Preparation method of manganese-based nano-enzyme, product and application of manganese-based nano-enzyme in antibiosis

Abstract

The invention discloses a manganese-based nano-enzyme preparation method, a manganese-based nano-enzyme product and an application of the manganese-based nano-enzyme product in antibiosis, and belongs to the technical field of nano-enzymes and biomedicine nanotechnology. According to the preparation method, KMnO4 and C18H34O2 are used as raw materials, and the manganese-based nano-enzyme is prepared through a water phase method. The nano-enzyme shows good application prospects in removing bacterial biofilms, inhibiting bacteria from forming biofilms and treating wound infection models. Animal experiments show that the hydrogel has excellent biocompatibility, is free of toxic accumulation, and can effectively treat mouse wound staphylococcus aureus infection and promote wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nano-enzyme and biomedical nanotechnology, and particularly relates to a preparation method of a manganese-based nano-enzyme, a product and application thereof in antibiosis. BACKGROUND

[0002] Planktonic and biofilm are two different life modes of bacteria. Most bacteria in the environment exist in the form of biofilm. Biofilm is a complex microbial structure wrapped on the surface of bacteria, which is composed of polysaccharides, extracellular DNA and proteins secreted by bacteria. The formation of biofilm is a complex process, including initial surface adhesion, microcolony formation, structure maturation and active diffusion of cells. The whole process is affected by external conditions, such as temperature fluctuation, pH change and quorum sensing. The formation of biofilm is a recognized mechanism for pathogenic bacteria to escape from antibacterial agents and immune response, which can promote the attachment of microorganisms to biological or non-biological surfaces to form a microbial community that provides protection for bacteria. In the biofilm, bacteria activate drug resistance genes, enhance their tolerance to antibiotics, and effectively resist the harmful effects of drugs on bacterial cells, leading to the emergence of multiple drug-resistant bacterial strains and persistent clinical infections.

[0003] Klebsiella pneumoniae is an important gram-negative opportunistic pathogen that can cause various infectious diseases, including pneumonia, urinary tract infection, bacteremia and liver abscess. In nosocomial sepsis, Klebsiella pneumoniae has a high mortality rate. Klebsiella pneumoniae is prone to form biofilm on medical devices such as ventilator catheters, urinary catheters and vascular stents, which can lead to serious iatrogenic infections. Staphylococcus aureus is a widely existing gram-positive coccus and is the main cause of hospital and community-acquired infections, which can cause a variety of diseases, including skin infections, respiratory tract infections, digestive tract infections and systemic infections. Staphylococcus aureus is prone to form biofilm, and the biofilm structure enhances the drug resistance of Staphylococcus aureus, leading to continuous recurrence of infection.

[0004] There are two methods to eliminate biofilm: inhibiting the formation of biofilm and removing the formed biofilm. Nano-silver, copper oxide and other nanomaterials have good bactericidal and biofilm removal performance, but nano-silver is easy to cause cytotoxicity and pollute the environment, which has certain biological safety problems and is not suitable for human use. Nanozymes, which are nanoparticles with enzyme-like catalytic activity, were discovered by Chinese scientists in 2007 and have been applied in the field of biological medicine. In the early stage of research, it was found that manganese-based nanozymes (MnOx) are a kind of simulated enzymes that not only have the characteristics of nanomaterials but also have catalytic function, and also have the advantages of good stability, low cost and high biological safety, and are therefore commonly used in cancer treatment and disease diagnosis. However, there is no related report on the application of this nanozyme in antibiosis and antibacterial biofilm. SUMMARY

[0005] To solve the above technical problems, the application provides a preparation method of manganese-based nanoscale enzyme, a product thereof and application thereof in antibiosis.

[0006] To achieve the above object, the application provides the following technical scheme.

[0007] One of the objects of the application is to provide a preparation method of manganese-based nanoscale enzyme, comprising the following steps: KMnO4 (potassium permanganate) and C 18 H 34 O2 (oleic acid) are used as raw materials, and manganese-based nanoscale enzyme is prepared by a water phase method.

[0008] KMnO4 is a strong oxidizing agent, which can provide oxidation ability in the reaction and promote the reaction. In an aqueous solution, KMnO4 can undergo a reduction reaction to generate Mn 2+ ions of low valence. These manganese ions can serve as the active center of the nanoscale enzyme and provide a basis for subsequent catalytic reactions. C 18 H 34 O2 acts as a reducing agent in the reaction and can reduce KMnO4, and the functional groups such as carboxyl groups in the molecule can coordinate with manganese ions. This coordination helps to control the growth and morphology of manganese-based nanoscale enzyme, so that it forms nanoparticles with specific structure and size, thereby affecting its catalytic performance. In the reaction process of the water phase method, KMnO4 is reduced, manganese ions are coordinated with C 18 H 34 O2 and gradually aggregate, and finally form manganese-based nanoscale enzyme. By controlling the reaction conditions such as raw material ratio, stirring time and reaction time, the physical and chemical properties of the nanoscale enzyme such as particle size and morphology can be controlled.

[0009] Further, the specific steps are as follows: KMnO4 is dissolved in deionized water and stirred (the whole process of stirring needs to be sealed), then C 18 H 34 O2 is added dropwise on the surface of the obtained liquid to react, and after the reaction is completed, the black sample is separated, washed and dried to obtain manganese-based nanoscale enzyme.

[0010] These detailed steps ensure the repeatability and stability of the preparation process, which helps to obtain manganese-based nanoscale enzyme products with uniform quality and stable performance. At the same time, by accurately controlling the reaction conditions, the physical and chemical properties of the nanoscale enzyme can be better controlled, so as to optimize its catalytic performance.

[0011] Further, the amount ratio of KMnO4, deionized water and C 18 H 34 O2 is 100 mg: 50 mL: 2 mL.

[0012] In this proportion, each component in the reaction system can fully react, so that the yield and performance of the manganese-based nanoszyme are optimal. This precise ratio control helps to improve the catalytic activity and stability of the nanoszyme, and is also conducive to the performance optimization of the nanoszyme in different application scenarios.

[0013] Further, the stirring time is 25 minutes.

[0014] It is determined that the stirring time is 25 minutes, which is long enough for KMnO4 to be fully dissolved. Proper stirring time can ensure the uniformity of the reaction system, thereby promoting the smooth progress of the reaction. If the stirring time is too short, KMnO4 may not be fully dissolved, affecting the generation and performance of the nanoszyme; and if the stirring time is too long, it may increase energy consumption without additional benefits.

[0015] Further, the reaction time is 48 hours.

[0016] It is clear that the reaction time is 48 hours, which is the best reaction time verified by experiments. In this time, the reaction can proceed fully, and the manganese-based nanoszyme generated has ideal physical and chemical properties and catalytic performance. If the reaction time is too short, the reaction may not be complete, and the performance of the generated nanoszyme may be poor; and if the reaction time is too long, impurities may be generated, affecting the purity and performance of the nanoszyme.

[0017] Further, the washing is: sequentially washing 6 times with anhydrous ethanol and sterile deionized water.

[0018] This washing method can effectively remove impurities and unreacted raw materials generated during the reaction, thereby improving the purity and performance of the manganese-based nanoszyme. Multiple washing can ensure the cleanliness of the nanoszyme surface and avoid interference of impurities in subsequent applications.

[0019] The second object of the present application is to provide a manganese-based nanoszyme prepared by the above preparation method. The diameter of the MnOx is about 100 nm, and the structure is a flower ball. It contains Mn, C and O three elements, and the valence of Mn is +2, +3 and +4, and the valence ratio is 17:37:46.

[0020] The manganese-based nanoszyme obtained by the above method has a diameter of about 100 nm and a flower ball structure, which is conducive to increasing the specific surface area and exposing active sites, thereby enhancing the catalytic performance. During the reaction, different valence manganese ions such as Mn 2+ , Mn 3+ and Mn 4+ are generated. These multi-valence manganese ions can synergistically act in the nanoszyme to produce enzyme-like activity. For example, Mn 2+and Mn 3+ The activity center of the natural enzyme can be simulated, and the ability of redox cycle is realized, so that the catalytic conversion of the substrate is realized. The synergistic effect of the multivalent manganese ions is one of the important factors for the manganese-based nanoscale enzyme to have high catalytic performance. The manganese-based nanoscale enzyme kills bacteria by regulating the intracellular ROS level. The catalytic activity can promote the generation or transformation of active oxygen substances (such as superoxide anion and hydrogen peroxide) in the bacteria, cause oxidative stress reaction in the bacteria, destroy the cell structure and biological macromolecules of the bacteria, and finally realize the antibacterial effect. At the same time, the nanoscale size of the nanoscale enzyme makes it easier to contact and act on the bacteria, further improving the antibacterial efficiency. The manganese-based nanoscale enzyme of the application has the advantages of good stability, low cost and high biological safety. Its unique physical and chemical properties make it have wide application prospects in the fields of antibacterial and antibiofilm.

[0021] The third object of the application is to provide an application of the manganese-based nanoscale enzyme in the preparation of an antibacterial product.

[0022] Further, the antibacterial product is a product for removing bacterial biofilm.

[0023] The application range of the manganese-based nanoscale enzyme is expanded, and it has important application value in removing bacterial biofilm. Biofilm is a complex structure of bacteria, which is difficult to be removed by traditional antibacterial agents. The manganese-based nanoscale enzyme can kill bacteria by regulating the intracellular ROS level, and effectively destroy the structure of the biofilm, providing a new strategy for solving the problem of bacterial drug resistance and biofilm-related infection.

[0024] Further, the antibacterial product is a product for inhibiting bacteria from forming biofilm.

[0025] The application field of the manganese-based nanoscale enzyme is further expanded, and it has a significant effect in inhibiting bacteria from forming biofilm. By adding the manganese-based nanoscale enzyme in the early stage of bacterial growth, the adhesion of bacteria and the formation of biofilm can be effectively prevented. This has important significance for preventing medical device-related infections and reducing the formation of biofilm.

[0026] Further, the antibacterial product is an antibacterial pharmaceutical preparation.

[0027] Compared with the prior art, the application has the following advantages and technical effects:

[0028] The preparation process method of the MnOx is scientific and reasonable, simple, low in cost, good in stability, good in biocompatibility, and can significantly remove the formed bacterial biofilm and inhibit the formation of the biofilm.

[0029] The manganese-based nanoenzyme is a kind of simulated enzyme with the characteristics of a nano material and catalytic function, and also has the advantages of good stability, low cost and high biological safety, and the manganese-based nanoenzyme can kill bacteria by regulating the intracellular ROS level of the bacteria, so the manganese-based nanoenzyme can be used as an antibacterial biofilm preparation and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description serve the purpose of explaining the present application. The present application is not limited by the accompanying drawings.

[0031] Figure 1 The transmission electron microscope and scanning electron microscope images of the MnOx prepared in Example 1 are shown, wherein Figure A is a transmission electron microscope image, and Figure B is a scanning electron microscope image;

[0032] Figure 2 The XPS analysis of the MnOx prepared in Example 1 is shown, wherein Figure A is an XPS total spectrum line graph, Figure B is an Mn2p subpeak graph, Figure C is an Mn, C and O atomic percentage graph, and Figure D is an Mn valence state ratio graph;

[0033] Figure 3 The evaluation of the bacterial biofilm removal of the MnOx prepared in Example 1 is shown, wherein Figure A is a scanning electron microscope image of the removal of the Klebsiella pneumoniae biofilm by the MnOx, and Figure B is a scanning electron microscope image of the removal of the Staphylococcus aureus biofilm by the MnOx;

[0034] Figure 4 The evaluation of the inhibition of the formation of the bacterial biofilm by the MnOx prepared in Example 1 is shown, wherein Figure A is a scanning electron microscope image of the inhibition of the formation of the Klebsiella pneumoniae biofilm by the MnOx, and Figure B is a scanning electron microscope image of the inhibition of the formation of the Staphylococcus aureus biofilm by the MnOx;

[0035] Figure 5 The in vivo safety evaluation of the MnOx prepared in Example 1 is shown, wherein Figure A is the body weight change of the mice treated with different concentrations of the MnOx, and Figure B is the survival rate of the mice treated with different concentrations of the MnOx;

[0036] Figure 6Figure of blood routine test after mice were treated with different concentrations of MnOx prepared in Example 1;

[0037] Figure 7 Figure of biochemical index test after mice were treated with different concentrations of MnOx prepared in Example 1;

[0038] Figure 8 Figure of weight change and wound size after mice treated with MnOx prepared in Example 1 were infected with Staphylococcus aureus, wherein Figure A is weight change after mice were treated with different drugs; Figure B is wound recovery and wound recovery trend after mice were treated with different drugs;

[0039] Figure 9 Transmission electron microscope image of MnOx prepared by traditional method. DETAILED DESCRIPTION

[0040] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The detailed description is intended only to provide a further example of the subject matter described in the application.

[0041] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. It is intended that the application not be limited to the details of the procedures set forth in the following description but include any and all modifications or equivalents thereof within the scope of the application.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. It is intended that the application not be limited to the details of the procedures set forth in the following description but include any and all modifications or equivalents thereof within the scope of the application. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0043] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples presented herein are meant as illustrative only and are not intended to limit the scope of the application. The scope of the application is limited only by the claims.

[0044] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0045] The present application uses KMnO4, C 18 H 34 O2 as raw materials to prepare manganese-based nanoszyme (MnOx). The ability of MnOx to remove bacterial biofilm and inhibit biofilm formation is evaluated, and the application of MnOx in wound infection is evaluated.

[0046] The technical solutions of the present application are further illustrated by the following examples.

[0047] Example 1

[0048] A method for preparing a manganese-based nanoszyme, the steps are as follows: 100 mg of KMnO4 is dissolved in 50 mL of deionized water and magnetically stirred for 25 minutes, then 2 mL of C 18 H 34 O2 is slowly added dropwise above the liquid surface, and after 48 hours of reaction, the black sample is separated and washed by centrifugation with anhydrous ethanol and sterile deionized water for 6 times in turn to remove any possible residual reactants; finally, the sample is dried at 60°C for 4 hours to obtain the product MnOx. The obtained product is sealed in a test tube and stored in a dry vessel for long-term preservation.

[0049] 1. Characterization of manganese-based nanoszyme (MnOx)

[0050] Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used to observe the morphology of MnOx.

[0051] Figure 1 The transmission electron microscope and scanning electron microscope images of MnOx prepared in Example 1 are shown in Figure A and Figure B, respectively. As shown in the figures, the diameter of MnOx is about 100 nm, and it has a flower ball structure, uniform size and uniform distribution.

[0052] 2. Analysis of manganese-based nanoszyme (MnOx)

[0053] An appropriate amount of MnOx is taken and analyzed by XPS to measure the element content and Mn valence state ratio of MnOx.

[0054] Figure 2XPS analysis of the MnOx prepared in Example 1, wherein Figure A is an XPS total spectrum, Figure B is a Mn2p sub-peak spectrum; Figure C is a Mn, C and O atomic percentage graph; and Figure D is a Mn valence state ratio graph. As shown in Figure A, the MnOx contains Mn, C and O three elements; as shown in Figure B, the Mn valence state in the MnOx is +2, +3 and +4 respectively; as shown in Figure C, the atomic percentage of Mn, C and O in the MnOx is 61.11:30.62:8.26; and as shown in Figure D, the ratio between the three valence states of Mn in the MnOx is 17:37:46.

[0055] 3. Evaluation of manganese-based nanoscale enzyme (MnOx) in removing bacterial biofilm

[0056] Test materials: anhydrous ethanol was purchased from Shanghai Test Company; KMnO4, C 18 H 34 O2 were purchased from Aladdin Company. LB broth medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; TSB medium was purchased from BD Pharmingen Company, USA.

[0057] Method: MnOx stock solution was freshly prepared before the experiment. The MnOx prepared in Example 1 was dissolved in LB broth and TSB solution containing 1% glucose respectively, and was completely dissolved by ultrasonic treatment, and was prepared into MnOx-LB solution and MnOx-TSB solution with a concentration of 2 mg / mL, which was used after high-pressure sterilization.

[0058] A single colony of Klebsiella pneumoniae was picked and inoculated in LB liquid medium, which was cultured at 37°C, 220 r / min for 12-18 h. The OD 600 of the bacteria in the LB broth was adjusted to 0.8, at which time the concentration of the bacterial solution was 5×10 8 CFU / mL. The above bacterial solution was diluted 1:10 with LB broth, which was incubated at 37°C for 24 h. The planktonic bacteria were removed, and 2 mg / mL of MnOx solution was added, which was incubated at 37°C for 2 h and 6 h. Then 2.5% glutaraldehyde was used for fixation at 4°C for 12 h, and the effect of MnOx on the formed Klebsiella pneumoniae biofilm was observed by scanning electron microscopy after dehydration with gradient alcohol, drying and gold spraying.

[0059] A single colony of Staphylococcus aureus was picked and inoculated in LB broth, which was cultured at 37°C, 220 r / min for 12-18 h. The OD 600 of the bacteria in the LB broth was adjusted to 0.5, at which time the concentration of the bacterial solution was 1×10 9CFU / mL. The above bacterial solution was diluted 1:30 with TSB liquid medium containing 1% glucose and inoculated, and after static culture at 37°C for 24 h, the planktonic bacteria were removed, and a MnOx solution with a concentration of 2 mg / mL was added, and incubated at 37°C for 2 h and 6 h. Then 2.5% glutaraldehyde was used for fixation at 4°C for 12 h, and after dehydration with gradient alcohol, drying, gold spraying, the effect of MnOx on the formed Staphylococcus aureus biofilm was observed by scanning electron microscopy.

[0060] Test results: Figure 3 The evaluation of the removal of bacterial biofilm by MnOx prepared in Example 1 is shown in Figure A, which is a scanning electron micrograph of the removal of Klebsiella pneumoniae biofilm by MnOx, and Figure B is a scanning electron micrograph of the removal of Staphylococcus aureus biofilm by MnOx. As shown in Figure A, after the interaction of MnOx with Klebsiella pneumoniae biofilm, it was found by scanning electron microscopy that the number of Klebsiella pneumoniae and the biofilm matrix were significantly reduced, indicating that MnOx can remove Klebsiella pneumoniae biofilm; as shown in Figure B, after the interaction of MnOx with Staphylococcus aureus biofilm, it was found by scanning electron microscopy that the number of bacteria in the biofilm and the biofilm matrix were significantly reduced, indicating that MnOx can remove Staphylococcus aureus biofilm.

[0061] 4. Evaluation of manganese-based nanoscale enzyme (MnOx) inhibiting bacteria from forming biofilm

[0062] Test materials: anhydrous ethanol was purchased from Shanghai Test Company; KMnO4, C 18 H 34 O2 were purchased from Aladdin Company. LB broth medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; TSB medium was purchased from BD Pharmingen Company, USA.

[0063] Method: MnOx stock solution was freshly prepared before the experiment. The MnOx prepared in Example 1 was dissolved in LB broth and TSB solution containing 1% glucose, respectively, and ultrasonically treated to completely dissolve, and prepared into MnOx-LB solution and MnOx-TSB solution with a concentration of 2 mg / mL. After autoclaving, the stock solution was diluted to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL of MnOx-LB broth and MnOx-TSB solution, respectively.

[0064] A single colony of Klebsiella pneumoniae was picked and inoculated in LB liquid medium, and after incubation at 37°C, 220 r / min for 12-18 h, the OD 600 was 0.8, at which time the concentration of the bacterial solution was 5 x 10 8CFU / mL. The above bacterial solution was diluted with MnOx solution at different concentrations at 1:10, and incubated at 37°C for 24 h. Then, 2.5% glutaraldehyde was used for fixation at 4°C for 12 h, and the gradient alcohol was used for dehydration, and then dried, sprayed with gold, and then the influence of MnOx on the formation of Klebsiella pneumoniae biofilm was observed by scanning electron microscopy.

[0065] A single colony of Staphylococcus aureus was picked and inoculated in LB broth, and then incubated at 37°C, 220 r / min for 12-18 h. The LB broth was adjusted to a bacterial OD 600 0.5, at which time the bacterial solution had a concentration of 1×10 9 CFU / mL. The above bacterial solution was diluted with MnOx solution at different concentrations at 1:10, and incubated at 37°C for 24 h. Then, 2.5% glutaraldehyde was used for fixation at 4°C for 12 h, and the gradient alcohol was used for dehydration, and then dried, sprayed with gold, and then the influence of MnOx on the formation of Klebsiella pneumoniae biofilm was observed by scanning electron microscopy.

[0066] Test results: Figure 4 The evaluation of MnOx prepared in Example 1 for inhibiting the formation of bacterial biofilm, wherein Figure A is a scanning electron micrograph of MnOx inhibiting the formation of Klebsiella pneumoniae biofilm, and Figure B is a scanning electron micrograph of MnOx inhibiting the formation of Staphylococcus aureus biofilm. As shown in Figure A, after different concentrations of MnOx were co-cultured with Klebsiella pneumoniae, it was found by scanning electron microscopy that the number of bacteria in the Klebsiella pneumoniae biofilm and the biofilm matrix were significantly reduced, indicating that MnOx can inhibit the formation of Klebsiella pneumoniae biofilm, and has a concentration-dependent effect; as shown in Figure B, after different concentrations of MnOx were co-cultured with Staphylococcus aureus, it was found by scanning electron microscopy that the number of bacteria in the Staphylococcus aureus biofilm and the biofilm matrix were significantly reduced, indicating that MnOx can inhibit the formation of Staphylococcus aureus biofilm, and has a concentration-dependent effect.

[0067] 5. In vivo safety evaluation of manganese-based nanoscale enzyme (MnOx)

[0068] Test materials: anhydrous ethanol was purchased from Shanghai Test Company; KMnO4, C 18 H 34 O2 were purchased from Aladdin Company. Female BALB / c mice were provided by the Comparative Medicine Center of Yangzhou University.

[0069] Methods: 6-week-old female BALB / c mice were selected. After the mice were anesthetized, a 10-mm full-thickness circular wound was constructed on the back of each mouse using sterile surgical scissors. The MnOx prepared in Example 1 was dissolved in PBS and sterilized by autoclaving. The mice were treated with 0 mg / kg, 25 mg / kg, 50 mg / kg, and 100 mg / kg of MnOx, respectively, to rinse the wound. The body weight change and survival rate were recorded daily, and the blood of the mice was taken on the 10th day for blood routine and biochemical index detection to observe whether MnOx had toxicity.

[0070] Test results: Figure 5 Example 1 MnOx was evaluated for its safety in vivo. Figure A shows the body weight change of mice treated with different concentrations of MnOx, and Figure B shows the survival rate of mice treated with different concentrations of MnOx. As shown in Figure A, there was no significant difference in body weight change of mice treated with MnOx. As shown in Figure B, all mice survived after being treated with different concentrations of MnOx. Figure 6 Figure shows the blood routine test results of mice treated with different concentrations of MnOx. As shown, there was no significant change in each type of blood routine index. Figure 7 Figure shows the biochemical index detection results of mice treated with different concentrations of MnOx. As shown, there was no significant change in each type of biochemical index, and the biocompatibility was excellent.

[0071] 6. Evaluation of manganese-based nanoscale enzyme (MnOx) in treating mouse wound infection with Staphylococcus aureus

[0072] Test materials: anhydrous ethanol was purchased from Shanghai Test Company; KMnO4, C 18 H 34 O2 were purchased from Aladdin Company. Female BALB / c mice were provided by the Comparative Medicine Center of Yangzhou University.

[0073] Methods: 6-week-old female BALB / c mice were selected. After the mice were anesthetized, a 10-mm full-thickness circular wound was constructed on the back of each mouse using sterile surgical scissors. The wound was inoculated with Staphylococcus aureus (5 x 10 7 CFU). The wound was rinsed with 1 mL of PBS and 1 mL of 50 mg / kg of MnOx prepared in Example 1, and the wound was cleaned 3 times a day for 3 days, for a total of 10 days of observation. The mice were weighed daily, and the wound size was recorded.

[0074] Test results: Figure 8The weight change and wound size of the mice treated with the MnOx prepared in Example 1 after the mice were infected with S. aureus are shown in Figure A and Figure B. As shown in Figure A, the MnOx can help the infected mice recover weight; as shown in Figure B, the MnOx effectively promotes wound healing and accelerates the wound recovery trend.

[0075] Comparative Example 1

[0076] The preparation method of the traditional manganese-based nanoszyme: 20 mg of KMnO4 was dissolved in 50 mL of deionized water and 20 mL of ethylene glycol, and was magnetically stirred under sealed conditions for 2 hours, followed by dropwise addition of 0.5 mL of C 18 H 34 O2, and after 2 hours of reaction, the reaction solution was centrifuged at 12000 r / min for 10 minutes to separate the black sample; then the sample was washed by centrifugation with anhydrous ethanol for 6 times; finally, the sample was dried at 60℃ for 4 hours to obtain the product MnOx. The obtained product was sealed in a test tube and stored in a dry vessel for long-term preservation.

[0077] Figure 9 The transmission electron microscope image of the MnOx prepared by the traditional method is shown in the figure. As shown in the figure, the morphology control of the manganese-based nanoszyme prepared by the traditional method of preparing manganese-based nanoszyme fails, and there is heterogeneity, uneven size, and aggregation phenomenon, and the dispersibility is poor; and the manganese-based nanoszyme prepared by this method has low yield, unstable synthesis, limits the functional characterization and performance research, affects the test repeatability and feasibility.

[0078] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a manganese-based nanoszyme, characterized in that, comprising the steps of: reacting KMnO4 and C 18 H 34 Manganese-based nanoszyme is prepared by aqueous phase method using O2 as raw material.

2. The method for preparing manganese-based nanozymes according to claim 1, characterized in that, The specific steps are as follows: KMnO4 is dissolved in deionized water, stirred, and then C 18 H 34 O2 is added dropwise, and after the reaction is completed, the black sample is separated by standing, washed, and dried to obtain a manganese-based nanoscale enzyme.

3. The method for preparing manganese-based nanozymes according to claim 2, characterized in that, The KMnO4 and C 18 H 34 The amount of O2 was 100 mg: 2 mL.

4. The method of claim 2, wherein the manganese-based nanoszyme is prepared by the steps of: The time of the stirring is 25 minutes.

5. The method for preparing manganese-based nanozymes according to claim 2, characterized in that, The time of the reaction is 48 hours.

6. The method of claim 2, wherein the manganese-based nanoszyme is prepared by the steps of: The washing is 6 washes with absolute ethanol and sterile deionized water, in this order.

7. A manganese-based nanoszyme characterized by, which is prepared by the method of any one of claims 1-6.

8. Use of a manganese-based nanoszyme according to claim 7 for the preparation of an antibacterial product.

9. Use according to claim 8, characterized in that, The antibacterial product is a product that inhibits the formation of bacterial biofilm.

10. Use according to claim 8, characterized in that, The antibacterial product is a product that eliminates bacterial biofilm.

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