Manganese-based nanoscale enzyme, preparation method and application thereof

Abstract

The application discloses a manganese-based nanoscale enzyme and a preparation method and application thereof. The preparation method comprises the following steps: (1) potassium permanganate and metal salt are respectively dissolved in water, and the potassium permanganate solution and the metal salt solution are formed by fully stirring; the metal salt is one of zinc salt, calcium salt, magnesium salt, aluminum salt, cerium salt, iron salt, strontium salt, cobalt salt, copper salt, barium salt and zirconium salt; (2) the potassium permanganate solution and the metal salt solution are fully mixed to form a mixed solution, the pH value of the system is adjusted, and the reaction is carried out under alkaline conditions; and (3) the precipitate after the reaction is washed and centrifuged by using water, and the manganese-based nanoscale enzyme is obtained after drying. The preparation method is simple in operation, does not need any heat treatment process, does not need any template agent or ligand, is low in production cost, does not need to use organic reagents, guarantees that the manganese-based nanoscale enzyme synthesized has good biological safety, and simultaneously has good antibacterial performance, anticancer performance and anti-inflammatory performance, and has a good application prospect in the technical field of biological medical materials.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a manganese-based nanozyme, its preparation method, and its application. Background Technology

[0002] Nanozymes are nanomaterials that mimic the catalytic function of natural enzymes, combining the characteristics of both nanomaterials and natural enzymes. They have the following advantages: high stability, low cost, simple processing, and diverse functions; broad-spectrum antibacterial and anticancer activity and stability; and ease of modification or combination with other materials to obtain more functions.

[0003] Nanozymes, as an antibiotic-free strategy, typically eliminate bacteria through three mechanisms: generating reactive oxygen species (ROS) in the bacterial microenvironment, generating hypohalous acids through the oxidation of halide ions, and eliminating bacterial extracellular DNA by mimicking DNases. Cancer development leads to tissue loss, decreased appetite, poorer quality of life, and reduced survival rates in patients. Nanozymes can enhance their killing effect on tumor cells by altering the hypoxic tumor microenvironment, consuming overexpressed glutathione in the tumor microenvironment, and generating ROS. Abnormal inflammatory responses produce excessive pro-inflammatory substances and ROS, excessively recruiting immune cells (such as macrophages) and causing tissue damage, ultimately leading to autoimmune diseases. Superoxide dismutase-like enzymes can dissolve toxic O2. - It can be converted into O2. Catalase-like enzymes can convert toxic H2O2 into O2, thereby eliminating the effects of inflammation.

[0004] Rapid, efficient, and green synthesis of nanozymes is an essential requirement in current production practices. Many current methods for preparing manganese-based nanozymes involve heat treatment, which increases energy consumption and requires specialized heated reaction vessels. In contrast, the production conditions of this invention are at room temperature, eliminating the need for heat treatment. Secondly, the preparation of many current manganese-based nanozymes requires the introduction of other substances as template agents or ligands, including but not limited to bovine serum albumin, Co3O4, silica, amino acids, oxalic acid, tannic acid, tartaric acid, and zucchini fiber, increasing production costs and steps. Furthermore, some manganese-based nanozyme preparation processes involve the use of organic reagents, including but not limited to methanol solutions, formaldehyde solutions, chloroform solutions, and N,N-dimethylformamide, which pose certain risks to biosafety. Summary of the Invention

[0005] To overcome the aforementioned shortcomings and deficiencies of the prior art, the present invention aims to provide a method for preparing manganese-based nanozymes, using potassium permanganate and metal salts as raw materials to form nanomaterials under alkaline conditions. The preparation method of the present invention is simple to operate, requires no heat treatment process, no template agent or ligand, has low production cost, does not require organic reagents, and ensures that the synthesized manganese-based nanozymes have good biosafety.

[0006] The present invention also provides a manganese-based nanozyme and its application. The manganese-based nanozyme has peroxidase-like activity, catalase-like activity and glutathione oxidase activity, and has low toxicity to normal cells. It also has good antibacterial, anticancer and anti-inflammatory properties, and has good application prospects in the field of biomedical materials technology.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a method for preparing manganese-based nanozymes, comprising the following steps:

[0009] (1) Dissolve potassium permanganate and metal salt separately in water and stir thoroughly to form potassium permanganate solution and metal salt solution; the metal salt is one of zinc salt, calcium salt, magnesium salt, aluminum salt, cerium salt, iron salt, strontium salt, cobalt salt, barium salt, copper salt and zirconium salt;

[0010] (2) Thoroughly mix the potassium permanganate solution and the metal salt solution to form a mixed solution, and adjust the pH value of the system so that the reaction can be carried out under alkaline conditions;

[0011] (3) The precipitate after the reaction was washed with water, centrifuged, and dried to obtain manganese-based nanozymes.

[0012] In some embodiments of the present invention, adjusting the pH value of the system specifically means adjusting the pH value of the system to 8-14.

[0013] In some embodiments of the present invention, the reaction time in step (2) is 10 hours to 48 hours.

[0014] In some embodiments of the present invention, the drying is one of freeze drying, natural drying, or oven drying; the drying temperature is 50~100℃.

[0015] In some embodiments of the present invention, the metal salt is a copper salt, and the molar ratio of potassium permanganate to copper salt is 1:(0.5~125).

[0016] In some embodiments of the present invention, the molar ratio of potassium permanganate to copper salt is 1:(0.8~1.2).

[0017] In some embodiments of the present invention, the copper salt is copper nitrate or copper chloride.

[0018] In some embodiments of the present invention, the nanozyme is a manganese-copper nanozyme, and is specifically prepared as follows:

[0019] Potassium permanganate and copper chloride were added to water in a molar ratio of 1:1 to form solutions. The two solutions were then mixed to form a mixed solution. The pH of the solution was adjusted to 10-12, and the reaction time was 20-24 hours. After centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained.

[0020] The present invention also provides a manganese-based nanozyme, which is prepared by the method described above.

[0021] The present invention also provides the application of the manganese-based nanozyme in the preparation of anti-inflammatory drugs and antibacterial drugs.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] (1) The preparation method of manganese-based nanozymes of the present invention does not require any heat treatment process or additional template agents or ligands, nor does it require the participation of any organic reagents. It can obtain manganese-based nanozymes, which is simple to operate, has low production cost, and ensures that the synthesized manganese-based nanozymes have good biosafety.

[0024] (2) The manganese-based nanozyme prepared by the present invention has nanoscale properties and good antibacterial properties.

[0025] (3) The manganese-based nanozyme prepared by the present invention has peroxidase-like activity, catalase-like activity and glutathione oxidase activity, and has low toxicity to normal cells, and has good application prospects in the field of biomedical materials technology. Attached Figure Description

[0026] Figure 1 The images show the physicochemical properties of the manganese-copper nanozyme prepared in Example 1 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0027] Figure 2 The images show the physicochemical properties of the manganese barium nanozyme prepared in Example 2 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0028] Figure 3 The images show the physicochemical properties of the manganese-magnesium nanozyme prepared in Example 3 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0029] Figure 4The images show the physicochemical properties of the manganese-zinc nanozyme prepared in Example 4 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0030] Figure 5 The images show the physicochemical properties of the manganese-aluminum nanozyme prepared in Example 5 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0031] Figure 6 The images show the physicochemical properties of the manganese-cobalt nanozyme prepared in Example 6 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0032] Figure 7 The images show the physicochemical properties of the manganese strontium nanozyme prepared in Example 7 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0033] Figure 8 The images show the physicochemical properties of the manganese-iron nanozyme prepared in Example 8 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0034] Figure 9 The images show the physicochemical properties of the manganese zirconium nanozyme prepared in Example 9 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0035] Figure 10 The images show the physicochemical properties of the manganese cerium nanozyme prepared in Example 10 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0036] Figure 11 The images show the physicochemical properties of the manganese-calcium nanozyme prepared in Example 11 of this invention; where A is a scanning electron microscope image, B is an XRD pattern, and C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen.

[0037] Figure 12 The antibacterial properties of the manganese-based nanozymes prepared in Examples 1-11 of the present invention are shown; where A is a photograph of the plate count method; B is a quantitative analysis of A, representing the antibacterial properties against Escherichia coli.

[0038] Figure 13 These are scanning electron microscope images of the manganese-copper nanozymes prepared in Examples 12-19 of the present invention.

[0039] Figure 14The physicochemical and catalytic properties of the manganese-copper nanozymes prepared in Examples 12-19 of the present invention are shown; wherein A is the XRD pattern of the nanozyme, B is the ability of the nanozyme to consume glutathione, C is the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen, and F and D are the abilities of the nanozyme to produce reactive oxygen species under pH conditions of 7.4, 6.4, and 5.0, respectively.

[0040] Figure 15 The catalytic performance of the manganese-based nanozymes prepared in Examples 1-11 and 14 of this invention is characterized by their peroxidase and catalase activities.

[0041] Figure 16 The antibacterial properties of the manganese-copper nanozymes prepared in Examples 12-19 of this invention are characterized. A and B are photographs using the plate count method; C is the quantitative analysis of A, representing the antibacterial properties against Staphylococcus aureus; D is the quantitative analysis of B, representing the antibacterial properties against Escherichia coli.

[0042] Figure 17 The antibacterial properties of the manganese-copper nanozyme prepared in Example 14 of this invention are characterized. A and B are photographs using the plate count method; C is the quantitative analysis of A, representing the antibacterial performance against Staphylococcus aureus; D is the quantitative analysis of B, representing the antibacterial performance against Escherichia coli. E shows the changes in membrane permeability of Staphylococcus aureus caused by different concentrations of nanozyme using the FDA staining method. F shows the changes in membrane permeability of Escherichia coli caused by different concentrations of nanozyme using the FDA staining method.

[0043] Figure 18 The cell compatibility and anticancer properties of the manganese-copper nanozymes prepared in Examples 12-19 of the present invention are shown in Figure 12; where A represents the cell compatibility of the nanozymes, B represents the anticancer properties of the nanozymes, C represents the cell compatibility of the nanozymes prepared in Example 14 of the present invention, and D represents the anticancer activity of the nanozymes prepared in Example 14 of the present invention.

[0044] Figure 19 This is a DCFH fluorescence image inside macrophages obtained by the manganese-copper nanozyme prepared in Example 14 of the present invention.

[0045] Figure 20 The manganese-copper nanozyme prepared in Example 14 of this invention promotes the expression of CD206 protein in macrophages. Detailed Implementation

[0046] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0047] Example 1:

[0048] Potassium permanganate and copper nitrate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 9, the reaction time was 24 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-copper nanozyme was obtained.

[0049] Its morphological characteristics were observed using a scanning electron microscope. Figure 1 The manganese-copper nanozyme exhibits a nanoparticle morphology. XRD patterns show that the product contains amorphous components and well-crystallized CuO. Hydrogen peroxide decomposition experiments demonstrate that the manganese-copper nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0050] Example 2:

[0051] Potassium permanganate and barium dichloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 12, the reaction time was 42 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-barium nanozyme was obtained.

[0052] Its morphological characteristics were observed using a scanning electron microscope. Figure 2 The manganese barium nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese barium nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0053] Example 3:

[0054] Potassium permanganate and magnesium chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 14, the reaction time was 36 hours, and the solution was centrifuged, washed, and dried at room temperature to obtain manganese-magnesium nanozyme.

[0055] Its morphological characteristics were observed using a scanning electron microscope. Figure 3 The manganese-magnesium nanozyme exhibits a nanosheet morphology. XRD analysis revealed the presence of amorphous products and well-crystallized Mg(OH)₂. Hydrogen peroxide decomposition experiments demonstrated that the manganese-magnesium nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0056] Example 4:

[0057] Potassium permanganate and zinc acetate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 8, the reaction time was 30 hours, and the solution was centrifuged, washed, and dried at room temperature to obtain manganese-zinc nanozyme.

[0058] Its morphological characteristics were observed using a scanning electron microscope. Figure 4 The manganese-zinc nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-zinc nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0059] Example 5:

[0060] Potassium permanganate and aluminum nitrate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 10, the reaction time was 20 hours, and the solution was centrifuged, washed, and dried at 100°C to obtain manganese-aluminum nanozyme.

[0061] Its morphological characteristics were observed using a scanning electron microscope. Figure 5 The manganese aluminum nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese aluminum nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0062] Example 6:

[0063] Potassium permanganate and cobalt chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 12, the reaction time was 10 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-cobalt nanozyme was obtained.

[0064] Its morphological characteristics were observed using a scanning electron microscope. Figure 6 The manganese-cobalt nanozyme exhibits a nanoparticle morphology. XRD patterns show that its crystal form is amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-cobalt nanozyme has the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the ability to continuously decompose hydrogen peroxide to produce oxygen.

[0065] Example 7:

[0066] Potassium permanganate and strontium acetate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 12, the reaction time was 20 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-strontium nanozyme was obtained.

[0067] Its morphological characteristics were observed using a scanning electron microscope. Figure 7 The manganese-strontium nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-strontium nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0068] Example 8:

[0069] Potassium permanganate and ferric chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and the solution was centrifuged, washed, and dried at room temperature to obtain manganese-iron nanozyme.

[0070] Its morphological characteristics were observed using a scanning electron microscope. Figure 8 The manganese-iron nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-iron nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0071] Example 9:

[0072] Potassium permanganate and zirconium oxychloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 10, the reaction time was 48 hours, and after centrifugation, washing, and natural drying at room temperature, manganese zirconium nanozyme was obtained.

[0073] Its morphological characteristics were observed using a scanning electron microscope. Figure 9 The manganese zirconium nanozyme exhibits a nanoparticle morphology. XRD patterns show that its crystal form is amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese zirconium nanozyme has the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the ability to continuously decompose hydrogen peroxide to produce oxygen.

[0074] Example 10:

[0075] Potassium permanganate and cerium nitrate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 14, the reaction time was 10 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-cerium nanozyme was obtained.

[0076] Its morphological characteristics were observed using a scanning electron microscope. Figure 10The manganese-cerium nanozyme exhibits a nanoparticle morphology. XRD patterns show it to be amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-cerium nanozyme possesses the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the capacity to continuously decompose hydrogen peroxide to generate oxygen.

[0077] Example 11:

[0078] Potassium permanganate and calcium nitrate were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 9, the reaction time was 10 hours, and after centrifugation, washing, and natural drying at room temperature, manganese-calcium nanozyme was obtained.

[0079] Its morphological characteristics were observed using a scanning electron microscope. Figure 11 The manganese-calcium nanozyme exhibits a nanoparticle morphology. XRD patterns show that its crystal form is amorphous. Hydrogen peroxide decomposition experiments demonstrated that the manganese-calcium nanozyme has the ability to decompose hydrogen peroxide to produce oxygen, exhibiting good catalase activity and the ability to continuously decompose hydrogen peroxide to produce oxygen.

[0080] Example 12:

[0081] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:0. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named Mn.

[0082] Example 13:

[0083] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:0.5. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu0.5.

[0084] Example 14:

[0085] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:1. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu1.

[0086] Example 15:

[0087] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:2. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu2.

[0088] Example 16:

[0089] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:5. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu5.

[0090] Example 17:

[0091] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:25. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu25.

[0092] Example 18:

[0093] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 1:125. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named MnCu125.

[0094] Example 19:

[0095] Potassium permanganate and copper chloride were added to water to form solutions, and then the two solutions were mixed to form a mixed solution with a final molar ratio of 0:1. The pH of the solution was adjusted to 10, the reaction time was 24 hours, and after centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained and named Cu.

[0096] Example 20:

[0097] The antibacterial properties were characterized using Gram-negative Escherichia coli ATCC 8739 as a model bacterium. Both bacteria were cultured separately in nutrient broth and incubated in a mold incubator for 24 hours before use. The antibacterial rate of different proportions of nanozymes was tested using the plate count method. The manganese-based nanozymes prepared in Examples 1-11 were sterilized by ultraviolet light and then added to the cultured E. coli bacterial suspensions to achieve a final bacterial concentration of 1 × 10⁻⁶. 6The final concentration of the nanozyme was 25 μg / mL, CFU / mL. After vortexing and incubation at 37℃ and humidity for 3 h, the nanozyme was plated onto agar plates. After another 18 hours of incubation, the colonies on the agar plates were counted and photographed. The antibacterial rate of the nanozyme was calculated using the following formula: R = (ABlank – An) / ABlank × 100%

[0098] Where R is the antibacterial rate of the nanozyme (%), ABLank is the number of colonies in the blank group, and An is the number of colonies in the nanozyme group.

[0099] Figure 12 The results show that the antibacterial rate was 87.60% in Example 1, 83.09% in Example 2, 45.77% in Example 3, 99.89% in Example 4, 75.54% in Example 5, 96.51% in Example 6, 94.48% in Example 7, 67.87% in Example 8, 70.91% in Example 9, 68.09% in Example 10, and 100% in Example 11. These data indicate that the manganese-based nanozymes synthesized in Examples 1-11 possess good antibacterial properties.

[0100] Example 21:

[0101] The manganese-copper nanozymes prepared in Examples 12-19 were observed using scanning electron microscopy. Figure 13 The results show that all nanozymes exhibit the shape of nanoparticles. Figure 14 As shown in Figure A, the MnO2 synthesized in this invention is amorphous. As the amount of copper chloride added increases, the crystal form of the nanozyme gradually changes from amorphous to crystalline, and its crystal forms include CuO and Cu2(OH)3Cl.

[0102] Example 22:

[0103] The glutathione peroxidase, catalase, and peroxidase activities of the nanozyme were characterized. Figure 14 As shown in Figure B, the manganese-copper nanozymes prepared in Examples 12-19 can degrade glutathione and have good glutathione peroxidase activity. Figure 14 As shown in Figure C, the manganese-copper nanozymes prepared in Examples 12-19 can decompose hydrogen peroxide to produce oxygen and have good catalase activity. Figure 14 The results from the Chinese DF study show that the manganese-copper nanozymes prepared in Examples 12-19 generate reactive oxygen species (ROS) at pH 5.0, while generating very little ROS at pH 6.4 and pH 7.4, indicating good peroxidase activity.

[0104] Example 23:

[0105] The catalase and peroxidase activities of the nanozymes were characterized. The maximum reaction rate and Michaelis constant of the manganese-based nanozymes prepared in Examples 1-11 and Example 14 were calculated using the Michaelis equation, and the enzyme activity was quantitatively characterized by the ratio of the maximum reaction rate to the Michaelis constant. Figure 15 In Example 1, the peroxidase activity was 5.62 × 10⁻⁶. -5 s -1 The catalase activity was 1.05 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 2 was 4.23 × 10⁻⁶. -5 s -1 The catalase activity was 2.65 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 3 was 2.90 × 10⁻⁶. -5 s -1 The catalase activity was 2.77 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 4 was 2.43 × 10⁻⁶. -5 s -1 The catalase activity was 1.68 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 5 was 2.62 × 10⁻⁶. -5 s -1 The catalase activity was 2.74 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 6 was 5.16 × 10⁻⁶. -5 s -1 The catalase activity was 1.83 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 7 was 3.97 × 10⁻⁶. -5 s -1 The catalase activity was 1.52 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 8 was 3.88 × 10⁻⁶. -5 s -1 The catalase activity was 3.19 × 10⁻⁶. -4 s -1 The peroxidase activity in Example 9 was 1.07 × 10⁻⁶. -5 s -1 The catalase activity was 1.92 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 10 was 1.27 × 10⁻⁶. -4s -1 The catalase activity was 1.50 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 11 was 6.17 × 10⁻⁶. -5 s -1 The catalase activity was 2.67 × 10⁻⁶. -3 s -1 The peroxidase activity in Example 14 was 1.06 × 10⁻⁶. -4 s -1 The catalase activity was 1.85 × 10⁻⁶. -3 s -1 Quantitative experiments have demonstrated that the nanozymes synthesized in this invention possess excellent peroxidase and catalase activities.

[0106] Example 24:

[0107] The antibacterial properties were characterized using Gram-negative Escherichia coli ATCC 8739 and Gram-positive Staphylococcus aureus ATCC 6538 as model bacteria. Both bacteria were cultured separately in nutrient broth and incubated in a mold incubator for 24 hours before use. The antibacterial rate of different proportions of nanozymes was tested using the plate count method. The manganese-copper nanozymes prepared in Examples 12-19 were sterilized by ultraviolet light and then added to the cultured Staphylococcus aureus and Escherichia coli cultures, respectively, to achieve a final bacterial concentration of 1 × 10⁻⁶ for both. 6 The final concentration of the nanozyme was 12 μg / mL, CFU / mL. After vortexing and incubation at 37℃ and humidity for 3 h, the nanozyme was plated onto agar plates. After another 18 hours of incubation, the colonies on the agar plates were counted and photographed. The antibacterial rate of the nanozyme was calculated using the following formula: R = (ABlank – An) / ABlank × 100%

[0108] Where R is the antibacterial rate of the nanozyme (%), ABLank is the number of colonies in the blank group, and An is the number of colonies in the nanozyme group.

[0109] The antibacterial rate of the manganese-copper nanozyme prepared in Example 14 was tested using the plate count method. The manganese-copper nanozyme prepared in Example 14 was sterilized and set aside. The manganese-copper nanozyme prepared in Example 14 was added to Staphylococcus aureus and Escherichia coli bacterial suspensions, respectively, to achieve a final bacterial concentration of 1 × 10⁻⁶. 6 The final concentrations of the manganese-copper nanozyme were 0, 3, 6, 12, 25, 50, and 100 μg / mL (CFU / mL). After vortexing and incubation at 37°C and humidity for 3 h, the nanozymes were plated onto agar plates. After another 18 hours of incubation, the colonies on the agar plates were counted and photographed. The antibacterial rate of different concentrations of manganese-copper nanozyme was calculated.

[0110] Figure 16 The results showed that for Staphylococcus aureus, the antibacterial rate of the Mn group was only 9.64%, while the antibacterial rates of MnCu0.5 were 94.53%, MnCu1 98.04%, MnCu2 99.59%, MnCu5 96.82%, MnCu25 98.44%, MnCu125 96.96%, and the Cu group 98.58%. For Escherichia coli, the antibacterial rate of the Mn group was only 54.86%, while the antibacterial rates of MnCu0.5, MnCu1, MnCu2, MnCu5, MnCu25, MnCu125 99.45%, and the Cu group 99.34%. This demonstrates that the manganese-copper nanozymes prepared in Examples 13-19 have good antibacterial properties.

[0111] Figure 17 The results showed that for Staphylococcus aureus, the bacterial survival rate was 82.36% in the 3 μg / mL group, while it was only 14.66% in the 6 μg / mL group. When the concentration of MnCu1 nanozyme was higher than 12 μg / mL, no colonies were observed on the agar plate, proving that its antibacterial rate reached 100%. For Escherichia coli, the bacterial survival rate was 3.86% in the 3 μg / mL group. When the concentration of MnCu1 nanozyme was higher than 6 μg / mL, no colonies were observed on the agar plate, proving that its antibacterial rate reached 100%. This demonstrates that the manganese-copper nanozyme prepared in Example 14 has good antibacterial properties. Furthermore, the results of the FDA experiment showed that the antibacterial effect of the nanozyme is achieved by disrupting the bacterial membrane.

[0112] Example 25:

[0113] Mouse fibroblasts (L929 cells) were selected as a cell model to study the cell compatibility of nanozymes. The manganese-copper nanozymes prepared in Examples 12-19 were immersed in basal culture medium at a concentration of 25 μg / mL, and then fetal bovine serum was added. Cell compatibility was determined using direct contact between the nanozymes and L929 cells. L929 cells were seeded at a density of 5000 / well in 96-well plates and cultured at 37 °C and 5% CO2 for 24 hours to allow complete cell adhesion. The original culture medium was then replaced with a prepared nanoparticle-containing medium, and the cells were co-cultured for 24 hours. The Blank group consisted of L929 cells not co-incubated with the nanozymes. The manganese-copper nanozymes prepared in Example 14 were immersed in basal culture medium at concentrations of 0, 1.5, 3, 6, 12, 25, 50, 100, and 200 μg / mL, and then fetal bovine serum was added. Cytotoxicity was determined using direct contact between the nanoparticles and L929 cells. L929 cells were seeded at a density of 5000 cells / well in 96-well plates and cultured at 37 °C and 5% CO2 for 24 hours to allow for complete cell adhesion and growth. The original culture medium was then replaced with a prepared nanoparticle-containing medium and co-cultured in an incubator for 24 hours.

[0114] Remove the culture medium from the wells and rinse with buffer. Cell viability was then assessed using the CCK8 reagent (Cell Counting Kit-8). Basal culture medium and CCK8 reagent were added to each well to bring the final concentration of CCK8 reagent to 10%, and the absorbance at 450 nm was measured using a microplate reader.

[0115] The results showed that the manganese-copper nanozymes prepared in batches 12-19 had good cell compatibility. Figure 18 The results in Figure A show that there was no significant difference in absorbance among all groups, proving that the nanozyme does not cause toxicity to L929 cells. Figure 18 The results in Figure C show that when the concentration of MnCu1 nanozyme is below 50 μg / mL, the absorbance of each material group is not significantly different from that of the Blank group, while the absorbance of groups with MnCu1 nanozyme concentrations above 100 μg / mL is much lower than that of the Blank group. The manganese-copper nanozyme prepared in Example 14 exhibits good cell compatibility.

[0116] Example 26:

[0117] Mouse breast cancer cells (4T1 cells) were selected as a cell model to study the in vitro anticancer activity of nanozymes. The manganese-copper nanozymes prepared in Examples 12-19 were immersed in basal culture medium at a concentration of 50 μg / mL, and then fetal bovine serum was added. Cytotoxicity was determined using a direct contact method between the nanoparticles and 4T1 cells. 4T1 cells were seeded at a density of 5000 / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours to allow complete cell adhesion. The original culture medium was then replaced with a prepared nanoparticle-containing medium, and the cells were co-cultured in an incubator for 24 hours. The Blank group consisted of 4T1 cells not co-incubated with the nanozymes. The manganese-copper nanozymes prepared in Example 14 were immersed in basal culture medium at concentrations of 0, 1.5, 3, 6, 12, 25, 50, 100, and 200 μg / mL, and then fetal bovine serum was added. Cytotoxicity was determined using a direct contact method between the nanoparticles and 4T1 cells. 4T1 cells were seeded at a density of 5000 cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours to allow complete cell adhesion. The original culture medium was then replaced with a prepared nanoparticle-containing medium and co-cultured in an incubator for 24 hours.

[0118] The culture medium was removed from the wells, and the plates were washed with PBS. Cell viability was then assessed using the CCK8 reagent (Cell Counting Kit-8). Basal culture medium and CCK8 reagent were added to each well to bring the final CCK8 reagent concentration to 10%. The absorbance at 450 nm was measured using a microplate reader. The half-maximal inhibitory concentration (IC50) of the manganese-copper nanozymes prepared in Examples 12-19 was calculated using the cell proliferation inhibition rate. The formula for calculating the cell proliferation inhibition rate is:

[0119] Proliferation inhibition rate = (ODBlank - ODn) / ODBlank × 100%

[0120] OD Blank The absorbance of the blank group at 450 nm, OD n The absorbance of the material group at 450 nm is given.

[0121] Figure 18 The results from CCK8 show that the Mn group has a good anti-tumor effect, while the Cu group does not have a significant anti-tumor effect. The anti-cancer activity of the manganese-copper nanozyme is higher than that of the Blank group, proving that the manganese-copper nanozyme prepared in Examples 13-18 has good anti-cancer properties.

[0122] Figure 18The results show that the manganese copper nanozyme prepared in Example 14 is toxic to 4T1 cells even at a low concentration, with an IC50 of approximately 25 μg / mL, indicating that the manganese copper nanozyme prepared in Example 14 is toxic to 4T1 cells.

[0123] Example 27:

[0124] The anti-inflammatory properties of the manganese-copper nanozyme prepared in Example 14 were verified using mouse mononuclear macrophage leukemia cells (RAW264.7 cells). RAW264.7 cells were seeded in 6-well plates at a concentration of 2 × 10⁻⁶ cells / well. 5 / well. After culturing at 37℃ for 24 h, lipopolysaccharide (LPS) was added to the wells to a final concentration of 1 μg / mL using LPS as an inducer. After culturing at 37℃ for 12 h, the original medium was replaced with medium containing nanoparticles. After culturing for 12 h, the medium was removed, the cells were gently washed with PBS, and 0.2% DCFH-DA medium solution was added. After incubation at 37℃ for 20 min, the medium was removed, and the cells were gently washed with fresh medium. Cells were fixed with 4% paraformaldehyde. After 15 min, the paraformaldehyde was removed, and 0.5% Triton X-100 cell permeation solution was added. After 5 min, the solution was removed, and an appropriate amount of DAPI staining solution was added to stain the cell nuclei. After 5 min, the staining solution was removed, and intracellular ROS were observed using laser confocal microscopy.

[0125] RAW264.7 cells treated with LPS and nanozymes were fixed with 4% paraformaldehyde for 15 min, followed by removal of paraformaldehyde and treatment with 0.5% Triton X-100 cell permeation buffer for 5 min. Then, a primary antibody (rabbit anti-CD206) was added to the system, and the cells were incubated overnight at 4°C. After washing with PBS, the cells were co-incubated with a secondary antibody (Cy3 conjugated to goat anti-rabbit IgG antibody) for 50 min. After washing with PBS, the cell nuclei were stained with an appropriate amount of DAPI staining solution. After 5 min, the staining solution was removed, and the expression of the anti-inflammatory protein CD206 was observed using laser confocal microscopy. Finally, the protein expression level was quantitatively analyzed using ImageJ software.

[0126] Figure 19The results showed that RAW264.7 cells induced by lipopolysaccharide exhibited significant green fluorescence, indicating the production of large amounts of reactive oxygen species (ROS) and a strong inflammatory response. In contrast, cells in the MnCu1 group did not show much green fluorescence, suggesting that few cells produced ROS. This indicates that the MnCu1 nanozyme inhibited ROS production in RAW264.7 cells, demonstrating a good anti-inflammatory effect. Furthermore, quantitative analysis of the fluorescence intensity of ROS production revealed that the fluorescence intensity in the MnCu1 group was less than 5% of that in the control group. This further confirms that the manganese-copper nanozyme prepared in Example 14 almost completely inhibited the inflammatory response of RAW264.7 cells, exhibiting a good anti-inflammatory effect.

[0127] Figure 20 The results showed that the Blank group exhibited less red fluorescence, while the MnCu1 group showed more, indicating that the MnCu1 group had more RAW264.7 cells expressing CD206 glycoprotein. Actin fluorescence also indicated that the RAW264.7 cells in the MnCu1 group were more spindle-shaped and possessed antennae, a characteristic of M2 macrophages. In contrast, the RAW264.7 cells in the Blank group were more rounded, a characteristic of M1 macrophages. Quantitative analysis of CD206 fluorescence intensity revealed that the CD206 expression level in the MnCu1 group was more than three times that in the Blank group, demonstrating that the manganese-copper nanozyme prepared in Example 14 promoted the differentiation of RAW264.7 cells into the M2 type.

[0128] Those skilled in the art will readily understand that the above description is merely an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a manganese-based nanozyme, characterized in that, Includes the following steps: (1) Dissolve potassium permanganate and metal salt separately in water and stir thoroughly to form potassium permanganate solution and metal salt solution; the metal salt is one of zinc salt, calcium salt, magnesium salt, aluminum salt, cerium salt, iron salt, strontium salt, cobalt salt, copper salt, barium salt and zirconium salt; (2) Thoroughly mix the potassium permanganate solution and the metal salt solution to form a mixed solution, and adjust the pH value of the system so that the reaction can be carried out under alkaline conditions; (3) The precipitate after the reaction was washed with water, centrifuged, and dried to obtain manganese-based nanozymes.

2. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, In step (2), adjusting the pH value of the system specifically means adjusting the pH value of the system to 8~14.

3. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, In step (2), the reaction time is 10 hours to 48 hours.

4. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, The drying process is one of freeze-drying, natural drying, or oven drying; the drying temperature is 50~100℃.

5. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, The metal salt is a copper salt, and the molar ratio of potassium permanganate to copper salt is 1:(0.5~125).

6. The method for preparing manganese-based nanozymes according to claim 5, characterized in that, The molar ratio of potassium permanganate to copper salt is 1:(0.8~1.2).

7. The method for preparing manganese-based nanozymes according to claim 6, characterized in that, The copper salt is copper nitrate or copper chloride.

8. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, The nanozyme is a manganese-copper nanozyme, and its specific preparation method is as follows: Potassium permanganate and copper chloride were added to water in a molar ratio of 1:1 to form solutions. The two solutions were then mixed to form a mixed solution. The pH of the solution was adjusted to 10-12, and the reaction time was 20-24 hours. After centrifugation, washing, and freeze-drying, manganese-copper nanozyme was obtained.

9. A manganese-based nanozyme, characterized in that, It is prepared by the method for preparing manganese-based nanozymes according to any one of claims 1 to 8.

10. The use of the manganese-based nanozyme according to claim 9 in the preparation of anti-inflammatory drugs and antibacterial drugs.

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