Ruthenium-doped Prussian blue nano-enzyme, preparation method and application thereof, SNP-PB nano-enzyme and application thereof

Abstract

The invention relates to the technical field of nano material application. The invention provides a ruthenium-doped Prussian blue nano-enzyme and a preparation method thereof. The ruthenium-doped Prussian blue nano-enzyme is obtained by reacting metal ruthenium with Prussian blue nano-particles at normal temperature. The invention further provides a nano-enzyme library containing the ruthenium-doped Prussian blue nano-enzyme and a construction method of the nano-enzyme library. The invention also provides the SNP-PB nano-enzyme obtained by loading sodium nitroprusside on the ruthenium-doped Prussian blue nano-enzyme. The ruthenium-doped Prussian blue nano-enzyme and the SNP-PB nano-enzyme both have good antibacterial and anti-inflammatory performance, after NO gas release capacity is given, the problem of resistance of a biological membrane is better solved, then oxidative stress resistance and periodontal bacterial infection resistance are better exerted, and a new treatment method is provided for periodontal diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterial application, and in particular to a ruthenium-doped Prussian blue nanozyme and a preparation method and application thereof, as well as a SNP-PB nanozyme and an application thereof. Background Art

[0002] Periodontitis is an infectious disease of periodontal tissues caused by plaque microorganisms. More than 750 million people suffer from the disease worldwide. It is one of the chronic diseases with a high incidence rate and the sixth most common disease in humans. The plaque biofilm formed by bacterial aggregation is considered to be the initiating factor of periodontitis. The periodontal pathogens in the plaque biofilm release virulence factors and enzymes to destroy the soft and hard tissues of the periodontium, leading to the risk of alveolar bone loss, loose roots and even tooth loss, which seriously affects the quality of life of patients. Clinically, auxiliary antibacterial agents are often used to treat periodontitis. However, the biofilm contains dense extracellular polymers, which are highly adhesive and can resist external mechanical scouring. In addition, the extracellular polymers in the biofilm prevent the inward penetration of traditional antibacterial agents, resulting in dormant bacteria deep in the biofilm, which greatly hinders the destructive effect of antibacterial agents on the plaque biofilm, causing the residual bacteria to re-aggregate, seriously affecting the clinical efficacy of periodontitis.

[0003] Nanozymes exhibit excellent antibacterial and antiviral activity by effectively catalyzing the production of a large amount of ROS (Reactive Oxygen Species) under physiological conditions, and have become a promising new type of "antibiotic". In addition to the characteristics of enzymes, as functionally rich inorganic nanomaterials, nanozymes can use their own characteristics such as high photothermal conversion efficiency and their catalytic properties to play the role of antimicrobial infection. In recent years, new antibacterial therapies based on nanozymes have gradually become a research hotspot. Previous studies have shown that existing nanozymes with photothermal properties have certain limitations in enhancing the antibacterial activity and catalytic properties of nanozymes by balancing ROS levels. Most of them are used to treat inflammation, promote osteogenesis or fight biofilms, and the treatment efficiency is not high. Therefore, it is very necessary to obtain a nanozyme with better antibacterial activity, anti-inflammatory, catalytic and other properties that can be used to treat periodontitis. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a ruthenium-doped Prussian blue nanozyme, which is obtained by reacting metal ruthenium with Prussian blue nanoparticles under room temperature conditions.

[0007] The present invention provides a method for preparing the ruthenium-doped Prussian blue nanozyme, comprising the following steps:

[0008] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution A;

[0009] (2) RuCl3 and KCl / HCl buffer were mixed at a ratio of (0.005-0.025) mmoL:(3-10) mL to obtain solution B;

[0010] (3) After mixing solution A and solution B, H2O2 and NaBH4 are added in sequence, ultrasonicated, stirred, dialyzed, and then dried to obtain the product.

[0011] The present invention provides a nanozyme library, comprising the ruthenium-doped Prussian blue nanozyme.

[0012] Preferably, it also includes one or more of metal iron-doped Prussian blue nanozymes, metal cobalt-doped Prussian blue nanozymes, metal nickel-doped Prussian blue nanozymes, metal copper-doped Prussian blue nanozymes, metal zinc-doped Prussian blue nanozymes, metal manganese-doped Prussian blue nanozymes, and metal cerium-doped Prussian blue nanozymes.

[0013] The present invention also provides a method for constructing the nanozyme library, comprising the following steps:

[0014] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution 1;

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

[0016] (3) After mixing solution 1 and solution 2, add H2O2, sonicate, stir, dialyze, and then dry to obtain;

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

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

[0019] The present invention also provides a SNP-PB nanozyme, which is obtained by loading sodium nitroprusside on the ruthenium-doped Prussian blue nanozyme.

[0020] The present invention also provides the use of the ruthenium-doped Prussian blue nanozyme or the SNP-PB nanozyme in the preparation of antioxidant drugs.

[0021] The present invention also provides the use of the ruthenium-doped Prussian blue nanozyme or the SNP-PB nanozyme in the preparation of antibacterial drugs.

[0022] The present invention also provides the use of the ruthenium-doped Prussian blue nanozyme or the SNP-PB nanozyme in preparing a drug for treating periodontitis.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects:

[0024] The technical solution of the present invention is technically optimized based on the synthesis technology of Prussian blue nanozyme. The metal-engineered Prussian blue nanozyme library can be synthesized at room temperature using different metal doping, and then the nanozyme library is screened according to the photothermal performance and antioxidant capacity to obtain a ruthenium-doped Prussian blue nanozyme with better performance. The ruthenium-doped Prussian blue nanozyme has relatively good antibacterial and anti-inflammatory properties, and also has a good therapeutic effect on combating periodontal bacterial infection. On this basis, ruthenium-doped Prussian blue nanozyme is used to load sodium nitroprusside to obtain SNP-PB nanozyme, and the antibacterial performance is enhanced by giving NO gas release ability. The SNP-PB nanozyme obtained by this photothermal response combined with gas therapy better plays the dual performance of anti-inflammatory and antibacterial, helps to solve the resistance problem of biofilm, and then better plays the role of anti-oxidative stress and combating periodontal bacterial infection, providing a new treatment method for periodontal disease.

[0025] The test examples also showed that while photothermal antibacterial treatment was used, NO gas therapy was released to enhance antibacterial properties and remove bacterial biofilms. At the same time, nanozymes simulated natural superoxide dismutase and catalase to remove excess ROS to achieve the purpose of anti-oxidative stress. This helps to solve the current problem of antibiotic resistance caused by biofilms, and effectively combats inflammation and bacterial infection of periodontitis through dual effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron microscopy morphology of Prussian blue nanozymes doped with different metals in the nanozyme library (scale bar = 200 nm);

[0027] Figure 2 The particle size characterization results of Prussian blue nanozymes doped with different metals in the nanozyme library;

[0028] Figure 3Radar charts of CAT, SOD, POD, DPPH, ABTS and enzyme activity of Prussian blue nanozymes doped with different metals in the nanozyme library;

[0029] Figure 4 The photothermal performance of Prussian blue nanozymes doped with different metals in the nanozyme library;

[0030] Figure 5 is the morphological characterization diagram of SPBzyme (scale bar = 200 nm);

[0031] Figure 6 The temperature rise curve and temperature rise image of 200 μg / mL Ru-PB and SPBzyme under 808 nm near-infrared light irradiation ( Figure 6 A is the temperature increase curve, and B is the temperature increase image);

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

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

[0034] Fig. 9 The effects of Ru-PB and SPBzyme on the cell activity of HGFs under H2O2-simulated inflammatory activation state;

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

[0036] Fig.11 Confocal fluorescence images of RAW264.7 after treatment with different treatment groups (scale bar = 60 μm);

[0037] Fig.12 Mitochondrial membrane potential of RAW264.7 cells after treatment with different treatment groups (scale bar = 60 μm);

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

[0039] Fig.14 SEM images of the effects of different treatment groups on F. nucleatum (scale bar = 5 μm);

[0040] Fig.15 Effects of different treatments on the formation and establishment of F. nucleatum biofilms ( Fig.15 A in the figure indicates the F. nucleatum biofilm formed by live / dead images, B indicates the F. nucleatum biofilm established by live / dead images, C indicates the dead / live ratio and average thickness of the formed F. nucleatum biofilm, and D indicates the dead / live ratio and average thickness of the established F. nucleatum biofilm; red indicates dead bacteria, green indicates live bacteria, and scale bar = 100 μm);

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

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

[0043] Fig.18 To evaluate the in vivo efficacy of different treatment groups in the treatment of periodontitis ( Fig.18 A represents the CEJ-ABC distance on the 3rd day, B represents the alveolar bone BV / TV measurement results on the 3rd day, C represents the CEJ-ABC distance on the 7th day, and D represents the alveolar bone BV / TV measurement results on the 7th day). DETAILED DESCRIPTION

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

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

[0046] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution A;

[0047] (2) RuCl3 and KCl / HCl buffer were mixed at a ratio of (0.005-0.025) mmoL:(3-10) mL to obtain solution B;

[0048] (3) After mixing solution A and solution B, H2O2 and NaBH4 are added in sequence, ultrasonicated, stirred, dialyzed, and then dried to obtain the product.

[0049] In the present invention, polyvinyl pyrrolidone (PVP) and potassium ferrocyanide (K4Fe(CN)6) are mixed, mixed evenly and dissolved in KCl / HCl buffer to obtain solution A. The ratio of polyvinyl pyrrolidone to potassium ferrocyanide in the present invention is preferably (0.001-0.01) mmoL:(0.01-0.03) mmoL, further preferably (0.003-0.008) mmoL:(0.015-0.025) mmoL, and further preferably 0.005 mmoL:0.02 mmoL. The concentration of the KCl / HCl buffer in the present invention is preferably 0.05-0.2M, further preferably 0.08-0.15M, and further preferably 0.1M; the amount of the KCl / HCl buffer is preferably 3-10mL, further preferably 4-8mL, and further preferably 5mL.

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

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

[0052] In the present invention, ultrasonic treatment is performed after uniform mixing, and the ultrasound is preferably water bath ultrasound, the temperature of the water bath is preferably room temperature, and the ultrasonic time is preferably 10-20 minutes, further preferably 12-18 minutes, and further preferably 15 minutes. In the present invention, stirring is performed after the water bath is completed, and the stirring method is preferably magnetic stirring; the stirring speed is preferably 200-400r / min, further preferably 250-350r / min, and further preferably 300r / min; the stirring time is preferably 2-4h, further preferably 2.5-3.5h, and further preferably 3h.

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

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

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

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

[0057] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution 1;

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

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

[0060] In the present invention, polyvinyl pyrrolidone (PVP) and potassium ferrocyanide (K4Fe(CN)6) are mixed, mixed evenly and dissolved in KCl / HCl buffer to obtain solution 1. The ratio of polyvinyl pyrrolidone to potassium ferrocyanide in the present invention is preferably (0.001-0.01) mmoL:(0.01-0.03) mmoL, further preferably (0.003-0.008) mmoL:(0.015-0.025) mmoL, and further preferably 0.005 mmoL:0.02 mmoL. The concentration of the KCl / HCl buffer in the present invention is preferably 0.05-0.2M, further preferably 0.08-0.15M, and further preferably 0.1M; the amount of the KCl / HCl buffer is preferably 3-10mL, further preferably 4-8mL, and further preferably 5mL.

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

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

[0063] In the present invention, ultrasonic treatment is performed after uniform mixing, and the ultrasound is preferably water bath ultrasound, the temperature of the water bath is preferably room temperature, and the ultrasonic time is preferably 10-20 minutes, further preferably 12-18 minutes, and further preferably 15 minutes. In the present invention, stirring is performed after the water bath is completed, and the stirring method is preferably magnetic stirring; the stirring speed is preferably 200-400r / min, further preferably 250-350r / min, and further preferably 300r / min; the stirring time is preferably 2-4h, further preferably 2.5-3.5h, and further preferably 3h.

[0064] In the present invention, dialysis is performed after stirring is completed, and the dialysis time is preferably 40-55h, further preferably 45-50h, and further preferably 48h. After the dialysis of the present invention is completed, freeze drying is preferably performed to obtain metal-doped Prussian blue nanozyme (Metal-PBzyme), that is, a nanozyme library is constructed. The freeze-drying temperature of the present invention is preferably -90°C to -60°C, further preferably -850°C to -70°C, and further preferably -80°C; the freeze-drying time is preferably 24h-72h, further preferably 36h-60h, and further preferably 48h.

[0065] The present invention also provides a SNP-PB nanozyme, which is obtained by loading sodium nitroprusside (SNP) with the ruthenium-doped Prussian blue nanozyme. The sodium nitroprusside loading treatment in the present invention can endow the ruthenium-doped Prussian blue nanozyme with NO release ability.

[0066] In the present invention, the preparation method of the SNP-PB nanozyme preferably includes the following: (1) polyvinyl pyrrolidone and potassium ferrocyanide are mixed in a ratio of (0.001-0.01) mmoL: (0.01-0.03) mmoL and dissolved in KCl / HCl buffer to obtain solution a; (2) RuCl3 and sodium nitroprusside are mixed and dissolved in KCl / HCl buffer to obtain solution b; (3) solution a and solution b are mixed and H2O2 and NaBH4 are added in sequence, ultrasonicated, stirred, dialyzed and dried to obtain.

[0067] In the present invention, the mass ratio of the RuCl3 and sodium nitroprusside mixed is preferably (0.005-0.025) mmoL:(0.01-0.05) mmoL, further preferably (0.01-0.022) mmoL:(0.01-0.022) mmoL, and further preferably 0.02 mmoL:0.02 mmoL.

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

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

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

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

[0072] The present invention also provides the use of the ruthenium-doped Prussian blue nanozyme or the SNP-PB nanozyme in preparing a drug for treating periodontitis.

[0073] In the present invention, it is preferred to treat periodontitis by anti-oxidative stress and / or anti-biofilm.

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

[0075] Example 1

[0076] A ruthenium-doped Prussian blue nanozyme:

[0077] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.005 mmol / L:0.02 mmol / L, and then dissolved in 5 mL of 0.1 M KCl / HCl buffer to obtain solution A;

[0078] (3) RuCl3 and 0.1M KCl / HCl buffer were mixed in a ratio of 0.02mmoL:5mL to obtain solution B;

[0079] (4) Solution A and solution B were mixed and stirred evenly, and 400 μL 12M H2O2 and 2 mg NaBH4 were added in sequence. Then, the mixture was ultrasonicated in a room temperature water bath for 15 min, magnetically stirred at 300 r / min for 3 h, dialyzed for 48 h, and freeze-dried at -80 °C for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0080] Example 2

[0081] A ruthenium-doped Prussian blue nanozyme:

[0082] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.001 mmol / L:0.03 mmol / L, and then dissolved in 3 mL of 0.05 M KCl / HCl buffer to obtain solution A;

[0083] (3) RuCl3 and 0.05M KCl / HCl buffer were mixed in a ratio of 0.025mmoL:3mL to obtain solution B;

[0084] (4) Solution A and solution B were mixed and stirred evenly, and 200 μL 15M H2O2 and 3 mg NaBH4 were added in sequence. Then, the mixture was ultrasonicated in a room temperature water bath for 10 min, magnetically stirred at 200 r / min for 4 h, dialyzed for 55 h, and freeze-dried at -80 °C for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0085] Example 3

[0086] A ruthenium-doped Prussian blue nanozyme:

[0087] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.01 mmoL:0.01 mmoL, and then dissolved in 10 mL of 0.2 M KCl / HCl buffer to obtain solution A;

[0088] (3) RuCl3 and 0.2M KCl / HCl buffer were mixed in a ratio of 0.01mmoL:10mL to obtain solution B;

[0089] (4) Solution A and solution B were mixed and stirred evenly, and 500 μL 8M H2O2 and 1 mg NaBH4 were added in sequence. Then, the mixture was ultrasonicated in a water bath at room temperature for 20 min, magnetically stirred at 400 r / min for 2 h, dialyzed for 40 h, and freeze-dried at -80 °C for 48 h to obtain ruthenium-doped Prussian blue nanozyme.

[0090] Example 4

[0091] A nanozyme library, consisting of ruthenium-doped Prussian blue nanozymes, metal iron-doped Prussian blue nanozymes, metal cobalt-doped Prussian blue nanozymes, metal nickel-doped Prussian blue nanozymes, metal copper-doped Prussian blue nanozymes, metal zinc-doped Prussian blue nanozymes, metal manganese-doped Prussian blue nanozymes, and metal cerium-doped Prussian blue nanozymes;

[0092] The preparation method is as follows:

[0093] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.005 mmol / L:0.02 mmol / L, and then dissolved 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 0.02mmoL:5mL to obtain solution 2 respectively;

[0095] (4) Solution 1 and solution 2 were mixed and stirred respectively, and 400 μL 12M H2O2 was added, wherein 2 mg NaBH4 was also required to be added in the reaction in which the metal compound was RuCl3, and then ultrasonicated in a room temperature water bath for 15 min, and then magnetically stirred at 300 r / min for 3 h, and then dialyzed for 48 h, and then freeze-dried at -80°C for 48 h, to obtain ruthenium-doped Prussian blue nanozyme, metal iron-doped Prussian blue nanozyme, metal cobalt-doped Prussian blue nanozyme, metal nickel-doped Prussian blue nanozyme, metal copper-doped Prussian blue nanozyme, metal zinc-doped Prussian blue nanozyme, metal manganese-doped Prussian blue nanozyme and metal cerium-doped Prussian blue nanozyme, respectively, to construct a nanozyme library.

[0096] Example 5

[0097] A nanozyme library, consisting of ruthenium-doped Prussian blue nanozymes, metal iron-doped Prussian blue nanozymes, metal cobalt-doped Prussian blue nanozymes, metal zinc-doped Prussian blue nanozymes, metal manganese-doped Prussian blue nanozymes, and metal cerium-doped Prussian blue nanozymes;

[0098] The preparation method is as follows:

[0099] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.01 mmol / L:0.03 mmol / L, and then dissolved 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 0.025mmoL:3mL to obtain solution 2 respectively;

[0101] (4) Solution 1 and solution 2 were mixed and stirred respectively, and 200 μL 15M H2O2 was added, wherein 3 mg NaBH4 was also required to be added in the reaction where the metal compound was RuCl3, and then ultrasonicated in a room temperature water bath for 10 min, and then magnetically stirred at 200 r / min for 4 h, and then dialyzed for 55 h, and then freeze-dried at -80°C for 48 h, to obtain ruthenium-doped Prussian blue nanozyme, metal iron-doped Prussian blue nanozyme, metal cobalt-doped Prussian blue nanozyme, metal zinc-doped Prussian blue nanozyme, metal manganese-doped Prussian blue nanozyme and metal cerium-doped Prussian blue nanozyme, respectively, to construct a nanozyme library.

[0102] Example 6

[0103] A SNP-PB nanozyme:

[0104] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly in a ratio of 0.005 mmol / L:0.02 mmol / L, and then dissolved in 5 mL of 0.1 M KCl / HCl buffer to obtain solution a;

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

[0106] (3) Solution a and solution b were mixed and added in sequence, and the mixture was stirred evenly. 400 μL 12 M H2O2 and 2 mg NaBH4 were added in sequence. Then, the mixture was ultrasonicated in a water bath at room temperature for 15 min, and then magnetically stirred at 300 r / min for 3 h. Then, the mixture was dialyzed for 48 h, and then freeze-dried at -80 °C for 48 h to obtain SNP-PB nanozyme.

[0107] Example 7

[0108] A SNP-PB nanozyme:

[0109] (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed uniformly at a ratio of 0.001 mmol / L:0.01 mmol / L, and then dissolved in 10 mL of 0.2 M KCl / HCl buffer to obtain solution a;

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

[0111] (3) Solution a and solution b were mixed and added in sequence, and the mixture was stirred evenly. 500 μL 8M H2O2 and 1 mg NaBH4 were added in sequence. Then, the mixture was ultrasonicated in a water bath at room temperature for 20 min, magnetically stirred at 400 r / min for 2 h, dialyzed for 48 h, and freeze-dried at -80 °C for 48 h to obtain SNP-PB nanozyme.

[0112] Test Example 1

[0113] (I) Construction of nanozyme library

[0114] 0.005mmoL of polyvinyl pyrrolidone and 0.02mmoL of K4Fe(CN)6 were dissolved in 5mL of 0.1M KCl / HCl buffer to form solution 1. 0.02mmoL of FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3 were dissolved in 5mL of 0.1M KCl / HCl buffer to form solution 2. Solution 1 and solution 2 were mixed and stirred, and 400μL of 12M H2O2 was added at the same time, wherein 2mg of NaBH4 was added in the reaction treatment of the metal compound being RuCl3. Water bath ultrasound was used for 15min, followed by magnetic stirring at a speed of 300r / min for 3h. Finally, after dialysis for 48 hours and freeze-drying at -80°C for 48 hours, ruthenium-doped Prussian blue nanozyme (Ru-PB), iron-doped Prussian blue nanozyme (Fe-PB), cobalt-doped Prussian blue nanozyme (Co-PB), nickel-doped Prussian blue nanozyme (Ni-PB), copper-doped Prussian blue nanozyme (Cu-PB), zinc-doped Prussian blue nanozyme (Zn-PB), manganese-doped Prussian blue nanozyme (Mn-PB) and cerium-doped Prussian blue nanozyme (Ce-PB) were obtained, respectively, and a nanozyme library was constructed. (two)

[0116] The products prepared above were characterized by electron microscopy, particle size, enzyme activity, photothermal performance, etc., and 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 results of the test are as follows Figure 1-4 Ruthenium-doped Prussian blue nanozyme (Ru-PB) has good uniform morphology, small particle size, relatively high superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), DPPH, and ABTS enzyme activities, and good photothermal performance.

[0118] Test Example 2

[0119] Based on the above-screened PBzyme with relatively better performance, ruthenium-doped Prussian blue nanozyme (Ru-PB) was further studied.

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

[0121] 44 mg of polyvinyl pyrrolidone was combined with 0.02 mmoL of K4Fe(CN)6 and dissolved in 5 mL of 0.1 M KCl / HCl buffer to form 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 form solution b. Solution A and solution B were mixed and stirred, and 400 μL of 12 M H2O2 and 2 mg of NaBH4 were added at the same time. Water bath ultrasound was used for 15 min, followed by magnetic stirring at 300 r / min for 3 h. Finally, SNP-PB nanozyme (SPBzyme) was obtained by freeze drying after dialysis for 48 h. (two)

[0123] The SNP-PB nanozyme prepared above was tested for morphology, photothermal performance, and loaded NO release capacity. In order to study the in vitro NO release capacity of nanozymes, this study used the Griess Reagent method to detect the concentration of NO in the system. First, Griess Reagent I and II were restored 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 a standard curve. According to 50 μL Griess Reagent I, 50 μL Griess Reagent II, and 50 μL NaNO2 / nanozyme system per well, a 96-well plate was added, and the absorbance at 540 nm was detected. A standard curve was drawn according to the absorbance of NaNO2 at different concentrations. The NO release concentration of SPBzyme nanozymes at different concentrations (25, 50, 100, 667 μg / mL) was substituted into the standard curve to calculate its ability to release NO.

[0124] according to Figure 5-7 It can be seen that the loading of sodium nitroprusside has no obvious effect on the morphology and photothermal performance of the nanozyme, and after loading with sodium nitroprusside, it has the ability to release NO gas.

[0125] Experimental Example 3 Verification of the safety and antioxidant properties of Ru-PB at the in vitro cell level (one)

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

[0128] HGFs cells were cultured in 96-well plates at a density of 8000 cells per well and adhered for 12 hours. Subsequently, the complete medium was mixed with SPBzyme in the experimental group and Ru-PB in the control group to form solutions with concentrations of 0, 10, 25, 50, 75, 100, 150, and 200 μg / mL, respectively, and incubated for 24 hours. Subsequently, the initial culture medium was discarded, and the cells were gently rinsed once or twice with PBS. The proliferation capacity of human gingival fibroblasts in each group of samples was quantitatively analyzed using a CCK-8 kit (purchased from Biyuntian Biotechnology Research Institute). The working solution was prepared according to a volume ratio of CCK-8 to the initial culture medium of 1:10, and then 100 μL of CCK-8 working solution was added to each well at 37°C for 1 hour. 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 have good safety in Ru-PB and SPBzyme environments. (two)

[0131] LPS / H2O2 was used to simulate oxidative stress, and the antioxidant stress capacity of Ru-PB and SPBzyme was detected.

[0132] HGFs cells were cultured in 96-well plates at a density of 8,000 cells per well and adhered for 12 h. H2O2 was used to simulate ROS-induced oxidative stress damage. The cell viability at 50% H2O2 concentration was selected for subsequent experiments. HGFs cells were cultured at a density of 8×10 3 Inoculate in a 96-well plate and set up PBS, H2O2, H2O2+Ru-PB and H2O2+SPBzyme groups. After the cells adhere to the wall, discard the original culture medium.

[0133] Culture media containing 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 hours, and then the cell culture supernatant was discarded and H2O2 was treated. Hydrogen peroxide was mixed with complete culture medium to 40 μM and added to the well plate for induction for 12 hours. Finally, the CCK-8 method was used to quantify cell viability.

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

[0136] Macrophages (RAW 264.7) (purchased from Wuhan Pronocell Biotechnology Co., Ltd.) were cultured at a density of 6 × 10 per well. 5 Cells were seeded in 6-well plates, and PBS, LPS, Ru-PB and SPBzyme groups were set up. After attachment, Ru-PB and SPBzyme were dissolved in complete medium at a final concentration of 100 μg / mL for 12 hours of pre-protection. Then, LPS was added to the LPS, Ru-PB and SPBzyme groups, respectively, so that the final concentration of LPS reached 30 μg / mL, and LPS, LPS+Ru-PB and LPS+SPBzyme groups were obtained. Then ROS induction was incubated for 12 hours. Incubate in serum-free medium containing 10 μM DCFH-DA reactive oxygen species probe for 30 minutes, and collect samples after washing three times with pre-cooled PBS. Flow cytometry was used to quantify the intracellular ROS content.

[0137] The results are as follows Fig.10 As shown, the 100 μg / mL LPS+Ru-PB and LPS+SPBzyme groups had better ROS scavenging ability, among which the LPS+SPBzyme group had the best ROS scavenging ability.

[0138] RAW264.7 cells were plated at a density of 5 × 10 5 Cells were inoculated on a slide in a 12-well plate, and PBS, LPS, LPS+Ru-PB, and LPS+SPBzyme groups were set up. After attachment, Ru-PB and SPBzyme were dissolved in complete culture medium at a final concentration of 100 μg / mL for 12 hours of pre-protection. Then, 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 hours. Incubate for 30 minutes in serum-free culture medium containing 10 μM DCFH-DA reactive oxygen species probe, wash three times with pre-cooled PBS, remove the slide, and use DAPI-containing anti-fluorescence quenching sealing agent and seal the slide. Observe the intracellular ROS content under a confocal microscope (green fluorescence represents the ROS level).

[0139] The results are as follows Fig.11 As shown, the green fluorescence intensity of 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, among which the LPS+SPBzyme group had the best effect.

[0140] Further detection of mitochondrial membrane potential to detect the recovery of ROS damage: Raw264.7 cells were plated at 1×10 5 The cells were placed on a cell slide in a 12-well plate and adhered to the wall in an incubator for 12 hours. The groups were set as PBS, LPS, LPS+Ru-PB and LPS+SPBzyme groups, and the cells were cultured in serum-containing medium containing drugs (PBzyme and SPBzyme groups) for 12 hours. Subsequently, they were induced in 30μg / mL LPS for 12 hours. The changes in mitochondrial membrane potential were evaluated using the JC-1 probe. The JC-1 probe included in the test kit (purchased from Biyuntian Biotechnology Research Institute) is usually used for staining at a temperature of 37°C for 30 minutes. After washing three times with PBS, the mitochondrial membrane potential was evaluated using a confocal laser microscope.

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

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

[0144] Subgingival plaque biofilm is the initiating factor of periodontal disease. Fusobacterium nucleatum (F.nucleatum) (purchased from Cyagen Biosciences) is a typical representative of periodontal pathogens. F.nucleatum biofilm was co-cultured with Ru-PB and SPBzyme to detect its bactericidal ability. The groups were set as PBS, Ru-PB, SPBzyme, Ru-PB+NIR and SPBzyme+NIR groups. After each group was treated, the antibacterial effect and colony forming unit (CFU) of F.nucleatum were determined.

[0145] like Fig.13 It can be seen that compared with the PBS group (control group), the F.nucleatum biofilm growth in the Ru-PB group and the SPBzyme group showed a significant downward trend regardless of whether it was treated with NIR. Among them, the F.nucleatum CFU in the Ru-PB group decreased by about 3 orders of magnitude, and the CFU in the SPBzyme group decreased by about 4 orders of magnitude. The CFU in the SPBzyme group was about 1 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 1 Log and 2 Log, respectively. Compared with the PBS group, the CFU in the NIR+SPBzyme group even dropped to 10 4 The decrease in CFU indicated that SPBzyme had a strong antibacterial ability under the condition of NIR participation, and it can also be indirectly speculated that SPBzyme released NO, thereby enhancing the antibacterial ability of nanozymes. (two)

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

[0148] according to Fig.14 It can be seen that in the PBS group, F. nucleatum was filamentous, while in the Ru-PB and SPBzyme groups, some bacteria began to show irregular linear morphology, and some bacteria in the SPBzyme group even began to break. After near-infrared treatment, the morphology of F. nucleatum changed significantly, especially in the SPBzyme group, indicating that its antibacterial effect was good. (three)

[0150] Live / dead staining of F. nucleatum biofilm formation after 96 h Live / dead staining images after biofilm establishment.

[0151] The results are as follows Fig.15As shown, the SPBzyme group had more red fluorescence compared with the Ru-PB group. The SPBzyme+NIR group had the most significant dead / live ratio and the smallest biofilm thickness due to the promotion of NO production. The Ru-PB group with synergistic NIR effect showed poor biofilm eradication ability in terms of dead / live ratio and biofilm thickness compared with the Ru-PB group and SPBzyme group, indicating that the release of NO in the SPBzyme group significantly increased the red staining ratio of bacteria (dead) in the entire biofilm, and the NIR treatment significantly accelerated the release of NO. The formation of biofilm thickness was also hindered by NO production, and it can be seen that its trend was similar to the live-dead ratio.

[0152] Experimental Example 5: Verification of therapeutic ability in rat periodontitis model (one)

[0154] Eight-week-old male SD rats (150-180 g / rat) (purchased from Vital River Corporation) were selected. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Peking University. The SD rats were anesthetized by inhalation (20 mg / kg body weight), and a ligature (orthodontic stainless steel wire with a diameter of 0.2 mm) was placed on the cervical part of the first maxillary molars on both sides of the rats to establish a periodontal inflammation model. The rats were closely observed after surgery. The rats were fed with sugar water from the day of the experiment, and high-sugar diet was fed every day after 12 hours (drinking water was 10% glucose water, and feed was regular feed, which was softened by soaking in 10% glucose water). The periodontal ligation was checked every week after surgery, and the periodontal tissue was observed. After 8 weeks, the rat periodontitis model was successfully established and the ligature was 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 was given the same concentration of Ru-PB and SPBzyme and was illuminated with NIR (808 nm, 1 W / cm 2 , 5min per day) on the 3rd and 7th days. The control group was given sterile PBS 0.2mL / rat. On the 3rd and 7th days, the rats were killed by excessive CO2 inhalation. The maxilla of SD rats was isolated and the soft tissue was removed. After fixation with 4% paraformaldehyde for 24h, the samples were dehydrated, decalcified, paraffin-embedded, and blocked. Then H&E staining and IHC staining were performed.

[0156] The results are as follows Fig.16As shown, the periodontal tissues were histologically evaluated by hematoxylin and eosin (H&E) staining to analyze the inflammatory response of the five groups after 3 and 7 days of treatment. A large number of immune cells were observed in the periodontal tissues of the PBS group, and the fibroblasts were irregularly arranged and the number was downregulated. This situation was alleviated after the use of Ru-PB and SPBzyme. In contrast, the inflammatory cells of Ru-PB and SPBzyme treated with NIR were significantly reduced, while SPBzyme treated with NIR appeared healthier. The results showed that the expression of IL-1β and TNF-α, the inflammation-related markers of periodontal tissues in each group, was detected by immunohistochemistry (IHC). In the IHC experiment, IL-1β and TNF-α positive cells were marked in brown-yellow. The IL-1β and TNF-α in the NIR group were significantly lower than those in the control group and the group without NIR. Among all the groups, the SPBzyme+NIR group had the lowest expression of IL-1β and TNF-α on the 3rd and 7th days of treatment, indicating that NIR-enhanced NO release can greatly inhibit the expression of inflammatory factors and alleviate the inflammatory response. (two)

[0158] Micro-CT and histomorphometric analysis were performed. The maxillae were isolated, fixed with 10% formalin for 24 h, and then analyzed using multimodal 3D visualization software.

[0159] The results are as follows Figure 17-18 As shown in Figure 2, regardless of the presence or absence of NIR, the CEJ-ABC distances of the SPBzyme group were smaller than those of the control group and the Ru-PB group after 3 and 7 days, and the CEJ-ABC distance of the SPBzyme+NIR group was the smallest ( Fig.17 The red dotted line in the middle indicates the area where trabecular bone analysis was performed, reflecting the alveolar bone loss. The alveolar bones of the first molars of the five groups were further analyzed, and the trabecular bone volume and bone volume fraction (BV / TV%) of the first molars of the five groups were measured. This trend is similar to the CEJ-ABC distance. This observation fully confirms that the treatment effect of SPBzyme+NIR is significantly better than that of other groups, indicating that NIR enhances the NO release ability of SPBzyme.

[0160] In summary, it can be seen that the ruthenium-doped Prussian blue nanozyme and SNP-PB nanozyme prepared by the technical scheme of the present invention both have good antibacterial and anti-inflammatory properties. After being endowed with the ability to release NO gas, they can better help solve the resistance problem of biofilm, and can better play an anti-oxidative stress and fight periodontal bacterial infection.

[0161] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ruthenium-doped Prussian blue nanozyme, characterized in that: It is obtained by reacting metallic ruthenium with Prussian blue nanoparticles at room temperature.

2. The method for preparing the ruthenium-doped Prussian blue nanozyme according to claim 1, characterized in that: The steps include: (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution A; (2) RuCl3 and KCl / HCl buffer were mixed at a ratio of (0.005-0.025) mmoL:(3-10) mL to obtain solution B; (3) After mixing solution A and solution B, H2O2 and NaBH4 are added in sequence, ultrasonicated, stirred, dialyzed, and then dried to obtain the product.

3. A nanozyme library, characterized in that: Including the ruthenium-doped Prussian blue nanozyme as described in claim 1.

4. The nanozyme library according to claim 3, characterized in that It also includes one or more of metal iron-doped Prussian blue nanozymes, metal cobalt-doped Prussian blue nanozymes, metal nickel-doped Prussian blue nanozymes, metal copper-doped Prussian blue nanozymes, metal zinc-doped Prussian blue nanozymes, metal manganese-doped Prussian blue nanozymes, and metal cerium-doped Prussian blue nanozymes.

5. The method for constructing the nanozyme library according to claim 3 or 4, characterized in that: The steps include: (1) polyvinyl pyrrolidone and potassium ferrocyanide were mixed in a ratio of (0.001-0.01) mmoL:(0.01-0.03) mmoL and dissolved in a KCl / HCl buffer solution to obtain a solution 1; (2) mixing the metal compound with KCl / HCl buffer in a ratio of (0.005-0.025) mmoL:(3-10) mL to obtain solution 2; (3) After mixing solution 1 and solution 2, add H2O2, sonicate, stir, dialyze, and then dry to obtain; The metal compounds include FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuCl2·5H2O, ZnCl2, MnCl2·4H2O, CeCl2·7H2O and RuCl3.

6. The construction method according to claim 5, characterized in that: When the metal compound is RuCl3, NaBH4 needs to be added after adding H2O2 in step (3); the amount of NaBH4 added is 1-3 mg.

7. A SNP-PB nanozyme, characterized in that: The method is obtained by loading sodium nitroprusside onto the ruthenium-doped Prussian blue nanozyme as described in claim 1.

8. Use of the ruthenium-doped Prussian blue nanozyme according to claim 1 or the SNP-PB nanozyme according to claim 7 in the preparation of antioxidant drugs.

9. Use of the ruthenium-doped Prussian blue nanozyme according to claim 1 or the SNP-PB nanozyme according to claim 7 in the preparation of antibacterial drugs.

10. Use of the ruthenium-doped Prussian blue nanozyme according to claim 1 or the SNP-PB nanozyme according to claim 7 in the preparation of a drug for treating periodontitis.

Images