A ruthenium-doped prussian blue nanoscale enzyme, a preparation method and application thereof, and an SNP-PB nanoscale enzyme and application thereof

Abstract

The present application relates to the technical field of nanomaterial application, and provides a ruthenium-doped Prussian blue nanoscale enzyme and a preparation method thereof, wherein the ruthenium-doped Prussian blue nanoscale enzyme is obtained by reacting metal ruthenium with Prussian blue nanoparticles under normal temperature conditions. The present application also provides a nanoscale enzyme library containing the ruthenium-doped Prussian blue nanoscale enzyme and a construction method thereof. The present application also provides SNP-PB nanoscale enzyme obtained by loading sodium nitroprusside into the ruthenium-doped Prussian blue nanoscale enzyme. The ruthenium-doped Prussian blue nanoscale enzyme and the SNP-PB nanoscale enzyme both have good antibacterial and anti-inflammatory properties, and can better solve the problem of biofilm resistance after being endowed with the ability to release NO gas, thereby better exerting antioxidant stress and fighting periodontal bacterial infection, and providing a new treatment method for periodontal disease.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial application technology, and in particular to a ruthenium-doped Prussian blue nanozyme, its preparation method and application, as well as an SNP-PB nanozyme and its application. Background Technology

[0002] Periodontitis is an infectious disease of the periodontal tissues caused by plaque microorganisms, affecting more than 750 million people worldwide. It is one of the most prevalent chronic diseases and the sixth most common disease in humans. Plaque biofilm, formed by bacterial aggregation, is considered the initiating factor of periodontitis. Periodontal pathogens within the plaque biofilm release virulence factors and enzymes that damage the soft and hard tissues of the periodontium, leading to alveolar bone loss, root loosening, and even tooth loss, severely impacting patients' quality of life. Clinically, adjunctive antibacterial agents are often used to treat periodontitis. However, the biofilm contains dense extracellular polymers with high adhesiveness, resisting external mechanical erosion. Furthermore, the extracellular polymers within the biofilm prevent the penetration of traditional antibacterial agents, resulting in dormant bacteria remaining deep within the biofilm. This significantly hinders the destructive effect of antibacterial agents on the plaque biofilm, leading to the re-aggregation of residual bacteria and severely affecting the clinical efficacy of periodontitis treatment.

[0003] Nanozymes, under physiological conditions, exhibit excellent antibacterial and antiviral activity by effectively catalyzing the production of large amounts of ROS (Reactive Oxygen Species), and have become a promising new type of "antibiotic." In addition to their enzymatic properties, as functionally rich inorganic nanomaterials, nanozymes can utilize their inherent properties, such as high photothermal conversion efficiency, combined with their catalytic properties to exert antimicrobial infection functions. In recent years, novel antibacterial therapies based on nanozymes have gradually become a research hotspot. Previous studies have shown that nanozymes with photothermal properties, through balancing ROS levels to enhance their antibacterial activity and catalytic performance, have certain limitations, mostly treating inflammation, promoting osteoogenesis, or combating biofilms with low therapeutic efficiency. Therefore, it is essential to obtain a nanozyme with superior antibacterial activity, anti-inflammatory properties, and catalytic properties for the treatment of periodontitis. Summary of the Invention

[0004] The purpose of this invention is to provide a ruthenium-doped Prussian blue nanozyme, its preparation method and application, as well as an SNP-PB nanozyme and its application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a ruthenium-doped Prussian blue nanozyme, which is obtained by reacting metallic ruthenium with Prussian blue nanoparticles at room temperature.

[0007] The application provides a preparation method of the ruthenium-doped Prussian blue nanoscale enzyme, and comprises the following steps:

[0008] (1) mixing polyvinylpyrrolidone and potassium ferrocyanide according to a ratio of (0.001-0.01) mmol:(0.01-0.03) mmol, and then dissolving the mixture in a KCl / HCl buffer solution to obtain solution A;

[0009] (2) mixing RuCl3 and the KCl / HCl buffer solution according to a ratio of (0.005-0.025) mmol:(3-10) mL to obtain solution B;

[0010] (3) mixing solution A and solution B, then adding H2O2 and NaBH4 in sequence, ultrasonicating, stirring, dialyzing and drying to obtain the ruthenium-doped Prussian blue nanoscale enzyme.

[0011] The application further provides a nanoscale enzyme library comprising the ruthenium-doped Prussian blue nanoscale enzyme.

[0012] As preferred, the nanoscale enzyme library further comprises one or more of a metal iron-doped Prussian blue nanoscale enzyme, a metal cobalt-doped Prussian blue nanoscale enzyme, a metal nickel-doped Prussian blue nanoscale enzyme, a metal copper-doped Prussian blue nanoscale enzyme, a metal zinc-doped Prussian blue nanoscale enzyme, a metal manganese-doped Prussian blue nanoscale enzyme and a metal cerium-doped Prussian blue nanoscale enzyme.

[0013] The application further provides a construction method of the nanoscale enzyme library, comprising the following steps:

[0014] (1) mixing polyvinylpyrrolidone and potassium ferrocyanide according to a ratio of (0.001-0.01) mmol:(0.01-0.03) mmol, and then dissolving the mixture in a KCl / HCl buffer solution to obtain solution 1;

[0015] (2) mixing a metal compound and the KCl / HCl buffer solution according to a ratio of (0.005-0.025) mmol:(3-10) mL to obtain solution 2;

[0016] (3) mixing solution 1 and solution 2, then adding H2O2, ultrasonicating, stirring, dialyzing and drying to obtain the nanoscale enzyme library.

[0017] The metal compound comprises FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3.

[0018] As preferred, when the metal compound is RuCl3, NaBH4 needs to be added after the addition of H2O2 in step (3); and the addition amount of the NaBH4 is 1-3 mg.

[0019] The application also provides a SNP-PB nano-enzyme, and the ruthenium-doped Prussian blue nano-enzyme is used to load sodium nitroprusside to obtain the SNP-PB nano-enzyme.

[0020] The application also provides application of the ruthenium-doped Prussian blue nano-enzyme or the SNP-PB nano-enzyme in preparation of an anti-oxidation drug.

[0021] The application also provides application of the ruthenium-doped Prussian blue nano-enzyme or the SNP-PB nano-enzyme in preparation of an anti-bacterial drug.

[0022] The application also provides application of the ruthenium-doped Prussian blue nano-enzyme or the SNP-PB nano-enzyme in preparation of a drug for treating periodontitis.

[0023] By adopting the technical scheme, the application has the following beneficial effects:

[0024] The technical scheme of the application is optimized on the basis of a Prussian blue nano-enzyme synthesis technology, different metal-doped Prussian blue nano-enzymes are synthesized at room temperature, then the nano-enzyme library is screened according to the photo-thermal performance and the anti-oxidation ability, and a ruthenium-doped Prussian blue nano-enzyme with better performance is obtained, which has relatively good anti-bacterial and anti-inflammatory performance and has a better treatment effect on fighting periodontal bacterial infection. On this basis, the SNP-PB nano-enzyme is obtained by loading sodium nitroprusside on the ruthenium-doped Prussian blue nano-enzyme, and the anti-bacterial performance is enhanced by giving the NO gas release ability. The SNP-PB nano-enzyme obtained by the photo-thermal response combined with the gas therapy better plays the dual performance of anti-inflammatory and anti-bacterial, helps to solve the problem of biofilm resistance, and better plays the anti-oxidation stress and fights the periodontal bacterial infection, thereby providing a new treatment method for periodontal disease.

[0025] The test examples also show that the anti-bacterial performance is enhanced by releasing the NO gas therapy while the photo-thermal anti-bacterial is performed, the bacterial biofilm is cleared, and the purpose of anti-oxidation stress is achieved by the nano-enzyme simulating the natural superoxide dismutase and catalase to remove the excess ROS, which helps to solve the problem of antibiotic resistance caused by the biofilm, and the periodontitis is effectively fought by the dual action of fighting inflammation and bacterial infection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 FIG. 4 is a transmission electron microscope morphology diagram of different metal-doped Prussian blue nano-enzymes in the nano-enzyme library (scale = 200 nm);

[0027] Figure 2 FIG. 5 is a particle size characterization result of different metal-doped Prussian blue nano-enzymes in the nano-enzyme library;

[0028] Figure 3The CAT, SOD, POD, DPPH, ABTS and enzyme activity radar chart of different metal-doped Prussian blue nanoszymes in the nanoszyme library;

[0029] Figure 4 The photothermal performance of different metal-doped Prussian blue nanoszymes in the nanoszyme library;

[0030] Figure 5 The morphology characterization graph of SPBzyme (scale = 200 nm);

[0031] Figure 6 The temperature rise curve and temperature rise image of Ru-PB and SPBzyme under 808 nm near-infrared light irradiation (A is the temperature rise curve, B is the temperature rise image in Figure 6 );

[0032] Figure 7 The NO release ability of SPBzyme (A represents the standard curve of NO release, B represents the NO release concentration of SPBzyme with or without the participation of H2O2, C represents the NO release concentration of SPBzyme under Laser or dark conditions in Figure 7 );

[0033] Figure 8 The safety evaluation results of Ru-PB and SPBzyme in HGFs cells;

[0034] Figure 9 The influence of Ru-PB and SPBzyme on the cell activity of HGFs under the simulation of H2O2 inflammatory activation state;

[0035] Figure 10 The influence of different treatment groups on the ROS content in RAW264.7 cells;

[0036] Figure 11 The confocal fluorescence image of RAW264.7 treated by different treatment groups (scale = 60 μm);

[0037] Figure 12 The mitochondrial membrane potential of RAW264.7 treated by different treatment groups (scale = 60 μm);

[0038] Figure 13 The influence of different treatment groups on the antibacterial effect of F. nucleatum (A is the colony situation in the culture medium, B is the number of colonies in Figure 13 );

[0039] Figure 14 The SEM image of the influence of different treatment groups on F. nucleatum (scale = 5 μm);

[0040] Figure 15 The effects of different treatment groups on the formation and establishment of F. nucleatum biofilm ( Figure 15 In the diagram, A represents the F. nucleatum biofilm formed by the live / dead image, B represents the F. nucleatum biofilm that has been established by the live / dead image, C represents the dead / live ratio and average thickness of the formed F. nucleatum biofilm, and D represents the dead / live ratio and average thickness of the established F. nucleatum biofilm; red represents dead bacteria, green represents live bacteria, and the scale bar is 100 μm.

[0041] Figure 16 The results of H&E staining and IHC staining of periodontal tissues in different treatment groups 3 and 7 days after treatment. Figure 16 In the figure, A represents the H&E staining results after 3 days of treatment, B represents the H&E staining results after 7 days of treatment, C represents the quantitative analysis of TNF-α and IL-1β positive cells in IHC staining after 3 days of treatment, and D represents the quantitative analysis of TNF-α and IL-1β positive cells in IHC staining after 7 days of treatment; the red arrows in the figure indicate the IL-1β and TNF-α positive areas (scale bar = 500 μm).

[0042] Figure 17 Micro-CT three-dimensional (3D) images of the maxillary first molar region in different treatment groups for periodontitis (red dashed line represents the distance between CEJ and ABC, scale bar = 1mm);

[0043] Figure 18 Evaluation of the in vivo efficacy of different treatment groups for periodontitis ( Figure 18 In the diagram, A represents the CEJ-ABC distance on day 3, B represents the alveolar bone BV / TV measurement result on day 3, C represents the CEJ-ABC distance on day 7, and D represents the alveolar bone BV / TV measurement result on day 7. Detailed Implementation

[0044] This invention provides a ruthenium-doped Prussian blue nanozyme. The ruthenium-doped Prussian blue nanozyme of this invention is preferably obtained by reacting metallic ruthenium with Prussian blue nanoparticles, and the reaction conditions are preferably at room temperature, specifically 20-30°C.

[0045] This invention provides a method for preparing the ruthenium-doped Prussian blue nanozyme, comprising the following steps:

[0046] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide in a ratio of (0.001-0.01) mmol / L:(0.01-0.03) mmol / L and dissolve in KCl / HCl buffer to obtain solution A;

[0047] (2) Mix RuCl3 with KCl / HCl buffer at a ratio of (0.005-0.025) mmol / L: (3-10) mL to obtain solution B;

[0048] (3) Mix solution A and solution B, then add H2O2 and NaBH4 in sequence, sonicate, stir, dialyze and dry to obtain the solution.

[0049] In this invention, polyvinyl pyrrolidone (PVP) and potassium ferrocyanide (K4Fe(CN)6) are mixed, thoroughly mixed, and then dissolved in a KCl / HCl buffer solution to obtain solution A. The preferred mixing ratio of PVP to potassium ferrocyanide in this invention is (0.001-0.01) mmol / L:(0.01-0.03) mmol / L, more preferably (0.003-0.008) mmol / L:(0.015-0.025) mmol / L, and even more preferably 0.005 mmol / L:0.02 mmol / L. The preferred concentration of the KCl / HCl buffer solution in this invention is 0.05-0.2 M, more preferably 0.08-0.15 M, and even more preferably 0.1 M; the preferred volume of the KCl / HCl buffer solution is 3-10 mL, more preferably 4-8 mL, and even more preferably 5 mL.

[0050] In this invention, RuCl3 is mixed with KCl / HCl buffer to obtain solution B. The concentration of the KCl / HCl buffer in this invention is preferably 0.05-0.2M, more preferably 0.08-0.15M, and even more preferably 0.1M. The mixing ratio of RuCl3 to KCl / HCl buffer in this invention is preferably (0.005-0.025) mmol / L:(3-10) mL, more preferably (0.01-0.023) mmol / L:(4-8) mL, and even more preferably 0.02 mmol / L:5 mL.

[0051] In this invention, solution A and solution B are mixed and stirred, and then H2O2 and NaBH4 are added sequentially and mixed thoroughly. The concentration of H2O2 in this invention is preferably 8-15M, more preferably 10-13M, and even more preferably 12M; the amount of H2O2 added is preferably 200-500 μL, more preferably 300-450 μL, and even more preferably 400 μL. The amount of NaBH4 added in this invention is preferably 1-3 mg, more preferably 1.5-2.5 mg, and even more preferably 2 mg. The purpose of adding NaBH4 in this invention is to better reduce the noble metal Ru.

[0052] In this invention, after uniform mixing, ultrasonic treatment is performed. The ultrasonic treatment is preferably water bath ultrasonic treatment, with the water bath temperature preferably at room temperature. The ultrasonic treatment time is preferably 10-20 minutes, more preferably 12-18 minutes, and even more preferably 15 minutes. After the water bath is completed, stirring is performed. The stirring method is preferably magnetic stirring. The stirring speed is preferably 200-400 r / min, more preferably 250-350 r / min, and even more preferably 300 r / min. The stirring time is preferably 2-4 hours, more preferably 2.5-3.5 hours, and even more preferably 3 hours.

[0053] In this invention, after stirring, dialysis is performed. The dialysis time is preferably 40-55 hours, more preferably 45-50 hours, and even more preferably 48 hours. After dialysis, freeze-drying is preferably performed to obtain ruthenium-doped Prussian blue nanozyme (Ru-PB). The freeze-drying temperature is preferably -80℃ to -50℃, more preferably -80℃ to -60℃, and even more preferably -80℃; the freeze-drying time is preferably 24-72 hours, more preferably 48-72 hours, and even more preferably 48 hours.

[0054] The present invention provides a nanozyme library comprising the above-mentioned ruthenium-doped Prussian blue nanozyme.

[0055] In this invention, the nanozyme library further includes one or more of the following: iron-doped Prussian blue nanozyme (Fe-PBzyme), cobalt-doped Prussian blue nanozyme (Co-PBzyme), nickel-doped Prussian blue nanozyme (Ni-PBzyme), copper-doped Prussian blue nanozyme (Cu-PBzyme), zinc-doped Prussian blue nanozyme (Zn-PBzyme), manganese-doped Prussian blue nanozyme (Mn-PBzyme), and cerium-doped Prussian blue nanozyme (Ce-PBzyme).

[0056] This invention provides a method for constructing the nanozyme library, comprising the following steps:

[0057] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide in a ratio of (0.001-0.01) mmol / L:(0.01-0.03) mmol / L and dissolve in KCl / HCl buffer to obtain solution 1;

[0058] (2) Mix the metal compound with KCl / HCl buffer at a ratio of (0.005-0.025) mmol / L: (3-10) mL to obtain solution 2;

[0059] (3) Mix solution 1 and solution 2, add H2O2, sonicate, stir, dialyze and dry to obtain the solution.

[0060] In this invention, polyvinyl pyrrolidone (PVP) and potassium ferrocyanide (K4Fe(CN)6) are mixed, and after thorough mixing, dissolved in a KCl / HCl buffer solution to obtain solution 1. The preferred mixing ratio of PVP to potassium ferrocyanide in this invention is (0.001-0.01) mmol / L:(0.01-0.03) mmol / L, more preferably (0.003-0.008) mmol / L:(0.015-0.025) mmol / L, and even more preferably 0.005 mmol / L:0.02 mmol / L. The preferred concentration of the KCl / HCl buffer solution in this invention is 0.05-0.2 M, more preferably 0.08-0.15 M, and even more preferably 0.1 M; the preferred volume of the KCl / HCl buffer solution is 3-10 mL, more preferably 4-8 mL, and even more preferably 5 mL.

[0061] In this invention, a metal compound is mixed thoroughly with a KCl / HCl buffer solution to obtain solution 2. The metal compound in this invention includes FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O, and RuCl3. The concentration of the KCl / HCl buffer solution in this invention is preferably 0.05-0.2M, more preferably 0.08-0.15M, and even more preferably 0.1M. The mixing ratio of the metal compound to the KCl / HCl buffer solution in this invention is preferably (0.005-0.025) mmol / L:(3-10) mL, more preferably (0.01-0.023) mmol / L:(4-8) mL, and even more preferably 0.02 mmol / L:5 mL.

[0062] In this invention, solution 1 and solution 2 are mixed and stirred, and then H2O2 is added and mixed evenly. The concentration of H2O2 in this invention is preferably 8-15M, more preferably 10-13M, and even more preferably 12M; the amount of H2O2 added is preferably 200-500 μL, more preferably 300-450 μL, and even more preferably 400 μL. In this invention, when the metal compound is RuCl3, NaBH4 is preferably added after adding H2O2. The amount of NaBH4 added is 1-3 mg, more preferably 1.5-2.5 mg, and even more preferably 2 mg. The purpose of adding NaBH4 in this invention is to better reduce the noble metal Ru.

[0063] In this invention, after uniform mixing, ultrasonic treatment is performed. The ultrasonic treatment is preferably water bath ultrasonic treatment, with the water bath temperature preferably at room temperature. The ultrasonic treatment time is preferably 10-20 minutes, more preferably 12-18 minutes, and even more preferably 15 minutes. After the water bath is completed, stirring is performed. The stirring method is preferably magnetic stirring. The stirring speed is preferably 200-400 r / min, more preferably 250-350 r / min, and even more preferably 300 r / min. The stirring time is preferably 2-4 hours, more preferably 2.5-3.5 hours, and even more preferably 3 hours.

[0064] In this invention, after stirring, dialysis is performed. The dialysis time is preferably 40-55 hours, more preferably 45-50 hours, and even more preferably 48 hours. After dialysis, the nanozyme is preferably freeze-dried to obtain metal-doped Prussian blue nanozyme (Metal-PBzyme), thus constructing a nanozyme library. The freeze-drying temperature is preferably -90℃ to -60℃, more preferably -850℃ to -70℃, and even more preferably -80℃; the freeze-drying time is preferably 24-72 hours, more preferably 36-60 hours, and even more preferably 48 hours.

[0065] This invention also provides an SNP-PB nanozyme, obtained by loading sodium nitroprusside (SNP) onto the ruthenium-doped Prussian blue nanozyme. The sodium nitroprusside loading treatment described in this invention endows the ruthenium-doped Prussian blue nanozyme with NO release capability.

[0066] In this invention, the preferred method for preparing the SNP-PB nanozyme includes the following steps: (1) mixing polyvinylpyrrolidone and potassium ferrocyanide in a ratio of (0.001-0.01) mmol / L:(0.01-0.03) mmol / L and dissolving them in KCl / HCl buffer to obtain solution a; (2) mixing RuCl3 and sodium nitroprusside and dissolving them in KCl / HCl buffer to obtain solution b; (3) mixing solution a and solution b and then adding H2O2 and NaBH4 sequentially, sonicating, stirring, dialyzing, and drying to obtain the final product.

[0067] In this invention, the preferred mass ratio of RuCl3 to sodium nitroprusside is (0.005-0.025) mmol / L:(0.01-0.05) mmol / L, more preferably (0.01-0.022) mmol / L:(0.01-0.022) mmol / L, and even more preferably 0.02 mmol / L:0.02 mmol / L.

[0068] In this invention, the concentration of the KCl / HCl buffer is preferably 0.05-0.2M, more preferably 0.08-0.15M, and even more preferably 0.1M; the amount of the KCl / HCl buffer is preferably 3-10mL, more preferably 4-8mL, and even more preferably 5mL.

[0069] In this invention, the specific parameters of step (3) of the preparation of the SNP-PB nanozyme are the same as above.

[0070] The present invention also provides the application of the ruthenium-doped Prussian blue nanozyme or SNP-PB nanozyme in the preparation of antioxidant drugs.

[0071] The present invention also provides the application of the ruthenium-doped Prussian blue nanozyme or SNP-PB nanozyme in the preparation of antibacterial drugs.

[0072] The present invention also provides the application of the ruthenium-doped Prussian blue nanozyme or SNP-PB nanozyme in the preparation of drugs for treating periodontitis.

[0073] In this invention, preferred methods include treating periodontitis by combating oxidative stress and / or biofilm.

[0074] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] A ruthenium-doped Prussian blue nanozyme:

[0077] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.005 mmol: 0.02 mmol, then dissolve in 5 mL of 0.1 M KCl / HCl buffer to obtain solution A;

[0078] (3) Mix RuCl3 with 0.1M KCl / HCl buffer at a ratio of 0.02 mmol: 5 mL to obtain solution B;

[0079] (4) Mix solution A and solution B evenly, add 400 μL of 12M H2O2 and 2 mg of NaBH4 in sequence, then sonicate in a water bath at room temperature for 15 min, then stir magnetically at 300 r / min for 3 h, then dialyze for 48 h, and then freeze dry at -80℃ for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0080] Example 2

[0081] A ruthenium-doped Prussian blue nanozyme:

[0082] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.001 mmol: 0.03 mmol, then dissolve in 3 mL of 0.05 M KCl / HCl buffer to obtain solution A;

[0083] (3) Mix RuCl3 with 0.05M KCl / HCl buffer at a ratio of 0.025 mmol: 3 mL to obtain solution B;

[0084] (4) Mix solution A and solution B evenly, add 200 μL of 15M H2O2 and 3 mg of NaBH4 in sequence, then sonicate in a water bath at room temperature for 10 min, then magnetically stir at 200 r / min for 4 h, then dialyze for 55 h, and then freeze dry at -80℃ for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0085] Example 3

[0086] A ruthenium-doped Prussian blue nanozyme:

[0087] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.01 mmol: 0.01 mmol, then dissolve in 10 mL of 0.2 M KCl / HCl buffer to obtain solution A;

[0088] (3) Mix RuCl3 with 0.2M KCl / HCl buffer at a ratio of 0.01 mmol: 10 mL to obtain solution B;

[0089] (4) Mix solution A and solution B evenly, add 500 μL of 8M H2O2 and 1 mg of NaBH4 in sequence, then sonicate in a water bath at room temperature for 20 min, then stir magnetically at 400 r / min for 2 h, then dialyze for 40 h, and then freeze dry at -80℃ for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0090] Example 4

[0091] A nanozyme library is composed of ruthenium-doped Prussian blue nanozymes, iron-doped Prussian blue nanozymes, cobalt-doped Prussian blue nanozymes, nickel-doped Prussian blue nanozymes, copper-doped Prussian blue nanozymes, zinc-doped Prussian blue nanozymes, manganese-doped Prussian blue nanozymes, and cerium-doped Prussian blue nanozymes.

[0092] The preparation method is as follows:

[0093] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.005 mmol: 0.02 mmol, then dissolve in 5 mL of 0.1 M KCl / HCl buffer to obtain solution 1;

[0094] (3) FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3 were mixed with 0.1M KCl / HCl buffer at a ratio of 0.02 mmol: 5 mL to obtain solution 2.

[0095] (4) Mix solutions 1 and 2 thoroughly, add 400 μL of 12M H2O2, and add 2 mg of NaBH4 in the reaction where the metal compound is RuCl3. Then sonicate in a water bath at room temperature for 15 min, stir magnetically at 300 r / min for 3 h, dialyze for 48 h, and freeze dry at -80℃ for 48 h to obtain ruthenium-doped Prussian blue nanozymes, iron-doped Prussian blue nanozymes, cobalt-doped Prussian blue nanozymes, nickel-doped Prussian blue nanozymes, copper-doped Prussian blue nanozymes, zinc-doped Prussian blue nanozymes, manganese-doped Prussian blue nanozymes, and cerium-doped Prussian blue nanozymes, and construct a nanozyme library.

[0096] Example 5

[0097] A nanozyme library is composed of ruthenium-doped Prussian blue nanozyme, iron-doped Prussian blue nanozyme, cobalt-doped Prussian blue nanozyme, zinc-doped Prussian blue nanozyme, manganese-doped Prussian blue nanozyme, and cerium-doped Prussian blue nanozyme.

[0098] The preparation method is as follows:

[0099] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.01 mmol: 0.03 mmol, then dissolve in 3 mL of 0.05 M KCl / HCl buffer to obtain solution 1;

[0100] (3) FeCl3·6H2O, CoCl2·6H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3 were mixed with 0.05M KCl / HCl buffer at a ratio of 0.025 mmol:3 mL to obtain solution 2.

[0101] (4) Mix solutions 1 and 2 thoroughly, add 200 μL of 15 M H2O2, and add 3 mg of NaBH4 in the reaction where the metal compound is RuCl3. Then sonicate in a water bath at room temperature for 10 min, stir magnetically at 200 r / min for 4 h, dialyze for 55 h, and freeze dry at -80℃ for 48 h to obtain ruthenium-doped Prussian blue nanozymes, iron-doped Prussian blue nanozymes, cobalt-doped Prussian blue nanozymes, zinc-doped Prussian blue nanozymes, manganese-doped Prussian blue nanozymes, and cerium-doped Prussian blue nanozymes, and construct a nanozyme library.

[0102] Example 6

[0103] A type of SNP-PB nanozyme:

[0104] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.005 mmol: 0.02 mmol, then dissolve in 5 mL of 0.1 M KCl / HCl buffer to obtain solution a;

[0105] (2) Mix RuCl3 and sodium nitroprusside at a ratio of 0.02 mmol: 0.001 mmol, then dissolve in 5 mL of 0.1 M KCl / HCl buffer to obtain solution b;

[0106] (3) After mixing solution a and solution b, add them one by one and stir until homogeneous. Then add 400 μL of 12M H2O2 and 2 mg of NaBH4 one by one. Then sonicate in a water bath at room temperature for 15 min, then stir magnetically at 300 r / min for 3 h, then dialyze for 48 h, and then freeze dry at -80℃ for 48 h to obtain SNP-PB nanozyme.

[0107] Example 7

[0108] A type of SNP-PB nanozyme:

[0109] (1) Mix polyvinylpyrrolidone and potassium ferrocyanide at a ratio of 0.001 mmol: 0.01 mmol, then dissolve in 10 mL of 0.2 M KCl / HCl buffer to obtain solution a;

[0110] (2) Mix RuCl3 and sodium nitroprusside at a ratio of 0.025 mmol:0.05 mmol, then dissolve in 10 mL of 0.2 M KCl / HCl buffer to obtain solution b;

[0111] (3) After mixing solution a and solution b, add them one by one and stir until homogeneous. Then add 500 μL of 8M H2O2 and 1 mg of NaBH4 one by one. Then sonicate in a water bath at room temperature for 20 min, then stir magnetically at 400 r / min for 2 h, then dialyze for 48 h, and then freeze dry at -80℃ for 48 h to obtain SNP-PB nanozyme.

[0112] Experimental Example 1

[0113] (I) Construction of Nanozyme Library

[0114] 0.005 mmol of polyvinylpyrrolidone and 0.02 mmol of K₄Fe(CN)₆ were combined and dissolved in 5 mL of 0.1 M KCl / HCl buffer to form solution 1. 0.02 mmol of each of the following compounds were dissolved in 5 mL of 0.1 M KCl / HCl buffer to form solution 2: FeCl₃·6H₂O, CoCl₂·6H₂O, NiCl₂·6H₂O, CuCl₂·5H₂O, ZnCl₂, MnCl₂·4H₂O, CeCl₂·7H₂O, and RuCl₃ respectively to form solution 2. Solutions 1 and 2 were mixed and stirred, and 400 μL of 12 M H₂O₂ was added simultaneously. In the reaction involving RuCl₃, 2 mg of NaBH₄ was added. The mixture was sonicated in a water bath for 15 min, followed by magnetic stirring at 300 rpm for 3 h. Finally, after dialysis for 48 hours, the nanozyme library was obtained by freeze-drying at -80℃ for 48 hours. The nanozyme libraries were constructed by obtaining ruthenium-doped Prussian blue nanozymes (Ru-PB), iron-doped Prussian blue nanozymes (Fe-PB), cobalt-doped Prussian blue nanozymes (Co-PB), nickel-doped Prussian blue nanozymes (Ni-PB), copper-doped Prussian blue nanozymes (Cu-PB), zinc-doped Prussian blue nanozymes (Zn-PB), manganese-doped Prussian blue nanozymes (Mn-PB), and cerium-doped Prussian blue nanozymes (Ce-PB). (two)

[0116] The products prepared above were characterized by electron microscopy, particle size, enzyme activity and photothermal properties, and the results were compared. For specific methods of relevant characterization measurements, please refer to "Bioinspired metal-organic framework nanozyme reinforced with thermosensitive hydrogel for regulating inflammatory responses in Parkinson's disease" (Fan X, Zhang T, Ding X, et al. Bioinspired metal-organic framework nanozyme reinforced with thermosensitive hydrogel for regulating inflammatory responses in Parkinson's disease [J]. Research, 2024,17(2):858-865.), "Personalized Carbon Monoxide-Loaded Biomimetic Single-AtomNanozyme for Ferroptosis-Enhanced FLASH Radioimmunotherapy" (Lyu M, Luo M, Li J, et al.Personalized Carbon Monoxide-Loaded Biomimetic Single-AtomNanozyme for Ferroptosis-Enhanced FLASH Radioimmunotherapy[J].AdvancedFunctional Materials,2023,33(51):2306930.), "Ablation of Gap Junction ProteinImproves the Efficiency of Nanozyme-Mediated Catalytic / Starvation / Mild-Temperature Photothermal Therapy” (Li Y, Zhang Y, Dong Y, et al.Ablation ofgapjunction protein improves the efficiency of nanozyme-mediated catalytic / starvation / mild-temperature photothermal therapy[J]. Advanced Materials, 2023, 35(22):2210464.). .

[0117] The measurement results are as follows Figures 1-4 Ruthenium-doped Prussian blue nanozymes (Ru-PB) exhibit good morphological uniformity, small particle size, relatively high enzyme activities such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), DPPH, and ABTS, and good photothermal properties.

[0118] Experimental Example 2

[0119] Further research will be conducted on the PBzyme with relatively superior performance selected above, namely ruthenium-doped Prussian blue nanozyme (Ru-PB).

[0120] (I) Preparation of SNP-PB nanozymes

[0121] 44 mg of polyvinylpyrrolidone was combined with 0.02 mmol of K₄Fe(CN)₆ and dissolved in 5 mL of 0.1 M KCl / HCl buffer to obtain solution a. 0.02 mmol of RuCl₃ and 0.02 mmol of SNP were dissolved in 5 mL of 0.1 M KCl / HCl buffer to obtain solution b. Solutions A and B were mixed and stirred, and 400 μL of 12 M H₂O₂ and 2 mg of NaBH₄ were added simultaneously. The mixture was sonicated in a water bath for 15 min, followed by magnetic stirring at 300 rpm for 3 h. Finally, after dialyzing for 48 h, the mixture was freeze-dried to obtain the SNP-PB nanozyme (SPBzyme). (two)

[0123] The morphology, photothermal properties, and NO release capacity of the prepared SNP-PB nanozyme were investigated. To study the in vitro NO release capacity of the nanozyme, the Griess Reagent method was used to detect the NO concentration in the system. First, Griess Reagent I and II were brought to room temperature, and NaNO2 was diluted with water to different concentrations (0, 1.25, 2.5, 5, 10, 20, 40, 60, 100 μM) to prepare standard curves. 50 μL of Griess Reagent I, 50 μL of Griess Reagent II, and 50 μL of NaNO2 / nanozyme systems were added to each well of a 96-well plate, and the absorbance at 540 nm was measured. Standard curves were plotted based on the absorbance of different NaNO2 concentrations. The NO release concentrations of different concentrations (25, 50, 100, 667 μg / mL) of SPBzyme nanozyme were substituted into the standard curves to calculate their NO release capacity.

[0124] according to Figures 5-7 It can be seen that loading sodium nitroprusside has no significant effect on the morphology and photothermal properties of nanozymes, and that loading sodium nitroprusside enables the release of NO gas.

[0125] Experiment 3: In vitro cellular level verification of the safety and antioxidant properties of Ru-PB (one)

[0127] Human gingival fibroblasts (HGFs) L929 (purchased from Wuhan Pronosai Biotechnology Co., Ltd.) were co-cultured with Ru-PB and SPBzyme, respectively, and their biocompatibility was tested.

[0128] HGFs cells were cultured in 96-well plates at a density of 8000 cells per well and allowed to adhere for 12 h. Subsequently, complete culture medium was mixed with SPBzyme (experimental group) and Ru-PB (control group) to prepare solutions with concentrations of 0, 10, 25, 50, 75, 100, 150, and 200 μg / mL, respectively, and incubated for 24 h. Afterward, the initial culture medium was discarded, and the cells were gently washed once or twice with PBS. The proliferation capacity of human gingival fibroblasts in each group was quantitatively analyzed using a CCK-8 assay kit (purchased from Beyotime Biotechnology Research Institute). Working solution was prepared at a CCK-8 to initial culture medium volume ratio of 1:10, and 100 μL of CCK-8 working solution was added to each well at 37°C for 1 h. Finally, the absorbance at 450 nm was measured using a GF-M3000 microplate reader.

[0129] The results are as follows Figure 8 As shown, HGFs cells are well-safe in Ru-PB and SPBzyme environments. (two)

[0131] The antioxidant capacity of Ru-PB and SPBzyme was tested by simulating oxidative stress using LPS / H2O2.

[0132] HGFs cells were cultured in 96-well plates at a density of 8000 cells per well and allowed to adhere for 12 h. H2O2 was used to simulate ROS-induced oxidative stress damage. Cell viability was selected for subsequent experiments at a 50% H2O2 concentration. HGFs cells were cultured at a density of 8 × 10⁶ cells / well. 3 Cells were seeded in 96-well plates and prepared in PBS, H2O2, H2O2+Ru-PB, and H2O2+SPBzyme groups. After cell adhesion, the original culture medium was discarded.

[0133] Different concentrations of Ru-PB and SPBzyme (25, 50, 75, 100, 150, 200 μg / mL) were added to 96-well plates for pre-protection for 12 h. The cell culture supernatant was then discarded, and the cells were treated with H2O2. Hydrogen peroxide was prepared to 40 μM using complete culture medium and added to the wells for induction for 12 h. Finally, cell viability was quantified using the CCK-8 assay.

[0134] The results are as follows Figure 9 As shown, SPBzyme at 100 μg / mL showed the best effect in alleviating oxidative stress. (three)

[0136] Macrophages (RAW 264.7) (purchased from Wuhan Pronosai Biotechnology Co., Ltd.) were used at a density of 6 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates, with PBS, LPS, Ru-PB, and SPBzyme groups selected. After adhesion, Ru-PB and SPBzyme were dissolved in complete culture medium at a final concentration of 100 μg / mL for 12 h of pre-protection. Next, LPS was added to the LPS, Ru-PB, and SPBzyme groups to achieve a final concentration of 30 μg / mL, resulting in LPS, LPS+Ru-PB, and LPS+SPBzyme groups, respectively. ROS induction incubation was then performed for 12 h. Cells were incubated for 30 min in serum-free medium containing 10 μM DCFH-DA reactive oxygen species probe, washed three times with pre-cooled PBS, and samples were collected. Intracellular ROS levels were quantitatively detected using flow cytometry.

[0137] The results are as follows Figure 10 As shown, the 100 μg / mL LPS+Ru-PB and LPS+SPBzyme groups exhibited good ROS scavenging abilities, with the LPS+SPBzyme group showing the best ROS scavenging ability.

[0138] RAW264.7 cells were spaced at a density of 5 × 10⁶ cells per well. 5 Cells were seeded onto 12-well plates using PBS, LPS, LPS+Ru-PB, and LPS+SPBzyme groups. After adhesion, Ru-PB and SPBzyme were dissolved in complete culture medium at a final concentration of 100 μg / mL for 12 h of pre-protection. Next, LPS was added to the LPS, LPS+Ru-PB, and LPS+SPBzyme groups to a final concentration of 30 μg / mL for ROS induction incubation for 12 h. Cells were then incubated for 30 min in serum-free medium containing 10 μM DCFH-DA reactive oxygen species probe. After washing three times with pre-cooled PBS, the smears were removed and mounted using a DAPI-containing anti-fluorescence quenching mounting medium. Intracellular ROS levels were observed under a confocal microscope (green fluorescence represents ROS levels).

[0139] The results are as follows Figure 11 As shown, the intensity of green fluorescence emitted by LPS-induced RAW264.7 cells was significantly reduced after treatment with 100 μg / mL Ru-PB or SPBzyme, indicating that they had good ROS scavenging ability, with the LPS+SPBzyme group showing the best effect.

[0140] Further detection of mitochondrial membrane potential to detect ROS damage response: Raw264.7 cells were loaded at 1 × 10⁻⁶ cells per well. 5 Cells were placed on cell spreaders in 12-well plates and incubated for 12 h. Cells were divided into groups: PBS, LPS, LPS+Ru-PB, and LPS+SPBzyme groups. Cells were cultured in serum-containing medium (PBzyme and SPBzyme groups) for 12 h. Subsequently, they were induced in 30 μg / mL LPS for 12 h. Changes in mitochondrial membrane potential were assessed using the JC-1 probe. The JC-1 probe included in the assay kit (purchased from Beyotime Biotechnology Research Institute) was typically used for staining at 37°C for 30 min. After washing three times with PBS, mitochondrial membrane potential was assessed using confocal laser microscopy.

[0141] according to Figure 12 It can be seen that the mitochondrial membrane potential was restored in the LPS+Ru-PB and LPS+SPBzyme groups, and the red fluorescence intensity increased significantly, with the LPS+SPBzyme group showing the most obvious effect.

[0142] Test Example 4: Verification of in vitro antibacterial activity (one)

[0144] Subgingival plaque biofilm is a key initiator of periodontal disease. *Fusobacterium nucleatum* (purchased from Cyagen Biosciences) is a typical representative of periodontal pathogens. *F. nucleatum* biofilms were co-cultured with Ru-PB and SPBzyme, respectively, to assess their bactericidal activity. Groups were established: PBS, Ru-PB, SPBzyme, Ru-PB+NIR, and SPBzyme+NIR. After treatment in each group, the antibacterial effect and colony-forming units (CFU) of *F. nucleatum* were measured.

[0145] like Figure 13 As can be seen, compared with the PBS group (control group), the growth of *F. nucleatum* biofilm in both the Ru-PB group and the SPBzyme group showed a significant decreasing trend, regardless of whether NIR treatment was applied. Specifically, the CFU of *F. nucleatum* in the Ru-PB group decreased by approximately three orders of magnitude, while the CFU in the SPBzyme group decreased by approximately four orders of magnitude. The CFU in the SPBzyme group was about one log less than that in the Ru-PB group, and the addition of NIR reduced the CFU in the Ru-PB group and the SPBzyme group by one and two logs, respectively. Compared with the PBS group, the CFU in the NIR+SPBzyme group even decreased to 10. 4 The decrease in CFU indicates that SPBzyme has a strong antibacterial ability under NIR-involved conditions, and it can also be indirectly inferred that SPBzyme releases NO, thereby enhancing the antibacterial ability of the nanozyme. (two)

[0147] To more intuitively observe the bacterial morphology after treatment, SEM was used to observe the morphology of F. nucleatum under different treatment conditions.

[0148] according to Figure 14 It can be seen that in the PBS group, *F. nucleatum* appeared as long filaments, while in the Ru-PB and SPBzyme groups, some bacteria began to exhibit irregular linear morphologies, and in the SPBzyme group, some bacteria even began to break apart. Near-infrared treatment significantly altered the morphology of *F. nucleatum*, especially in the SPBzyme group, indicating its good antibacterial effect. (three)

[0150] Images of F. nucleatum biofilm formation after 96 hours of biofilm establishment were obtained by live / dead staining.

[0151] The results are as follows Figure 15As shown, the SPBzyme group exhibited more red fluorescence compared to the Ru-PB group. The SPBzyme+NIR group, due to its promotion of NO production, showed the most significant increase in the ratio of dead to live bacteria and the smallest biofilm thickness. The Ru-PB group, with its synergistic NIR effect, demonstrated poorer biofilm eradication ability than both the Ru-PB and SPBzyme groups in terms of both the dead / live bacteria ratio and biofilm thickness. This indicates that NO release from the SPBzyme group significantly upregulated the proportion of (dead) bacteria in the overall biofilm, and that NIR treatment significantly accelerated NO release. Biofilm formation was also hindered by NO production, showing a trend similar to that of the live / dead bacteria ratio.

[0152] Experiment 5: Validation of therapeutic efficacy in a rat model of periodontitis (one)

[0154] Eight-week-old male SD rats (150-180g / rat) were used (purchased from Vital River Corporation). All animal experiments in this study were approved by the Institutional Animal Welfare and Utilization Committee of Peking University. SD rats were anesthetized by inhalation (20mg / kg body weight), and a periodontal inflammation model was established by ligating the necks of the bilateral maxillary first molars with 0.2mm diameter orthodontic stainless steel wire. The rats were closely monitored post-operatively. From the day of the experiment, they were fed a sugar-containing solution, and 12 hours later, they were fed a high-sugar diet daily (drinking water was 10% glucose solution, and the feed was the regular feed, softened by soaking in 10% glucose solution). The periodontal ligation was checked weekly post-operatively, and the periodontal tissue condition was observed. After 8 weeks, the rat periodontitis model was successfully established, and the ligatures were removed.

[0155] Ru-PB and SPBzyme (100 μg / mL) were injected in situ at a dose of 0.2 mL / rat on days 3 and 7. The NIR group received the same concentrations of Ru-PB and SPBzyme and was treated with NIR (808 nm, 1 W / cm²). 2 Rats were irradiated for 5 minutes daily on days 3 and 7. The control group received 0.2 mL of sterile PBS per rat. On days 3 and 7, rats were euthanized by excessive CO2 inhalation. The maxillae of SD rats were isolated, and soft tissue was removed. After fixation with 4% paraformaldehyde for 24 hours, the samples were dehydrated, decalcified, embedded in paraffin, and sealed. Then, H&E staining and IHC staining were performed.

[0156] The results are as follows Figure 16As shown, periodontal tissues were histologically evaluated using hematoxylin and eosin (H&E) staining, and the inflammatory response in the five groups was analyzed after 3 and 7 days of treatment. In the PBS group, a large number of immune cells were observed in the periodontal tissues, with irregularly arranged and downregulated fibroblasts. This condition was alleviated after the use of Ru-PB and SPBzyme. In contrast, Ru-PB and SPBzyme treated with NIR showed a significant reduction in inflammatory cells, while SPBzyme treated with NIR appeared healthier. The results indicated that immunohistochemistry (IHC) was used to detect the expression of inflammatory markers IL-1β and TNF-α in the periodontal tissues of each group. In the IHC experiment, IL-1β and TNF-α positive cells were marked as brownish-yellow. IL-1β and TNF-α levels in the NIR group were significantly lower than those in the control group and the non-NIR group. Among all groups, the SPBzyme+NIR group had the lowest expression of IL-1β and TNF-α on days 3 and 7 of treatment, indicating that NIR-enhanced NO release can significantly inhibit the expression of inflammatory factors and alleviate the inflammatory response. (two)

[0158] Micro-CT and histological analysis were performed. The maxilla was dissected, fixed with 10% formalin for 24 hours, and then analyzed using multimodal three-dimensional visualization software.

[0159] The results are as follows Figures 17-18 As shown, regardless of whether NIR was present, the CEJ-ABC distance in the SPBzyme group was smaller than that in the control group and the Ru-PB group after 3 and 7 days, with the SPBzyme+NIR group having the smallest CEJ-ABC distance. Figure 17 The red dashed line indicates the area where trabecular bone analysis was performed, reflecting alveolar bone loss. Further analysis of the alveolar bone in the five groups of first molars was conducted, measuring the trabecular bone volume and bone volume fraction (BV / TV%). This trend is similar to the CEJ-ABC distance. This observation strongly confirms that the SPBzyme+NIR treatment was significantly more effective than other groups, indicating that NIR enhanced the NO release capacity of SPBzyme.

[0160] In summary, the ruthenium-doped Prussian blue nanozymes and SNP-PB nanozymes prepared by the technical solution of this invention both have good antibacterial and anti-inflammatory properties. After being endowed with NO gas release capability, they better help to solve the resistance problem of biofilms and can better exert their antioxidant stress resistance and fight against periodontal bacterial infection.

[0161] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A SNP-PB nanozyme, characterized in that, The ruthenium-doped Prussian blue nanoscale enzyme is obtained by reacting metal ruthenium with Prussian blue nanoparticles at room temperature. The ruthenium-doped Prussian blue nanoscale enzyme is obtained by reacting metal ruthenium with Prussian blue nanoparticles at room temperature. The preparation method of the ruthenium-doped Prussian blue nanoscale enzyme comprises the following steps: (1) polyvinylpyrrolidone and potassium ferrocyanide are mixed in a ratio of (0.001-0.01) mmol:(0.01-0.03) mmol, and then dissolved in KCl / HCl buffer solution to obtain solution A; (2) RuCl3 and KCl / HCl buffer solution are mixed in a ratio of (0.005-0.025) mmol:(3-10) mL to obtain solution B; (3) solution A and solution B are mixed, and then H2O2 and NaBH4 are added in sequence, ultrasonic stirring, dialysis and drying to obtain the ruthenium-doped Prussian blue nanoscale enzyme; The concentration of H2O2 is 8-15 M, and the addition amount of H2O2 is 200-500 μL; The addition amount of NaBH4 is 1-3 mg.

2. The method of claim 1, wherein the ruthenium-doped Prussian blue nanoszyme is prepared by the method comprising: The preparation method comprises the following steps: (1) polyvinylpyrrolidone and potassium ferrocyanide are mixed in a ratio of (0.001-0.01) mmol:(0.01-0.03) mmol, and then dissolved in KCl / HCl buffer solution to obtain solution A; (2) RuCl3 and KCl / HCl buffer solution are mixed in a ratio of (0.005-0.025) mmol:(3-10) mL to obtain solution B; (3) solution A and solution B are mixed, and then H2O2 and NaBH4 are added in sequence, ultrasonic stirring, dialysis and drying to obtain the ruthenium-doped Prussian blue nanoscale enzyme.

3. A nanoscale enzyme library, characterized in that, The ruthenium-doped Prussian blue nanoscale enzyme prepared by the preparation method of claim 2.

4. The nanoscale enzyme library of claim 3, wherein, It also includes one or more of metal iron-doped Prussian blue nanoscale enzyme, metal cobalt-doped Prussian blue nanoscale enzyme, metal nickel-doped Prussian blue nanoscale enzyme, metal copper-doped Prussian blue nanoscale enzyme, metal zinc-doped Prussian blue nanoscale enzyme, metal manganese-doped Prussian blue nanoscale enzyme, and metal cerium-doped Prussian blue nanoscale enzyme.

5. The method for constructing the nanoscale enzyme library according to claim 3 or 4, characterized in that, The preparation method comprises the following steps: (1) polyvinylpyrrolidone and potassium ferrocyanide are mixed in a ratio of (0.001-0.01) mmol:(0.01-0.03) mmol, and then dissolved in KCl / HCl buffer solution to obtain solution 1; (2) the metal compound and KCl / HCl buffer solution are mixed in a ratio of (0.005-0.025) mmol:(3-10) mL to obtain solution 2; (3) solution 1 and solution 2 are mixed, and then H2O2 is added, ultrasonic stirring, dialysis and drying to obtain the ruthenium-doped Prussian blue nanoscale enzyme; The metal compound includes FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3; When the metal compound is RuCl3, NaBH4 needs to be added after the addition of H2O2 in step (3); the addition amount of NaBH4 is 1-3 mg; The concentration of H2O2 is 8-15 M, and the added amount of H2O2 is 200-500 μL.

6. The use of the SNP-PB nanoscale enzyme of claim 1 or the ruthenium-doped Prussian blue nanoscale enzyme prepared by the preparation method of claim 2 in the preparation of an anti-F. nucleatum drug.

7. The use of the SNP-PB nanoscale enzyme of claim 1 or the ruthenium-doped Prussian blue nanoscale enzyme prepared by the preparation method of claim 2 in the preparation of a drug for treating periodontitis.

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