Two-dimensional bimetal organic framework nano-enzyme hydrogel as well as preparation method and application thereof

Abstract

The invention provides two-dimensional bimetal organic framework nano-enzyme hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological materials. According to the preparation method, a FeCuMOF bimetal organic framework is adopted as a nano-enzyme, tannic acid and lipoic acid are used for constructing a composite hydrogel system with antibacterial and antioxidant capacities, and the composite hydrogel is combined with FeCuMOF to form the two-dimensional bimetal organic framework nano-enzyme hydrogel. The two-dimensional bimetal organic framework nano-enzyme hydrogel disclosed by the invention has antibacterial and anti-biofilm properties, and can be used for removing various ROS (Reactive Oxygen Species) by simulating superoxide dismutase and the capability of removing ABTS free radicals thereof. In addition, proinflammatory cytokines can be reduced, and anti-inflammatory cytokines can be increased, so that periodontitis inflammation is relieved, and tissue regeneration is accelerated. The two-dimensional bimetal organic framework nano-enzyme hydrogel disclosed by the invention is used as a multifunctional nano-enzyme hydrogel slow-release platform, and an ideal strategy is provided for treating periodontitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a two-dimensional bimetallic organic framework nanozyme hydrogel and its preparation method and application. Background Art

[0002] Periodontitis is a common chronic oral bacterial infection caused by microorganisms in dental plaque. Many systemic diseases, including diabetes and cardiovascular diseases, are affected by periodontitis. Non-surgical treatment remains the first choice. However, in some severe cases, surgical treatment may be necessary. Inflammation can be controlled by conventional treatment; however, promoting periodontal tissue regeneration and managing inherent periodontal infections are challenging. Although periodontal regeneration surgery has certain clinical benefits, there are problems such as poor predictability and limited clinical indications. The hydrogel drug delivery system has unique structures and properties, which can promote the gradual and regulated release of drugs with antibacterial, anti-inflammatory, and periodontal tissue regeneration properties, and shows great potential in the treatment of periodontitis due to its safety, practicality, and effectiveness. The integration of complex nanoparticles and innovative treatment methods now represents the most dynamic research field in the treatment of periodontitis.

[0003] However, despite the progress in the treatment techniques and drugs for periodontitis, there are still problems in the research and treatment of periodontitis, such as antibiotic resistance, short drug half-life, and lack of antioxidant and antibacterial integrated drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a two-dimensional bimetallic organic framework nanozyme hydrogel and its preparation method and application. The two-dimensional bimetallic organic framework nanozyme hydrogel can effectively combat periodontitis with inflammation and bacterial infection and can be used as an antioxidant and antibacterial integrated drug.

[0005] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of a two-dimensional bimetallic organic framework nanozyme hydrogel, including the following steps:

[0007] Mix tannic acid, FeCu MOF, lipoic acid, Tris, and water, and carry out a gelation reaction to obtain a viscous solution;

[0008] Freeze and thaw the viscous solution in sequence to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel.

[0009] Preferably, the preparation method of the FeCu MOF includes the following steps:

[0010] Mix a copper salt, an iron salt, and a first solvent to obtain a mixed solution;

[0011] Mix the mixture, organic ligand, second solvent and triethylamine, and carry out a coordination reaction to obtain FeCuMOF.

[0012] Preferably, the organic ligand includes terephthalic acid; the mass ratio of the copper salt, iron salt to the organic ligand is (100 - 200):(100 - 200):(150 - 200).

[0013] Preferably, the dosage ratio of the organic ligand to triethylamine is (150 - 200) mg:(20 - 200) μL; the temperature of the coordination reaction is 20 - 30 °C, and the time is 1 - 5 h.

[0014] Preferably, the mass ratio of tannic acid, FeCu MOF and zinc sulfate is (200 - 500):(100 - 300):(1000 - 3000).

[0015] Preferably, the temperature of the gelation reaction is 50 - 80 °C, and the time is 1 - 10 min.

[0016] Preferably, the temperature of freezing is -20 °C ± 3 °C, and the time is 1 - 5 h; the temperature of thawing is 20 - 30 °C, and the time is 1 - 5 h.

[0017] The present invention provides a two-dimensional bimetallic organic framework nanozyme hydrogel prepared by the preparation method described in the above technical solution.

[0018] The present invention provides the application of the two-dimensional bimetallic organic framework nanozyme hydrogel described in the above technical solution in the preparation of antioxidant drugs and antibacterial drugs.

[0019] The present invention provides the application of the two-dimensional bimetallic organic framework nanozyme hydrogel described in the above technical solution in the preparation of drugs for treating periodontitis.

[0020] The present invention provides a method for preparing a two-dimensional bimetallic organic framework nanozyme hydrogel. The FeCuMOF bimetallic organic framework is used as a nanozyme, and tannic acid (antioxidant) and lipoic acid (antioxidant and antibacterial) are used as the hydrogel matrix to construct a composite hydrogel platform with antibacterial and antioxidant capabilities. The composite hydrogel is combined with the FeCuMOF nanozyme to form a two-dimensional bimetallic organic framework nanozyme hydrogel. The two-dimensional bimetallic organic framework nanozyme hydrogel of the present invention has antibacterial and anti-biofilm properties. At the same time, as a nanozyme, the Cu-based nanozyme has high superoxide dismutase (SOD)-like enzyme activity, and the release of Fe ions can cause ferroptosis antibacterial in bacteria. This nanozyme hydrogel can scavenge various reactive oxygen species (ROS) by mimicking superoxide dismutase (SOD) and its ability to scavenge ABTS radicals. The FeCuMOF nanozyme can regulate the level of reactive oxygen species (ROS) to achieve the antioxidant and antibacterial effects of treating periodontitis. In addition, the two-dimensional bimetallic organic framework nanozyme hydrogel platform can reduce pro-inflammatory cytokines and increase anti-inflammatory cytokines, thereby alleviating periodontitis inflammation and accelerating tissue regeneration. The two-dimensional bimetallic organic framework nanozyme hydrogel of the present invention contains water, a gel matrix, and a nanozyme (the FeCuMOF metal-organic framework is injected into the tannic acid-lipoic acid adhesive hydrogel platform) at the same time, which can promote the anti-inflammatory and antibacterial properties of tannic acid and lipoic acid, and gradually release the FeCuMOF nanozyme at the same time to enhance the eradication of periodontal bacteria and relieve the oxidative stress associated with periodontitis. As a multifunctional nanozyme hydrogel sustained-release platform (pTL-FeCuMOF), it provides an ideal strategy for treating periodontitis.

[0021] The results of the test examples show that at the cellular level, the hydrogel platform enhances the mitochondrial bioenergy of inflammatory periodontal ligament stem cells (PDLSCs) and human gingival fibroblasts (HGFs), while reducing the ROS level; the gradual release of the nanozyme hydrogel, combined with its inherent anti-inflammatory properties, can exhibit antibacterial effects and promote the remission of periodontitis. In-situ tissue regeneration was achieved in rat inflammatory periodontal bone defects, which helps to solve the current problem of antibiotic resistance caused by biofilms, and effectively combats periodontitis with inflammation and bacterial infection through dual actions. Description of the Drawings

[0022] Figure 1 Transmission electron microscopy morphology diagram (A, scale bar = 200 nm) and local enlarged diagram (B, scale bar = 20 nm) of the FeCuMOF nanozyme prepared in Example 1;

[0023] Figure 2 Superoxide dismutase enzyme activity inhibition rate curve and enzyme activity results of the FeCuMOF nanozyme prepared in Example 1;

[0024] Figure 3ESR electron spin resonance spectrum of the FeCuMOF nanozyme prepared in Example 1 for scavenging hydroxyl radicals;

[0025] Figure 4 ESR electron spin resonance spectrum of the FeCuMOF nanozyme prepared in Example 1 for scavenging superoxide anions;

[0026] Figure 5 Scanning electron microscopy (SEM) image of the pTL-FeCuMOF nanozyme hydrogel prepared in Example 1 (scale bar = 20 μm);

[0027] Figure 6 Rheological properties of the pTL hydrogel prepared in Comparative Example 1;

[0028] Figure 7 Rheological properties of the pTL-FeCuMOF nanozyme hydrogel prepared in Example 1;

[0029] Figure 8 Safety evaluation results of Ru-PBzyme and SPBzyme in BMSCs cells; among them, (A) Representative live cell images of BMSCs on the 3rd day after MOF, pTL, and pTL-FeCuMOF at concentrations of 0, 2.5, 5, 10, 15, and 20 μg / mL, scale bar = 200 μm; (B-E) CCK-8 method was used to determine the activity of BMSCs on the 1st, 3rd, 5th, and 7th days after treatment with MOF, pTL, and pTL-FeCuMOF in the concentration range of 0-20 μg / mL; (F-J) Cell viability of BMSCs cultured with MOF, pTL, and pTL-FeCuMOF at specific concentrations of (F) 2.5, (G) 5, (H) 10, (I) 15, and (J) 20 μg / mL;

[0030] Figure 9 Biocompatibility and ROS scavenging activity of pTL-FeCuMOF prepared in Example 1 in HGF; among them, (A) CCK-8 method was used to determine the cell viability of HGF after 3 days of culture with different treatments; (B) CCK-8 method was used to evaluate the protective effect of different treatments on HGF after H2O2 exposure; (C) Quantitative analysis of intracellular ROS levels in HGF cells after LPS treatment based on fluorescence images; (D) Live / dead fluorescence images of HGF on the 3rd day, scale bar = 200 μm; (E) Activity of MOF, pTL, and pTL-FeCuMOF in scavenging ROS in HGF was evaluated by fluorescence images of intracellular ROS levels after LPS treatment, scale bar = 200 μm, n = 3;

[0031] Figure 10Biocompatibility and ROS scavenging activity of pTL-FeCuMOF prepared in Example 1 in PDLSCs. Among them, (A) Cell viability of PDLSCs cultured for 3 days after different treatments was determined by CCK-8 method; (B) Protective effect of different treatments on PDLSCs after H2O2 exposure was evaluated by CCK-8 method; (C) Intracellular ROS level in PDLSCs after LPS treatment was quantitatively analyzed based on fluorescence images; (D) Live / dead fluorescence images of PDLSCs on the 3rd day, scale bar = 200 μm; (E) ROS scavenging activity of MOF, pTL and pTL-FeCuMOF in PDLSCs was evaluated by fluorescence images of intracellular ROS level after LPS treatment, scale bar = 200 μm, n = 3;

[0032] Figure 11 Biocompatibility and reactive oxygen species scavenging activity of pTL-FeCuMOF prepared in Example 1 in L929; Among them, (A) Cell viability of L929 cultured for 24 h after different treatments was determined by CCK-8 method; (B) Quantitative analysis of intracellular ROS level in L929 cells after H2O2 treatment based on fluorescence images; (C) Protective effect of different treatments on L929 after H2O2 exposure was evaluated by CCK-8 method; (D) DCFH-DA fluorescence images of L929, scale bar = 61.4 μm; (E) Activity of MOF, pTL and pTL-FeCuMOF in scavenging ROS in L929 cells, intracellular ROS level after H2O2 treatment was detected by flow cytometry;

[0033] Figure 12 Antibacterial effect of pTL-FeCuMOF prepared in Example 1 against Fusobacterium nucleatum and Porphyromonas gingivalis; (A) Colony forming units (CFUs) of Fusobacterium nucleatum and Porphyromonas gingivalis treated with PBS, MOF, pTL and pTL-FeCuMOF at the same concentration; (B,C) Quantitative analysis of CFU counts of Fusobacterium nucleatum (B) and Porphyromonas gingivalis (C);

[0034] Figure 13 SEM images of Fusobacterium nucleatum and Porphyromonas gingivalis after different treatments, showing the overall view (scale bar = 5 μm) and enlarged area (scale bar = 1 μm), and the red arrows indicate damaged bacteria;

[0035] Figure 14 Anti-biofilm activity of pTL-FeCuMOF prepared in Example 1 against Fusobacterium nucleatum and Porphyromonas gingivalis; (A,B) Three-dimensional reconstruction and superimposed images of biofilms of Fusobacterium nucleatum (A) and Porphyromonas gingivalis (B) treated with PBS, MOF, pTL and pTL-FeCuMOF; (C,D) Survival rates of Fusobacterium nucleatum (C) and Porphyromonas gingivalis (D) in biofilms after different treatments; (E) Schematic diagram of antibacterial ability;

[0036] Figure 15 Therapeutic effect of pTL-FeCuMOF prepared in Example 1 on a rat periodontitis model; (A) Schematic diagram of the establishment of a rat periodontitis model and the treatment process with pTL-FeCuMOF; (B) Micro-CT images and 3D reconstructions of the maxillary molar region 3 days and 7 days after treatment. The red dotted line indicates the distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC), and the red circle indicates the region for analyzing the bone volume fraction (BV / TV); (C) Quantitative analysis of the CEJ-ABC distance 3 days (C) and 7 days after treatment; (D) Quantitative analysis of the periapical alveolar bone BV / TV of the first maxillary molar 3 days (D) and 7 days after treatment.

[0037] Figure 16 Histological analysis of the anti-inflammatory effect of pTL-FeCuMOF prepared in Example 1 on a periodontitis model; among them, (A, B) H&E staining and IHC staining of TNF-α and IL-1β in periodontal tissues 3 days (A) and 7 days (B) after treatment, scale bar = 500 μm; the brown-yellow area is the positive area of TNF-α and IL-1β, marked with red arrows; (C, D) Quantitative analysis of the TNF-α-positive area (C) and IL-1β-positive area (D) in periodontal tissues 3 days after treatment; (E, F) Quantitative analysis of the TNF-α-positive area (E) and IL-1β-positive area (F) in periodontal tissues 7 days after treatment. Detailed implementation manners

[0038] In the present invention, unless otherwise specified, the raw materials or reagents required for preparation are all commercially available products well-known to those skilled in the art.

[0039] The present invention provides a preparation method of a two-dimensional bimetallic organic framework nanozyme hydrogel, comprising the following steps:

[0040] Mix tannic acid, FeCu MOF, lipoic acid, Tris and water, and carry out a gelation reaction to obtain a viscous solution;

[0041] Freeze and thaw the viscous solution in sequence to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel.

[0042] In the present invention, the preparation method of the FeCuMOF preferably comprises the following steps:

[0043] Mix a copper salt, an iron salt and a first solvent to obtain a mixed solution;

[0044] Mix the mixed solution, an organic ligand, a second solvent and triethylamine, and carry out a coordination reaction to obtain FeCu MOF.

[0045] In the present invention, the organic ligand preferably includes terephthalic acid; the copper salt is preferably copper nitrate; the iron salt is preferably ferric chloride hexahydrate; the mass ratio of the copper salt, the iron salt to the organic ligand is preferably (100 - 200):(100 - 200):(150 - 200), more preferably (150 - 190):(150 - 190):(180 - 200), and further preferably 180:180:200.

[0046] In the present invention, the dosage ratio of the organic ligand to triethylamine is preferably (150 - 200) mg:(20 - 200) μL, more preferably (150 - 200) mg:(100 - 200) μL.

[0047] In the present invention, the first solvent is preferably dimethylformamide (DMF); the dosage ratio of the copper salt to the first solvent is preferably (100 - 200) mg:(5 - 25) mL, more preferably (150 - 180) mg:(10 - 20) mL.

[0048] In the present invention, the second solvent is preferably ethanol and water; the volume ratio of ethanol to water is preferably (3 - 15):(3 - 15), more preferably (5 - 10):(5 - 10); the volume ratio of ethanol to triethylamine is preferably (3 - 15) mL:(20 - 200) μL, more preferably (3 - 15) mL:(50 - 100) μL.

[0049] In the present invention, the temperature of the coordination reaction is preferably 20 - 30 °C, more preferably 25 °C, and the time is preferably 1 - 5 h, more preferably 2 - 3 h.

[0050] In the present invention, the copper salt and the iron salt are preferably added to the first solvent according to the mass ratio and stirred until dissolved, then terephthalic acid is added, and then ethanol, water (preferably ddH2O) and triethylamine are added. After stirring for the coordination reaction at room temperature, a uniform colloidal suspension is obtained. After centrifugation, the precipitate is washed three times with absolute ethanol, the product is collected, and dried in a vacuum drying oven at 40 - 80 °C (more preferably 50 - 60 °C) to obtain FeCu MOF. In the present invention, the rotation speed of the centrifugation is preferably 8000 - 12000 rpm, more preferably 8000 - 10000 rpm, and the time is preferably 5 - 15 min, more preferably 10 min.

[0051] In the present invention, the mass ratio of tannic acid, FeCu MOF and zinc sulfate is preferably (200 - 500):(100 - 300):(1000 - 3000), more preferably (300 - 400):(150 - 200):(2000 - 2500); the mass ratio of tannic acid to Tris is (200 - 500):(500 - 2000), more preferably (300 - 400):(1000 - 1500). The present invention has no special limitation on the amount of water used in this step, and it can be adjusted according to actual needs to ensure the smooth progress of the reaction.

[0052] In the present invention, the temperature of the gelation reaction is preferably 50 - 80°C, more preferably 60 - 70°C, and the time is preferably 1 - 10 min, more preferably 1 - 5 min.

[0053] The present invention preferably dissolves FeCuMOF in water (preferably ddH2O) at 50 - 80°C (preferably 60 - 70°C), adds tannic acid and Tris, stirs and dissolves at 60°C, then adds lipoic acid and stirs vigorously to dissolve, and conducts the gelation reaction under stirring conditions to obtain a viscous solution.

[0054] In the present invention, the temperature of freezing is preferably -20°C ± 3°C, and the time is preferably 1 - 5 h, more preferably 2 - 3 h; the temperature of thawing is preferably 20 - 30°C, more preferably 25°C, and the time is preferably 1 - 5 h, more preferably 1 - 2 h.

[0055] Tannic acid (TA) has antibacterial and antioxidant properties and good biocompatibility. The pyrogallol and catechol groups on the TA molecule can establish physical crosslinks with polymers through mechanisms such as hydrogen bonding, π-π interaction, and hydrophobic interaction, thereby enhancing the mechanical properties and viscosity of the hydrogel. Lipoic acid (LA) is an important coenzyme with a dithiolane ring structure, high electron density, significant electrophilicity, and the ability to react with free radicals. LA can inhibit mitochondrial aging and dysfunction caused by oxidative stress. The present invention constructs a hydrogel platform by compounding tannic acid and lipoic acid, which can assist in improving the antioxidant and anti-inflammatory effects.

[0056] In the present invention, the antibacterial and anti-inflammatory effects of FeCu MOF nanozyme mainly rely on the regulation of the level of reactive oxygen species. Usually, it is used to reduce the concentration of ROS in the inflammatory area, including superoxide dismutase and hydrogen peroxide, thereby reducing the levels of H2O2 and superoxide anions. Hydroxyl radicals (·OH) are generated through the activities of oxidase, peroxidase, and enzyme-like activities to destroy the bacterial cell membrane. At the same time, according to the selectivity of metal ion release, there is also a bactericidal effect. Copper ions (Cu 2+) has high reactivity and significant oxidation potential. It can interact with biomolecules such as microbial proteins, DNA, RNA, etc., destroy the integrity and stability of microbial cell membranes, and cause the death of microorganisms, thereby playing an antibacterial role.

[0057] The present invention provides a two-dimensional bimetallic organic framework nanozyme hydrogel prepared by the preparation method described in the above technical scheme, which has adhesion properties.

[0058] The present invention provides the use of the two-dimensional bimetallic organic framework nanozyme hydrogel described in the above technical solution in the preparation of antioxidant drugs and antibacterial drugs.

[0059] The present invention provides the use of the two-dimensional bimetallic organic framework nanozyme hydrogel described in the above technical solution in the preparation of drugs for treating periodontitis.

[0060] The present invention has no particular limitation on the above-mentioned application method, and the application can be carried out according to methods well known in the art.

[0061] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0062] The following experimental methods and detection methods, unless otherwise specified, are conventional methods; the following reagents and raw materials, unless otherwise specified, are commercially available.

[0063] Example 1

[0064] Preparation of two-dimensional bimetallic organic framework FeCu MOF nanozyme:

[0065] 180 mg Cu(NO3)2 and 180 mg FeCl3·6H2O were added to 20 mL dimethylformamide (DMF) and stirred to dissolve, then 200 mg terephthalic acid was added, followed by 10 mL ethanol, 10 mL ddH2O and 100 μL triethylamine. After stirring at room temperature for 1 h, a uniform colloidal suspension was obtained, which was then centrifuged (10000 rpm for 10 min), the precipitate was washed three times in ethanol, and the product was collected and finally dried in a vacuum oven at 50 °C to obtain FeCuMOF;

[0066] Preparation of pTL-FeCuMOF hydrogel:

[0067] Dissolve 200 mg of FeCu MOF in 100 mL of ddH2O at 60 °C. Add 400 mg of tannic acid and 1 g of Tris, stir to dissolve at 60 °C, then add 2 g of lipoic acid and stir vigorously to dissolve. Then heat to 70 °C and stir vigorously for 1 min to obtain a viscous solution. Freeze at -20 °C for 1 h and thaw at 25 °C for 1 h to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel, denoted as pTL-FeCuMOF, and store at 4 °C.

[0068] Example 2

[0069] Preparation of two-dimensional bimetallic organic framework FeCu MOF nanozyme:

[0070] Add 190 mg of Cu(NO3)2 and 190 mg of FeCl3·6H2O to 30 mL of dimethylformamide (DMF) and stir to dissolve. Then add 150 mg of terephthalic acid, followed by 20 mL of ethanol, 20 mL of ddH2O, and 200 μL of triethylamine. After stirring at room temperature for 2 h, a uniform colloidal suspension can be obtained. Then centrifuge (10000 rpm for 10 min), take the precipitate and wash it three times with ethanol. Finally, collect the product and dry it in a vacuum drying oven at 50 °C to obtain FeCuMOF;

[0071] Preparation of pTL-FeCuMOF hydrogel:

[0072] Dissolve 300 mg of FeCu MOF in 200 mL of ddH2O at 60 °C. Add 500 mg of tannic acid and 1.5 g of Tris, stir to dissolve at 60 °C, then add 2.5 g of lipoic acid and stir vigorously to dissolve. Then heat to 70 °C and stir vigorously for 5 min to obtain a viscous solution. Freeze at -20 °C for 1 h and thaw at 25 °C for 1 h to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel, and store at 4 °C.

[0073] Example 3

[0074] Preparation of two-dimensional bimetallic organic framework FeCu MOF nanozyme:

[0075] Add 200 mg of Cu(NO3)2 and 200 mg of FeCl3·6H2O to 40 mL of dimethylformamide (DMF) and stir to dissolve. Then add 400 mg of terephthalic acid, followed by 20 mL of ethanol, 15 mL of ddH2O, and 200 μL of triethylamine. After stirring at room temperature for 3 h, a uniform colloidal suspension can be obtained. Then centrifuge (10000 rpm for 10 min), take the precipitate and wash it three times with ethanol. Finally, collect the product and dry it in a vacuum drying oven at 50 °C to obtain FeCuMOF;

[0076] Preparation of pTL-FeCuMOF hydrogel:

[0077] Dissolve 400 mg of FeCu MOF in 200 mL of ddH2O at 60 °C, add 600 mg of tannic acid and 1.8 g of Tris, stir and dissolve at 60 °C, then add 3 g of lipoic acid and stir vigorously to dissolve. Then heat to 70 °C and stir vigorously for 10 min to obtain a viscous solution. Freeze at -20 °C for 2 h and thaw at 25 °C for 2 h to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel, and store it at 4 °C.

[0078] Comparative Example 1

[0079] Tannic acid-lipoic acid hydrogel:

[0080] Add 400 mg of tannic acid and 1 g of Tris to ddH2O at 60 °C and stir to dissolve. Then add 2 g of lipoic acid and stir to dissolve. Then heat the solution to 70 °C and stir for 1 min to obtain a viscous solution. Freeze at -20 °C ± 3 °C for 1 h and thaw at 25 °C for 1 h to obtain a hydrogel, denoted as pTL, and store it at 4 °C.

[0081] The following test examples use the FeCuMOF nanozyme and pTL-FeCuMOF hydrogel in Example 1, and use the pTL hydrogel in Comparative Example 1 as a comparison.

[0082] Test Example 1

[0083] The FeCu MOF nanozymes prepared in Example 1 were characterized and measured by electron microscopy, particle size, enzyme activity, etc., and compared. The specific methods of the relevant characterization and measurement refer to "Bioinspired metal-orga nic framework nanozymereinforced with thermosensitive hydrogel for regul ating inflammatoryresponses in Parkinson’s disease" (Fan X,Zhang T,Ding X,et al.Bioinspiredmetal-organic framework nanozyme reinforced with thermosensitive hydrogel forregulating inflammatory responses in Parkinson’s disease[J].Nano Research,2024,17(2):858-865.), "Personalized Carbon Monoxide-Loaded Biomimetic Single-Atom Nanozyme 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 of gapjunction protein improves the efficiency of nanozyme-mediated catalytic / starvation / mild-temperature photothermal therapy[J].Advanced Materials,2023,35(22):2210464.)。.

[0084] The measurement results are as Figure 1-4 , the FeCuMOF nanozyme has a uniform and good morphology, a small particle size, relatively high superoxide dismutase (SOD)-like and ABTS enzyme activities, and the ability to scavenge hydroxyl radicals and superoxide anions.

[0085] Experimental Example 2

[0086] The pTL-FeCuMOF nanozyme hydrogel prepared in Example 1 was subjected to basic characterization of its morphology and rheological properties. The specific methods for the relevant characterization measurements refer to "Self-Assembled Eutectogel with Cell Permeationand Multiple Anti-Inflammatory Abilities for Treating Chronic Periodontitis.(Z.Cheng,M.Kang,X.Peng,L.Ren,J.Xie,Q.Yuan,X.Xu,J.Li,Self-Assembled Eutectogelwith Cell Permeation and Multiple Anti-InflammatoryAbilities forTreatingChronic Periodontitis.Adv.Mater.2024,37,2412866.)。

[0087] According to Figure 5-7 It can be seen that the morphology of the nanozyme hydrogel conforms to the appearance characteristics of the hydrogel, and the rheological property is shear thinning, which is in line with the current application trend.

[0088] Experimental Example 3

[0089] Verify the safety and antioxidant properties of the pTL-FeCuMOF nanozyme hydrogel prepared in Example 1 at the in vitro cell level

[0090] (1) Co-culture bone marrow mesenchymal stem cells (BMSCs) / human gingival fibroblasts (HGFs) / periodontal ligament stem cells (PDLSCs) / mouse fibroblasts L929 (all purchased from Wuhan Pusaisai Biotechnology Co., Ltd.) with FeCu MOF nanozyme and pTL-FeCuMOF nanozyme hydrogel respectively, and detect their biosafety. L-929 cells were cultured in 96-well plates for 12 h at a density of 8000 cells per well. Then complete medium was mixed with pTL-FeCuMOF extract and control pTL extract at ratios of 0, 0.097656, 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100% respectively, and incubated for 24 h. Subsequently, the initial medium was discarded and the cells were gently washed twice with PBS. The proliferation ability of L929 in each group was quantitatively analyzed by CCK-8 assay. The working solution was prepared at a volume ratio of CCK-8:medium 1:10, and 100 μL of CCK-8 working solution was incubated at 37 °C for 1 h in each well. Finally, the absorbance at 450 nm was measured using a GF-M3000 microplate reader. The biocompatibility of FeCuMOF, pTL and pTL-FeCuMOF was evaluated using BMSCs, HGFs and PDLSCs. Cells were seeded into 96-well plates at a density of 1000 cells per well and treated with different concentrations (0, 2.5, 5, 10, 15 and 20 μg / mL) of Fe-CuMOF, pTL and pTL-FeCuMOF. The cell viability was evaluated using a CCK-8 detection kit (Aoqing Biotechnology, China) at days 0, 1, 3, 5 and 7. Briefly, 100 μL of medium containing 10% CCK-8 solution was added to each well, and then incubated at 37 °C in the dark for 1 h. The absorbance at 450 nm was measured using an ELx800 microplate reader (BioTek, USA), and a growth curve was generated based on the results. For live / dead cell staining, BMSCs, hgf and PDLSCs were seeded into 24-well plates at a density of 1×10 4 cells per well. After cell attachment, the cells were treated with FeCu MOF, pTL or pTL-FeCuMOF at concentrations of 0, 2.5, 5, 10, 15, 20 μg / mL for 3 days. Subsequently, the cells were gently washed three times with phosphate buffered saline (PBS). Then Calcein-AM / PI Live / Dead Assay Kit (Kaiji, China) was added, and the cells were incubated at 37 °C in the dark for 30 min, followed by three more PBS washes. Cell viability was observed under a fluorescence microscope (Nikon, Japan), and images were captured for analysis.

[0091] (2) To simulate in vitro oxidative stress, H2O2 was used to induce oxidative damage to evaluate the protective effect of nanozymes on cells. L-929 cells were cultured in a 96-well plate for 12 h at a density of 8000 cells / well. H2O2 was used to simulate ROS-induced oxidative stress damage. L-929 cells were seeded in a 96-well plate at a density of 8×10 3 , and PBS, H2O2, H2O2 + pTL, and H2O2 + pTL-FeCuMOF groups were set. After cell adhesion, the original culture medium was discarded, and MEM complete medium containing extracts of different concentrations of pTL and pTL-FeCuMOF (25, 50, 75, 100, 150, 200 μg / mL) was added to the 96-well plate for pre-protection for 12 h. The cell culture supernatant was discarded and treated with H2O2. After mixing the complete medium with hydrogen peroxide to a certain concentration, it was added to the well plate to induce for 12 h. Finally, the cell viability was measured by the CCK-8 method. HGF and PDLSCs were seeded into a 24-well plate at a density of 1×104 cells per well. When the cell density reached 80%, the cells were stimulated with 30 μg / mL lipopolysaccharide (LPS, Sigma-Aldrich, USA) to generate ROS for 48 h (negative stimulation), and then co-incubated with MOF, pTL, and pTL-FeCuMOF at concentrations of 2.5, 5, 10, 15, and 20 μg / mL for 24 h (positive stimulation). The positive control group was added with 50 μg / mL Rosup for 30 min to further stimulate ROS production. After treatment, all cells were washed twice with serum-free medium and incubated with a 5 μM DCFH-DA probe for 30 min. The excess dye was removed by washing with serum-free medium. Fluorescence was captured with a fluorescence microscope (Nikon, Japan), and the fluorescence intensity was quantified with ImageJ software.

[0092] The results are as Figure 8-11 shown. The safety at the cellular level was good in the FeCu MOF, pTL, and pTL-FeCuMOF environments, and pTL-FeCuMOF had the best effect on alleviating oxidative stress.

[0093] Test Example 4

[0094] Verification of the in vitro antibacterial ability of the pTL-FeCuMOF nanozyme hydrogel prepared in Example 1

[0095] (1) The strains Fusobacterium nucleatum (ATCC 25586) and Porphyromonas gingivalis (ATCC 33277) were obtained from the China General Microbiological Culture Collection Center. Among periodontal pathogens, Pseudomonas nucleatum and Pseudomonas gingivalis play key roles. F. nucleatum is a "bridge bacterium" that promotes biofilm formation and the progression of periodontal disease by connecting early colonizers and late pathogens. To evaluate the antibacterial effect of pTL-FeCuMOF, F. nucleatum and P. gingivalis were treated with PBS, FeCuMOF, pTL, and pTL-FeCuMOF (20 μg / mL), respectively. After treatment, CFU was quantified using the plate counting method.

[0096] As Figure 12 shown, the CFU count in the PBS group was significantly higher than that in other groups, while the CFU reduction of the two bacteria in the pTL-FeCuMOF group was the largest. Among them, the CFU of F. nucleatum treated with FeCuMOF decreased by 2 logs, the CFU of the pTL group decreased by 3 logs, and the antibacterial effect of the pTL-FeCuMOF group was the best. In P. gingivalis, pTL-FeCuMOF showed the strongest antibacterial effect, reducing the CFU by 4 logs.

[0097] As Figure 13 shown, scanning electron microscopy further revealed the morphological changes of bacteria after treatment. In the PBS group, P. nucleatum was smooth and slender fusiform, and P. gingivalis was short rod-shaped. In contrast, bacteria treated with FeCuMOF or pTL showed wrinkled and collapsed surfaces. The pTL-FeCuMOF treatment group showed severe damage, with cracks and irregularities on the bacterial surface, indicating enhanced bactericidal effects.

[0098] As Figure 14As shown, to evaluate the activity of the antibacterial film, live / dead fluorescence staining was performed using the live / dead BacLight Bacterial Viability Kit (ThermoFisher, USA). Four experimental groups were set up: PBS, FeCuMOF, pTL, and pTL-FeCuMOF. The bacterial suspension was added to a 24-well plate containing a sterilized glass cover, and cultured for 96 hours to form a biofilm. Subsequently, 20 μg / mL of FeCu MOF, pTL, and pTL-FeCuMOF were added to the corresponding wells, and the control group was given an equal volume of sterile PBS. After 72 hours of treatment, the biofilm was washed with PBS and stained with a working solution containing 1.5 μL of SYTO 9 and 1.5 μL of propidium iodide (PI) per mL of PBS. The coverslip was incubated in the dark at room temperature for 15 minutes and washed three times with PBS. The biofilm was visualized and imaged using a confocal laser scanning microscope (Leica TCS-SP8, Germany). The obtained images were reconstructed and analyzed using NIS-Elements Viewer software.

[0099] Test Example 5

[0100] Verification of the Therapeutic Ability of the pTL-FeCuMOF Nanozyme Hydrogel Prepared in Example 1 in a Rat Periodontitis Model

[0101] (I) Six-week-old Sprague-Dawley (SD) rats were selected for the experiment and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal procedures were reviewed and approved by the Peking University Institutional Animal Care and Use Committee (PUIRB-LA2023282) and carried out under its supervision. All surgeries and animal husbandry were performed under specific pathogen-free (SPF) conditions. The SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital to establish a periodontitis model. Stainless steel orthodontic ligature wires with a diameter of 0.2 mm were tied around the cervical regions of the bilateral maxillary first molars. After the surgery, the rats were closely monitored until they fully recovered. Once a week, the ligature wires were checked under isoflurane gas anesthesia to ensure correct placement and the changes in the periodontal tissues were observed. After 8 weeks, a periodontitis model was successfully established and the ligature wires were removed. The periodontitis rats were randomly divided into 4 groups, and 200 μL of PBS, FeCuMOF, pTL, and pTL-FeCuMOF (each dissolved in PBS to a concentration of 10 μg / mL) were injected into the periodontal pockets of the maxillary first molars, respectively. The drugs were administered daily for 3 days and 7 days. At 3 days and 7 days after treatment, the rats were euthanized by excessive inhalation of carbon dioxide and tissue samples were collected.

[0102] The results are as Figure 15As shown, according to in vitro biocompatibility and antibacterial studies, 10 μg / mL of pTL-FeCuMOF was selected as the concentration for subsequent treatment. 200 μL of PBS, FeCu MOF, pTL, and pTL-FeCuMOF were respectively injected into the periodontal pockets of the first maxillary molars of periodontitis-induced rats for 3 and 7 consecutive days. Samples were collected on days 3, 7, and 28 after treatment for analysis. Micro-CT scanning and 3D reconstruction of the samples collected on day 28 showed significant bone loss in the periodontitis group. However, the FeCu MOF, pTL, and pTL-FeCuMOF groups all showed therapeutic effects, with the pTL-FeCuMOF group showing the most obvious improvement. Quantitative analysis of the distance from the cementum to the alveolar bone crest (CEJ-ABC) and bone volume fraction (BV / TV) further confirmed these results. The pTL-FeCuMOF group had the largest decrease in CEJ-ABC and the largest increase in BV / TV on days 3 and 7 of treatment. The FeCu MOF group and the pTL group also showed improvement compared to the PBS group. These results indicate that pTL-FeCuMOF can effectively inhibit alveolar bone loss and promote periodontal recovery.

[0103] As Figure 16 shown, maxillary bone tissues were collected after 3 and 7 days of treatment for H&E staining to evaluate the inflammation level and immune cell infiltration. On day 3, extensive inflammatory cell infiltration, epithelial tissue damage, and obvious pathological changes were observed in the PBS group. Although the inflammatory cell infiltration decreased in the FeCu MOF and pTL groups, the inflammation was still obvious. In contrast, the pTL-FeCuMOF group showed a significant reduction in infiltration and a decrease in the inflammatory response. A similar trend was observed on day 7, with the pTL-FeCuMOF group having the lowest inflammation level.

[0104] In summary, the pTL-FeCuMOF hydrogel prepared by the present invention has good biocompatibility and broad-spectrum antibacterial properties. At the same time, the pTL-FeCuMOF hydrogel can scavenge various ROS by simulating the SOD cascade process to counteract oxidative stress. It is worth noting that it further promotes periodontal tissue regeneration by further removing stubborn biofilms resistant to antibiotics, thereby reducing pro-inflammatory factors and upregulating anti-inflammatory factors.

[0105] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a two-dimensional bimetallic organic framework nanozyme hydrogel, characterized in that: The following steps are involved: Tannic acid, FeCu MOF, lipoic acid, Tris and water were mixed to undergo a gelation reaction to obtain a viscous solution; The viscous solution is sequentially frozen and thawed to obtain a two-dimensional bimetallic organic framework nanozyme hydrogel.

2. The preparation method according to claim 1, characterized in that: The preparation method of the FeCu MOF comprises the following steps: Mixing the copper salt, the iron salt and the first solvent to obtain a mixed solution; The mixed solution, the organic ligand, the second solvent and triethylamine are mixed to carry out coordination reaction to obtain FeCuMOF.

3. The preparation method according to claim 2, characterized in that: The organic ligand includes terephthalic acid; the mass ratio of the copper salt, iron salt and organic ligand is (100-200):(100-200):(150-200).

4. The preparation method according to claim 3, characterized in that: The dosage ratio of the organic ligand to triethylamine is (150-200) mg: (20-200) μL; the temperature of the coordination reaction is 20-30° C., and the time is 1-5 hours.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the tannic acid, FeCu MOF and zinc sulfate is (200-500):(100-300):(1000-3000).

6. The preparation method according to claim 5, characterized in that: The temperature of the gelation reaction is 50-80° C. and the time is 1-10 minutes.

7. The preparation method according to claim 6, characterized in that: The freezing temperature is -20°C ± 3°C, and the time is 1 to 5 hours; the thawing temperature is 20 to 30°C, and the time is 1 to 5 hours.

8. The two-dimensional bimetallic organic framework nanozyme hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the two-dimensional bimetallic organic framework nanozyme hydrogel according to claim 8 in the preparation of antioxidant drugs and antibacterial drugs.

10. Use of the two-dimensional bimetallic organic framework nanozyme hydrogel according to claim 8 in the preparation of drugs for treating periodontitis.

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