some embodiments, the step of calcining the nanomaterial precursor in an inert atmosphere to obtain the Mn-based multifunctional nanozyme specifically includes the following steps:

[0019] The nanomaterial precursor is heated to 800-1200° C. at a heating rate of 3-6° C. / min under a nitrogen atmosphere and kept warm for 1-3 hours; then the temperature is naturally cooled to room temperature to obtain the Mn-based multifunctional nanozyme.

[0020] In some embodiments, the molar ratio of Mn in the Mn-based multifunctional nanozyme is 40%.

[0021] The second purpose of the present application is to provide a Mn-based multifunctional nanozyme, which is prepared by any of the preparation methods of the Mn-based multifunctional nanozyme described in one item.

[0022] The third purpose of this application is to provide the use of the Mn-based multifunctional nanozyme in treating gout and inhibiting inflammation.

[0023] In some embodiments, the following steps are also included:

[0024] Grinding the Mn-based multifunctional nanozyme to prepare a Mn-based multifunctional nanozyme solution and mixing it with the PEG solution and stirring it for 24 hours to obtain a mixed solution, wherein the Mn-based multifunctional nanozyme solution and the PEG solution have the same concentration and volume;

[0025] The mixed solution was washed with double distilled water and centrifuged at 10000 rpm for 10 min at room temperature. The precipitate was resuspended with the original volume of double distilled water, ultrasonically dispersed and centrifuged again. After washing, the precipitate was dried.

[0026] In some embodiments, the Mn-based multifunctional nanozyme solution and the PEG solution have the same concentration and volume.

[0027] This application adopts the above technical solution, and its beneficial effects are as follows:

[0028] The present application provides a preparation method, a Mn-based multifunctional nanozyme and an application thereof. The metal framework ZIF-8 is used as a template to uniformly disperse Mn in the metal organic framework, and then calcined in an inert atmosphere to successfully achieve uniform dispersion of the Mn catalyst on the nitrogen-doped dodecahedral carbon material derived from ZIF-8 to form a Mn-based multifunctional nanozyme. The optimal Mn valence ratio and manganese coordination structure are obtained by regulating the Mn doping amount and temperature, thereby obtaining a Mn-based multifunctional nanozyme with good UOD, CAT and SOD properties to achieve the purpose of rapidly reducing uric acid and hydrogen peroxide and eliminating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 Scanning electron microscope images of Mn-NC and its precursor MnZIF-8 with different doping ratios provided in the examples of the present application.

[0031] Figure 2 The performance comparison of Mn-NC with different Mn ion doping ratios provided in the embodiments of the present application is shown in Figure 1, wherein (A) represents UOD performance, (B) represents CAT performance, and (C) represents SOD performance.

[0032] Figure 3 X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) of 40% Mn-NC provided in the examples of the present application.

[0033] Figure 4 The UOD performance of Mn-NC at different temperatures provided in the embodiments of the present application.

[0034] Figure 5 The degradation diagrams of uric acid by Mn-NC at different concentrations provided in the examples of the present application, wherein (A) is the degradation diagram of UA detected by UV-vis, and (B) is the degradation kinetic curve of UA.

[0035] Figure 6 The SEM images of Mn-NC degrading MSU at different time periods provided in the examples of the present application.

[0036] Figure 7 The antioxidant performance diagram of Mn-NC provided in the examples of the present application, wherein (A) is the dissolved oxygen content curve of Mn-NC (H2O2: 10mM), and (B) is the SOD inhibition rate diagram of Mn-NC.

[0037] Figure 8 (A) is a CCK-8 test of P-Mn-NC cell biocompatibility provided in the example of the present application, (B) is a hemolysis test of P-Mn-NC, (C) is a fluorescence image of ROS generated by human umbilical vein cell fusion cells (EA-hy926) after different treatments, Hoechst (cell nucleus, blue) and 2,7-dichlorofluorescein (DCFH-DA) (ROS, green), positive control: H2O2 treatment; negative control: phosphate buffered saline (PBS) treatment. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0039] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0040] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0041] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0042] The method for preparing the Mn-based multifunctional nanozyme provided in the embodiment of the present application specifically includes the following steps S110 to S120, and its implementation method is described in detail below.

[0043] Step S110: using the metal framework ZIF-8 as a template and uniformly dispersing Mn in the metal framework ZIF-8 to obtain a nanomaterial precursor.

[0044] In this embodiment, the step of using the metal framework ZIF-8 as a template and uniformly dispersing Mn in the metal framework ZIF-8 to obtain a nanomaterial precursor specifically includes the following steps:

[0045] Step S111: mixing a manganese chloride solution, a zinc nitrate solution and a dimethylimidazole solution, and then sealing and stirring to react, wherein the molar ratio of manganese chloride in the manganese chloride solution to zinc nitrate in the zinc nitrate solution is 5% to 50%.

[0046] Specifically, the manganese chloride solution and the zinc nitrate solution are mixed, sealed and stirred at room temperature for 30 minutes, and then the dimethylimidazole solution is added to the mixed solution, and the stirring state is maintained during the process; after the addition of the dimethylimidazole solution is completed, the mixture is sealed again and stirred for reaction at room temperature for 3 hours.

[0047] Step S112: centrifuging the stirred mixed solution to obtain a precipitate.

[0048] Specifically, the stirred mixed solution was centrifuged at 10000 rpm for 7 min at room temperature to obtain a precipitate.

[0049] Step S113: washing and drying the precipitate to obtain the nanomaterial precursor.

[0050] Specifically, the precipitate is washed with anhydrous methanol, centrifuged at 10,000 rpm for 7 min at room temperature, and the supernatant is removed; then centrifuged once at 1,000 rpm for 2-3 min, retaining the milky white suspension and removing the brown-black precipitate; then centrifuged 3-5 times at 10,000 rpm for 7 min, and after each centrifugation, the precipitate is placed on an oscillator to loosen the precipitate, and ultrasonically dispersed the material, and then the original volume of the anhydrous methanol is added to resuspend it, and finally the washed material is placed in a 40°C oven to dry the precipitate to obtain the nanomaterial precursor.

[0051] It should be noted that the concentration and volume of the manganese chloride solution, zinc nitrate solution and dimethylimidazole solution provided in the above embodiments of the present application can be adjusted. By adjusting the concentration and volume of the above substances and combining the process flow, different Mn valence ratios and manganese coordination structures can be obtained to better meet the actual application needs.

[0052] In addition, the raw materials provided in the above embodiments of the present application can be directly purchased commercially, the raw materials are conventional, low-priced, economical and practical; and there is no need to modify or purify the raw materials, they can be used directly, the operation is simple, the repeatability is strong, and they have mass production value.

[0053] Step S120: calcining the nanomaterial precursor in an inert atmosphere to obtain the Mn-based multifunctional nanozyme.

[0054] In some embodiments, the step of calcining the nanomaterial precursor in an inert atmosphere to obtain the Mn-based multifunctional nanozyme specifically includes the following steps:

[0055] The nanomaterial precursor is heated to 800-1200° C. at a heating rate of 3-6° C. / min under a nitrogen atmosphere and kept warm for 1-3 hours; then the temperature is naturally cooled to room temperature to obtain the Mn-based multifunctional nanozyme.

[0056] In the above embodiments of the present application, in view of the clinical existence of "excessive uric acid", "hydrogen peroxide byproducts" and "excessive accumulation of sodium urate crystals in joints causing inflammatory environment" in acute gout, a multifunctional manganese-based nanozyme is designed to simultaneously achieve rapid degradation of uric acid and cascade removal of hydrogen peroxide, anti-inflammatory and degradation of sodium urate crystals. The metal framework ZIF-8 is used as a template to uniformly disperse Mn in the metal organic framework, and then calcined in an inert atmosphere to successfully achieve uniform dispersion of the Mn catalyst on the nitrogen-doped dodecahedral carbon material derived from ZIF-8 to form a Mn-based multifunctional nanozyme. The optimal Mn valence ratio and manganese coordination structure are obtained by regulating the Mn doping amount and temperature, thereby obtaining a Mn-based multifunctional nanozyme with good UOD, CAT and SOD properties to achieve the purpose of rapidly reducing uric acid and hydrogen peroxide and eliminating inflammation.

[0057] The above technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0058] (1) Preparation, configuration and precautions of each solution and reagent

[0059] a. Manganese chloride solution: Weigh 0.792g MnCl2·4H2O pellets and dissolve in 60ml anhydrous methanol. Seal with a sealing film to prevent excessive exposure to air (methanol is volatile), and dissolve completely. Prepare the solution before use.

[0060] b. Zinc nitrate solution: Weigh 3g Zn(NO3)2·6H2O pellets, dissolve in 60ml anhydrous methanol, seal with sealing film to avoid excessive exposure to air (methanol is volatile), and dissolve completely. The solution should be prepared before use. Note: Zinc nitrate easily absorbs water, so the surrounding environment should be kept dry during the weighing process, and the action should be quick. After weighing, seal it and store it in a drying cabinet.

[0061] c. Polyethylene glycol solution (PEG) (Mw: 8000): Weigh 0.020 g of PEG powder and dissolve it in 5 ml of double distilled water. After it is completely dissolved, dilute to 5 ml. The solution should be prepared before use.

[0062] d. Dimethylimidazole solution: Weigh 6.6g of dimethylimidazole and dissolve it completely in 120ml of anhydrous methanol. Note: Dimethylimidazole easily absorbs water. Keep the surrounding environment dry during the weighing process and move quickly. After weighing, seal it and store it in a drying cabinet. After preparing the solution, seal it with a sealing film in time (methanol is volatile) and wait for use. It is usually prepared immediately before use.

[0063] (2) Preparation of Mn-based multifunctional nanozymes

[0064] a. Mix the prepared manganese chloride solution and zinc nitrate solution, seal with a sealing film to avoid excessive exposure to the air (methanol is volatile), and stir at room temperature for 30 minutes;

[0065] b. Slowly pour the prepared dimethylimidazole solution into the mixed solution of manganese chloride and zinc nitrate through a glass rod, while keeping the rotor rotating during this process;

[0066] c. After adding the dimethylimidazole solution, seal it again with a sealing film to prevent methanol from volatilizing, and react at room temperature for 3 hours while keeping the rotor rotating;

[0067] d. After stirring, centrifuge at 10000 rpm for 7 min at room temperature to obtain a precipitate, which is the prepared nanomaterial precursor;

[0068] e. Cleaning and drying of nanoparticles: Use anhydrous methanol to clean, centrifuge at 10000rpm for 7min at room temperature, remove the supernatant; then centrifuge once at 1000rpm for 2-3min, retain the milky suspension, and remove the brown-black precipitate; centrifuge again at 10000rpm for 7min 3-5 times, place on an oscillator to shake loose the precipitate after each centrifugation, and ultrasonicate to disperse the material, then add the original volume of anhydrous methanol to resuspend. Finally, place the washed material precursor in a 40℃ oven to dry the precipitate.

[0069] f. After the precursor is dried, put the material into a tube furnace, heat it up to 1000℃ at a rate of 5℃ / min under nitrogen atmosphere, and keep it warm for 2h; then turn off the equipment, cool it down naturally, and take out the naturally cooled material 24h after adding the material. Note: This process involves high-temperature working equipment, which may have potential safety issues. During operation, carefully check whether the equipment can operate normally and whether there is enough gas in the nitrogen bottle.

[0070] (3) PEG modification of Mn-based multifunctional nanozymes

[0071] a. After the dried nanoparticles were ground in a mortar, a 2 mg / ml solution was prepared, and ultrasonication was performed for 30 min to fully disperse the material. Then, the prepared 2 mg / ml PEG solution was added in a 1:1 ratio and stirred at room temperature for 24 h.

[0072] b. Washing of PEG-modified nanoparticles: Wash with double distilled water, centrifuge at room temperature at 10,000 rpm for 10 minutes, resuspend the precipitate with the original volume of double distilled water, then disperse with ultrasound and centrifuge again. Wash 4-5 times in this way. After the last wash, place the precipitate in a 40°C oven to dry it.

[0073] The present invention has been verified by preliminary experiments, and the results are as follows:

[0074] In order to determine whether the unPEG-modified manganese-doped metal organic framework (hereinafter referred to as Mn-NC) in the present invention has the performance of decomposing uric acid and sodium urate and resisting oxidative stress, a series of characterization methods were used to detect its enzyme-like activity: 500 μg / ml Mn-NC solution (PBS (pH 7.2-7.4)) was prepared and ultrasonically dispersed for 30 minutes; 1000 μM uric acid (UA) solution (PBS) was prepared and stored in the dark. Take 1 ml of UA solution, then add 8 ml of PBS, mix well, add 1 ml of Mn-NC solution, and stir slowly at the same time. Pay attention to sealing and avoiding light, and measure the absorbance after 2 hours. Use UV-vis to test the absorbance of 270-350nm (UA characteristic absorption peak is at 292nm).

[0075] Similarly, the CAT activity of the material was characterized by the Leici JPB-608 dissolved oxygen meter, and the SOD activity of the material was evaluated by the Biyuntian SOD enzyme activity reagent test kit.

[0076] This example first explored different Mn doping ratios of Mn-NC and evaluated its UOD, CAT and SOD activities to determine the optimal doping ratio. Mn-NC doped with different molar ratios (10, 20, 30, 40, 50%) was prepared, and its morphology is shown in Figure 2. Figure 1 , UOD, CAT and SOD activities Figure 2 .

[0077] Depend on Figure 2 and Figure 3 It can be seen that as the Mn doping ratio increases, the particle size of the Mn-NC nanomaterials increases. Through the characterization of the uricase and catalase activities of the nanomaterials, it was finally determined that the 40% Mn-doped Mn-NC (40% Mn-NC) had the best multi-enzyme catalytic performance, which may be due to its Mn 4 + / Mn2+. Studies have shown that by adjusting the ratio of ions with different valence states, the electron transfer process and electronic structure of nanozymes can be controlled, thereby regulating their catalytic activity.

[0078] Furthermore, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) of 40% Mn-NC were evaluated to further analyze the reasons for the changes in material properties, see Figure 3 .

[0079] according to Figure 3 The X-ray diffraction results show that the (002) and (101) crystal planes correspond to the diffraction peaks of amorphous carbon, which means that Mn-NC has an amorphous carbon structure. The X-ray photoelectron spectrum of 642.20 eV and 640.72 eV are respectively attributed to Mn 4+ and Mn2+ Characteristic peaks, the ratio of which is 50.5%:49.5%. This may be the source of its excellent enzyme activity, and the appropriate valence ratio is conducive to the rapid progress of the reaction.

[0080] Literature has shown that changes in temperature during carbonization can cause changes in material properties, and it has been proven that this change is due to changes in the coordination structure of the metal active site. Therefore, this example further explores the effect of temperature on the activity of 40% Mn-NC nanozymes. First, 40% Mn-NC nanozymes with three different carbonization temperatures (900, 1000, 1100°C) were prepared, and then their uricase activity (UOD) was tested to determine the optimal carbonization temperature. Please refer to Figure 4 .

[0081] pass Figure 4 It was found that the material carbonized at 1000℃ had the best UOD activity, which is similar to the results reported in the literature. The enhancement of catalytic activity may come from the change of the coordination structure of manganese from the original Mn-N4 coordination to the Mn-N3-C structure at 1000℃, which enables the material to obtain a better electronic conduction efficiency and thus enhances the enzyme-like activity of the material. Therefore, this example selected 40% Mn doped and 1000℃ carbonized Mn-NC as the final material preparation parameters, and characterized the degradation ability and kinetic curve of UA by different concentrations of 40% Mn-NC nanomaterials, please refer to Figure 5 .

[0082] In addition to demonstrating that nanomaterials have excellent uric acid-lowering properties, this example also has important significance for the degradation of sodium urate (MSU) in the macroscopic treatment of acute gout. Scanning electron microscopy results show that ( Figure 6 ), after the nanomaterial interacted with sodium urate crystals for 10 minutes, the typical needle-shaped MSU crystals were

[0083] 40% Mn-NC nanomaterials surround it, and at 30 and 60 min, the needle-shaped MSU crystals are gradually decomposed by 40% Mn-NC into fine small crystal fragments and wrapped on the nanomaterials to make the surface rougher. As the time extends to 120 min, the needle-shaped MSU basically disappears completely, and the surface of the 40% Mn-NC material returns to its original state. We speculate that this adsorption may come from the physical adsorption of its amorphous carbon structure.

[0084] The above results show that Mn-NC can effectively eliminate uric acid in in vitro tests. In the treatment of gout, inhibiting inflammatory response is also an indispensable step. Overexpression of reactive oxygen species (ROS) is an important part of inflammatory response. Therefore, removing excessive ROS is expected to relieve inflammation. -Therefore, the activities of catalase (CAT) and superoxide dismutase (SOD) of 40% Mn-NC were investigated. Figure 7 It can be seen that 40% Mn-NC has excellent CAT and SOD properties, which indicates that the material has excellent ROS scavenging effect.

[0085] The biocompatibility of 40% Mn-NC (denoted as P-Mn-NC) was enhanced by modifying it with PEG, and its ability to clear oxidative stress at the cellular level was tested. Figure 8 As shown in A, we evaluated the biosafety of P-Mn-NC in EA-hy926 and RAW264.7 cells. The results showed that at a concentration of 10 μg / ml, P-Mn-NC did not show obvious cytotoxicity to both cells. We further evaluated the blood compatibility of P-Mn-NC by a mouse erythrocyte hemolysis experiment. By monitoring the absorbance at 540 nm (corresponding to hemoglobin released by ruptured erythrocytes), no obvious hemolysis was observed even at a P-Mn-NC concentration of 200 μg / ml, indicating that it has good blood compatibility ( Figure 8 B).

[0086] The results are as follows Figure 8 As shown in Figure C, the ROS level in cells not treated with H2O2 was low, while after H2O2 treatment, the intracellular ROS level increased significantly. It is worth noting that after RAW264.7 cells were treated with 2.5 μg / ml P-Mn-NC, the intracellular ROS fluorescence intensity decreased, and with the increase of nanozyme concentration, the ROS fluorescence intensity further decreased, indicating that its ROS scavenging ability gradually increased. This confirms that P-Mn-NC can effectively scavenge ROS in RAW264.7 cells.

[0087] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A method for preparing a Mn-based multifunctional nanozyme, characterized in that: The steps include: Using a metal framework ZIF-8 as a template and uniformly dispersing Mn in the metal framework ZIF-8 to obtain a nanomaterial precursor; The nanomaterial precursor is calcined in an inert atmosphere to obtain the Mn-based multifunctional nanozyme.

2. The method for preparing the Mn-based multifunctional nanozyme according to claim 1, characterized in that: The step of using the metal framework ZIF-8 as a template and uniformly dispersing Mn in the metal framework ZIF-8 to obtain a nanomaterial precursor specifically includes the following steps: A manganese chloride solution, a zinc nitrate solution and a dimethyl imidazole solution are mixed and sealed and stirred for reaction, wherein the molar ratio of manganese chloride in the manganese chloride solution to zinc nitrate in the zinc nitrate solution is 5% to 50%; The stirred mixed solution is centrifuged to obtain a precipitate; The precipitate is washed and dried to obtain the nano material precursor.

3. The method for preparing the Mn-based multifunctional nanozyme according to claim 2, characterized in that: The step of mixing the manganese chloride solution, the zinc nitrate solution and the dimethylimidazole solution and then sealing and stirring the mixture for reaction specifically comprises the following steps: After mixing the manganese chloride solution and the zinc nitrate solution, seal the mixture and stir at room temperature for 30 minutes. Then add the dimethylimidazole solution to the mixed solution and keep stirring during the process. After the addition of the dimethylimidazole solution is completed, seal the mixture again and stir the mixture for 3 hours at room temperature.

4. The method for preparing the Mn-based multifunctional nanozyme according to claim 2, characterized in that: The step of centrifuging the stirred mixed solution to obtain a precipitate specifically includes the following steps: centrifuging the stirred mixed solution at 10000 rpm for 7 minutes at room temperature to obtain a precipitate.

5. The method for preparing the Mn-based multifunctional nanozyme according to claim 2, characterized in that: The step of washing and drying the precipitate to obtain the nanomaterial precursor specifically includes the following steps: The precipitate was washed with anhydrous methanol, centrifuged at 10000 rpm for 7 min at room temperature, and the supernatant was removed; then centrifuged once at 1000 rpm for 2-3 min, retaining the milky white suspension and removing the brown-black precipitate; then centrifuged 3-5 times at 10000 rpm for 7 min, and after each centrifugation, the precipitate was placed on an oscillator to loosen the precipitate, and ultrasonically dispersed the material, and then the original volume of the anhydrous methanol was added to resuspend it, and finally the washed material was placed in a 40°C oven to dry the precipitate to obtain the nanomaterial precursor.

6. The method for preparing the Mn-based multifunctional nanozyme according to claim 1, characterized in that: The step of calcining the nanomaterial precursor in an inert atmosphere to obtain the Mn-based multifunctional nanozyme specifically includes the following steps: The nanomaterial precursor is heated to 800-1200° C. at a heating rate of 3-6° C. / min under a nitrogen atmosphere and kept at this temperature for 1-3 hours; and then cooled in the furnace to obtain the Mn-based multifunctional nanozyme.

7. The method for preparing the Mn-based multifunctional nanozyme according to claim 1, characterized in that: The molar ratio of Mn in the Mn-based multifunctional nanozyme is 40%.

8. A Mn-based multifunctional nanozyme, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Mn-based multifunctional nanozyme as claimed in claim 8 in treating gout and inhibiting inflammation.

10. The use of the Mn-based multifunctional nanozyme in treating gout and inhibiting inflammation as claimed in claim 9, characterized in that: The following steps are also included: Grinding the Mn-based multifunctional nanozyme to prepare a Mn-based multifunctional nanozyme solution and mixing it with the PEG solution and stirring it for 24 hours to obtain a mixed solution, wherein the Mn-based multifunctional nanozyme solution and the PEG solution have the same concentration and volume; The mixed solution was washed with double distilled water and centrifuged at 10000 rpm for 10 min at room temperature. The precipitate was resuspended with the original volume of double distilled water, ultrasonically dispersed and centrifuged again. After washing, the precipitate was dried.

11. The use of the Mn-based multifunctional nanozyme in treating gout and inhibiting inflammation as claimed in claim 10, characterized in that: The Mn-based multifunctional nanozyme solution has the same concentration and volume as the PEG solution.

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