Bioactive glass nano-enzyme with lactonase catalytic performance as well as preparation method and application of bioactive glass nano-enzyme

Abstract

The invention belongs to the technical field of preparation of nano biological materials, and discloses a bioactive glass nano enzyme with lactonase catalytic performance as well as a preparation method and application of the bioactive glass nano enzyme. The preparation method comprises the following steps: carrying out coordination reaction on 4-imidazole formaldehyde and zinc-doped bioactive glass prepared by a microemulsion method, and carrying out heat treatment to obtain the bioactive glass nano-enzyme with lactonase-like activity. The bioactive glass nano-enzyme obtained by the invention has a Zn-N coordination structure similar to a natural lactonase catalytic activity center, not only has good biocompatibility and intrinsic osteoinductivity of bioactive glass, but also can specifically hydrolyze bacterial quorum sensing signal molecules and efficiently inhibit the formation of a biological membrane, so that the bioactivity of the bioactive glass nano-enzyme is improved. The antibacterial-osteogenesis dual synergistic effect is achieved, and the antibacterial-osteogenesis composite material has important application value in the fields of infectious bone defect repair, periodontitis and other disease treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterial preparation technology, and relates to a bioactive glass nanozyme with lactone enzyme catalytic properties, its preparation method, and its application. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by specific pathogenic bacteria. Its main pathological features are the destruction of periodontal supporting tissues (gingiva, alveolar bone, and periodontal ligament) and persistent local infection. As the leading cause of tooth loss in adults, it severely impacts oral function. Studies have shown that bacterial biofilms formed in subgingival plaque play a crucial role in the development of periodontitis. These biofilms not only protect pathogens from host defenses and drug effects but also continuously activate inflammatory responses, causing tissue damage and accelerating periodontal tissue destruction by disrupting bone metabolic balance. Currently, mechanical debridement combined with antibiotics is the main clinical treatment strategy for periodontitis. However, due to limitations in tissue regeneration regulation, low antibiotic penetration through biofilms, and increasing bacterial resistance, its treatment efficacy is unsatisfactory. Therefore, developing novel treatment strategies that combine highly effective antibacterial activity with bone regeneration promotion is of significant clinical importance.

[0003] Bioactive glass nanoparticles (BGN) are amorphous silicate materials composed of SiO2, CaO, and other components, possessing a unique mesoporous structure, high specific surface area, good porosity, and excellent biocompatibility. As an artificial material capable of forming chemical bonds with bone tissue, BGN promotes hydroxyapatite deposition and osteoblast differentiation by continuously releasing active components such as silicon and calcium ions, exhibiting a significant regulatory role in bone and periodontal tissue regeneration. Current research primarily focuses on doping BGN with Ag. + Zn 2+ Cu 2+ Antibacterial ions or antibiotic loading can be used to enhance the antibacterial properties of BGNs, thereby increasing their antibacterial-osteogenic synergistic effect in the treatment of periodontitis. However, the uncontrolled release of metal ions, the burst release effect of drugs, and bacterial resistance to antibiotics mean that existing antibacterial BGNs still cannot achieve long-lasting, safe antibacterial effects and multiple functions of inducing bone tissue regeneration, thus limiting their efficient application in the treatment of periodontitis.

[0004] Nanozymes are a class of novel functional materials that combine the catalytic activity of natural enzymes with the physicochemical properties of nanomaterials. Compared with traditional natural enzymes, nanozymes have significant advantages such as simple preparation, high stability, and low cost, showing broad application prospects in the biomedical field. Currently, nanomaterials with peroxidase-like and peroxidase-like activities have been extensively studied in the field of anti-infection. However, the antibacterial mechanism of these nanozymes mainly relies on the strong oxidizing properties of reactive oxygen species (ROS) to directly or indirectly damage cell structure, biomolecules, and metabolic functions, lacking specificity for bacteria. Furthermore, the short lifetime and limited diffusion distance of ROS severely restrict their effectiveness. Notably, the bacterial quorum sensing system (QS) plays a crucial role in regulating biofilm formation. As a core signaling molecule in the QS system, acyl-homoserine lactone (AHL) mediates interbacterial communication and virulence factor expression. Acyl-homoserine lactoneases can interfere with bacterial quorum sensing by specifically degrading AHL, thereby inhibiting biofilm formation and achieving antibacterial effects. Based on this, the development of acyl-homoserine lactonease mimics holds promise for achieving highly efficient biofilm inhibition, representing a promising new antibacterial strategy. This quorum sensing-quenching antibacterial mechanism differs significantly from the action modes of traditional redox nanozymes and antibiotics, effectively avoiding the side effects of ROS and the risks of bacterial resistance. In conclusion, the development of bioactive glass nanozymes with lactonease catalytic activity holds promise for achieving a synergistic "anti-infection-promoting regeneration" effect by integrating highly efficient antibacterial and bone regeneration functions, opening new avenues for precise and efficient treatment of periodontitis. Summary of the Invention

[0005] To address the limitations of existing BGNs (biotinylated glass nanozymes) in periodontitis treatment due to their limited functionality and insufficient antibacterial activity, this invention, based on the crucial role of acylhomoserine lactoneases in inhibiting bacterial growth and biofilm formation, aims to provide a bioactive glass nanozyme with lactonease catalytic properties, along with its preparation method and applications. Considering that the catalytic active center of natural lactoneases is a binuclear Zn... 2+ The present invention relates to a Zn-N structure formed by coordination with amino acids, through the doping of Zn into BGN. 2+ Zinc-doped bioactive glass nanoparticles (ZnBGN) with a porous structure were constructed, and 4-imidazolium formaldehyde (4-ICA) ligand molecules were used to bind Zn... 2+Through coordination, a catalytically active center with precise Zn-N coordination structure and spatial configuration is constructed within ZnBGN. This preparation method is characterized by mild reaction conditions, requires no complex equipment, and is simple to operate. The prepared bioactive glass nanozyme (IM-ZnBGN) can specifically hydrolyze the key bacterial quorum sensing signal molecule AHL, effectively inhibiting biofilm formation. It holds promise for achieving synergistic regulation of both antibacterial and osteogenic functions, providing an innovative solution for periodontal tissue regeneration and infection control, and demonstrating significant application potential in the treatment of periodontitis, infected bone defects, and other diseases.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] A method for preparing a bioactive glass nanozyme with lactonease catalytic properties includes the following steps:

[0008] An aqueous solution containing hexadecyl pyridine bromide and urea is subjected to a micelle self-assembly reaction with one of cyclohexane and n-hexane, and one of isopropanol and n-butanol, to obtain a micelle self-assembled microemulsion.

[0009] Tetraethyl orthosilicate was added to a micelle self-assembled microemulsion, followed by hydrolysis and polycondensation. Then, calcium nitrate tetrahydrate and zinc nitrate hexahydrate were added to carry out a sol-gel conversion reaction, followed by drying and calcination to obtain ZnBGN.

[0010] 4-ICA and ZnBGN were mixed in a reaction solvent and stirred to obtain an imidazole-coordinated ZnBGN (ZnBGN@4-ICA) composite material.

[0011] Heat treatment of ZnBGN@4-ICA composite material yields bioactive glass nanozymes with lactone enzyme catalytic properties.

[0012] Furthermore, the mass ratio of urea to hexadecylpyridine bromide is 0.3:1 to 0.8:1;

[0013] The volume ratio of cyclohexane to water is 0.8:1 to 1.2:1;

[0014] The volume ratio of n-hexane to water is 0.8:1 to 1.2:1;

[0015] The volume ratio of isopropanol to water is 1:20-1:30;

[0016] The volume ratio of n-butanol to water is 1:20-1:30.

[0017] Furthermore, the ratio of the amount of tetraethyl orthosilicate to the total amount of calcium nitrate tetrahydrate and zinc nitrate hexahydrate is 7:3, and the molar ratio of calcium nitrate tetrahydrate to zinc nitrate hexahydrate is (3-x):x, 1≤x≤3.

[0018] Furthermore, the self-assembly reaction temperature of micelles is 20-30℃, and the reaction time is 1-3 hours; the hydrolysis-condensation reaction temperature is 65-75℃, and the reaction time is 6-12 hours; the sol-gel conversion reaction temperature is 65-75℃, and the reaction time is 12-20 hours.

[0019] Furthermore, the mass ratio of ZnBGN to 4-ICA is 3:0.1-3:5.

[0020] Furthermore, the reaction solvent is water. After mixing 4-imidazolium formaldehyde and zinc-doped bioactive glass in the reaction solvent, the reaction is stirred at a temperature of 20-30°C for 24-48 hours.

[0021] Furthermore, the heat treatment temperature is 550-750℃, and the time is 2 hours.

[0022] Furthermore, the heat treatment atmosphere is argon, and the temperature is increased to 550-750℃ at a heating rate of 5℃ / min, and then cooled to room temperature at a cooling rate of 5℃ / min after the reaction.

[0023] A bioactive glass nanozyme with lactone catalytic properties.

[0024] Application of a bioactive glass nanozyme with lactonease catalytic properties in the preparation of antibacterial drugs.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention is the first to utilize Zn 2+ Precise coordination with imidazole ligands constructs Zn-N catalytic active centers within bioactive glass nanoparticles. Combined with calcination treatment to optimize the coordination structure, this achieves an organic integration of biomimetic design of the enzyme active center and material multifunctionality. This design retains the inherent properties of bioactive glass while endowing the material with novel enzyme-like catalytic functions. This invention features a simple preparation process and mild reaction conditions, facilitating large-scale production. The resulting material not only exhibits good biocompatibility and physiological stability but also avoids the complexity and potential safety issues associated with using multiple materials in combination through its multifunctional integrated design. Furthermore, the design concept of combining catalytic function with biomaterial properties in this invention provides an innovative solution for periodontal tissue regeneration and infection control, and also offers valuable reference for the development of other infectious tissue repair materials, demonstrating broad development potential in the biomedical field and clinical applications.

[0027] The IM-ZnBGN prepared in this invention exhibits multiple functional properties. Its ability to specifically hydrolyze bacterial quorum sensing signaling molecules can effectively block bacterial communication and inhibit biofilm formation, while its retained osteoinductive activity can promote periodontal tissue regeneration, achieving synergistic regulation of antibacterial and osteogenic functions. This dual efficacy breaks through the limitations of the single-function treatment modality and provides a new approach to the regulation of complex pathological environments. Attached Figure Description

[0028] Figure 1 These are scanning electron microscope (SEM) images of ZnBGN (Examples 1-3) with different metal ion doping ratios in this invention.

[0029] Figure 2 These are transmission electron microscope (TEM) images and elemental distributions of the IM-ZnBGN prepared in Example 2 of this invention. (a) is a TEM image of IM-ZnBGN, (b) is the energy dispersive spectroscopy (EDS) scan region of IM-ZnBGN, and (c), (d), (e), (f), and (g) are the elemental distributions of Si, O, Ca, Zn, and N, respectively.

[0030] Figure 3 These are the characterization results of the catalytic activity of the IM-ZnBGN (Example 2, Comparative Example 1 and Comparative Example 2) lactone-like enzymes synthesized using different imidazole ligand molecules in this invention. Among them, (a) is the characterization result of catalytic activity, and (b) is the fluorescence image of different reaction systems.

[0031] Figure 4 The catalytic activity characterization results are those of the IM-ZnBGN lactone enzymes synthesized in Examples 2, 4 and Comparative Example 3 of this invention, obtained by calcining ZnBGN@4-ICA at 600℃, 700℃ and 800℃ respectively.

[0032] Figure 5 This is the characterization result of the hydrolysis effect of IM-ZnBGN synthesized in Example 2 of this invention on AHL molecules;

[0033] Figure 6 This describes the effect of IM-ZnBGN synthesized in Example 2 of this invention on inhibiting biofilm formation. Detailed Implementation

[0034] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0035] This invention prepares ZnBGN nanozymes with lactone-like catalytic properties by in-situ constructing Zn-N catalytic active centers with precise coordination configurations within BGN. These nanozymes can specifically hydrolyze bacterial quorum sensing signal molecules, achieving a dual regulatory effect of antibacterial and osteogenic activity, providing an innovative solution for the treatment of periodontitis and infected bone defects. This invention selects bioactive BGN as a carrier and introduces Zn into the BGN glass network by optimizing the metal ion doping ratio and preparation conditions. 2+ Based on its rich mesoporous / macroporous structure, imidazole organic ligands 4-ICA and Zn were further selected. 2+ Coordination assembly was performed, and the reactant feed ratio was systematically controlled to precisely construct Zn-N catalytic active centers within the BGN. Finally, calcination was used to optimize the coordination space structure of the active centers, resulting in the synthesis of IM-ZnBGN nanozymes with lactonease catalytic properties. The ZnBGN nanozyme material prepared in this invention possesses dual functional characteristics: on the one hand, it maintains the inherent excellent biocompatibility and osteoinductive activity of BGN materials; on the other hand, based on the Zn-N catalytic active centers, it achieves highly efficient lactonease-like catalytic function, thus exhibiting unique biofilm inhibition properties. This demonstrates significant application value and development potential in the clinical treatment of infectious bone defects such as periodontitis.

[0036] The present invention discloses a method for preparing a bioactive glass nanozyme with lactonease catalytic properties, comprising the following steps:

[0037] 1) Preparation of ZnBGN: ZnBGN was prepared using a microemulsion method. Hexadecyl bromopyridine was used as a surfactant and mixed with urea in an aqueous solution. Cyclohexane (or n-hexane) and isopropanol (or n-butanol) were then added to initiate a micelle self-assembly reaction. Vigorous stirring resulted in a homogeneous and stable micelle self-assembled microemulsion. Tetraethyl orthosilicate was added to the micelle self-assembled microemulsion for hydrolysis and polycondensation. Then, calcium nitrate tetrahydrate and zinc nitrate hexahydrate were added sequentially for metal ion doping. Following a sol-gel conversion reaction, a milky white bioactive glass gel was finally formed. The product was washed, purified, vacuum dried, and calcined at high temperature to obtain ZnBGN.

[0038] The mass ratio of urea to hexadecylpyridine is 0.3:1-0.8:1, preferably 0.5:1;

[0039] The volume ratio of cyclohexane to water is 0.8:1-1.2:1, preferably 1:1;

[0040] The volume ratio of n-hexane to water is 0.8:1-1.2:1, preferably 1:1.

[0041] The volume ratio of isopropanol to water is 1:20-1:30, preferably 1:25;

[0042] The volume ratio of n-butanol to water is 1:20-1:30, preferably 1:25.

[0043] The molar ratio of tetraethyl orthosilicate to nitrate (total amount of calcium nitrate tetrahydrate and zinc nitrate hexahydrate) is 7:3, and the molar ratio of calcium nitrate tetrahydrate to zinc nitrate hexahydrate is (3-x):x, 1≤x≤3, preferably 2:1, 1:2 or 0:3.

[0044] The self-assembly reaction temperature of micelles is 20-30℃, the reaction time is 1-3 hours, and the preferred reaction time is 2 hours;

[0045] The hydrolysis-condensation reaction temperature is 65-75℃, the reaction time is 6-12 hours, and the preferred reaction time is 8 hours;

[0046] The temperature for the sol-gel conversion reaction is 65-75℃, and the reaction time is 12-20 hours, with the preferred reaction time being 16 hours.

[0047] After the final reaction solution was cooled to room temperature, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes. The centrifuged product was washed three times with acetone, ethanol and deionized water in sequence, and then freeze-dried.

[0048] The product was calcined for 5 hours at a heating rate of 1℃ / min to remove the template agent.

[0049] 2) Preparation of ZnBGN@4-ICA: Using 4-ICA as the organic functional ligand, it was mixed with the prepared ZnBGN and subjected to vigorous stirring. The resulting product was purified by centrifugation and freeze-dried to obtain ZnBGN@4-ICA with Zn-N coordination active centers.

[0050] Among them, 5 mg ZnBGN should be dispersed in 500 μL of deionized water and ultrasonically treated for 5-15 minutes before adding 4-ICA;

[0051] The mass ratio of ZnBGN to 4-ICA is 3:0.1-3:5, preferably 3:3.2;

[0052] ZnBGN and 4-ICA were stirred vigorously at room temperature, with deionized water as the reaction solvent, for 24-48 hours.

[0053] The final reaction solution was centrifuged at 10,000 rpm for 10 minutes and the precipitate was washed twice with deionized water to remove unbound 4-ICA and then freeze-dried.

[0054] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA was heat-treated by high-temperature calcination under inert gas protection. By precisely controlling the Zn-N coordination spatial configuration, ZnBGN nanozyme IM-ZnBGN with lactone enzyme-like catalytic performance was obtained.

[0055] The ZnBGN@4-ICA heat treatment atmosphere is argon, with the temperature increased to 550-750℃ at a heating rate of 5℃ / min, followed by a cooling rate of 5℃ / min to room temperature. The preferred calcination temperature is 600 or 700℃.

[0056] The following are specific examples.

[0057] Example 1

[0058] 1) Preparation of ZnBGN: First, 0.5 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. Then, 30 mL of cyclohexane and 1.2 mL of isopropanol were slowly added. The mixture was stirred vigorously at 25 °C for 2 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise. After hydrolysis for 30 minutes, the temperature was raised to 70 °C and the mixture was stirred for another 8 hours. Then, 0.512 g of calcium nitrate tetrahydrate was added. After reacting for 15 minutes, 0.804 g of zinc nitrate hexahydrate was added and the mixture was stirred for another 16 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0059] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:3.2. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 5 minutes to ensure complete dispersion. Then, 5.37 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 24 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0060] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 700℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0061] Example 2

[0062] 1) Preparation of ZnBGN: First, 0.5 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. Then, 30 mL of cyclohexane and 1.2 mL of isopropanol were slowly added. The mixture was stirred vigorously at 25 °C for 2 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise. After hydrolysis for 30 minutes, the temperature was raised to 70 °C and the mixture was stirred for another 8 hours. Then, 0.402 g of calcium nitrate tetrahydrate was added. After reacting for 15 minutes, 1.02 g of zinc nitrate hexahydrate was added and the mixture was stirred for another 16 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0063] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:3.2. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 5 minutes to ensure complete dispersion. Then, 5.37 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 24 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0064] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 700℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0065] Example 3

[0066] 1) Preparation of ZnBGN: First, 0.5 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. Then, 30 mL of cyclohexane and 1.2 mL of isopropanol were slowly added. The mixture was stirred vigorously at 25 °C for 2 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise. After hydrolysis for 30 minutes, the temperature was raised to 70 °C and the mixture was stirred for another 8 hours. Then, 1.53 g of zinc nitrate hexahydrate was added and the mixture was stirred for another 16 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0067] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:3.2. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 5 minutes to ensure complete dispersion. Then, 5.37 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 24 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0068] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 700℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0069] Example 4

[0070] 1) Preparation of ZnBGN: First, 0.5 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. Then, 30 mL of cyclohexane and 1.2 mL of isopropanol were slowly added. The mixture was stirred vigorously at 25 °C for 2 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise. After hydrolysis for 30 minutes, the temperature was raised to 70 °C and the mixture was stirred for another 8 hours. Then, 0.512 g of calcium nitrate tetrahydrate was added. After reacting for 15 minutes, 0.804 g of zinc nitrate hexahydrate was added and the mixture was stirred for another 16 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0071] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:3.2. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 5 minutes to ensure complete dispersion. Then, 5.37 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 24 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0072] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 600℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0073] Example 5

[0074] 1) Preparation of ZnBGN: First, 0.6 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. Then, 36 mL of n-hexane and 1.2 mL of isopropanol were slowly added. The mixture was stirred vigorously at 20 °C for 3 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise. After hydrolysis for 30 minutes, the temperature was raised to 65 °C and the mixture was stirred for another 12 hours. Then, 0.512 g of calcium nitrate tetrahydrate was added. After reacting for 15 minutes, 0.804 g of zinc nitrate hexahydrate was added and the mixture was stirred for another 12 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0075] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:0.1. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 10 minutes to ensure complete dispersion. Then, 0.168 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 40 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0076] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 550℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0077] Example 6

[0078] 1) Preparation of ZnBGN: First, 0.3 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. 24 mL of cyclohexane and 1.2 mL of n-butanol were slowly added, and the mixture was stirred vigorously at 30 °C for 1 hour to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise, and after hydrolysis for 30 minutes, the temperature was raised to 75 °C and the reaction was continued for 6 hours. Then, 0.402 g of calcium nitrate tetrahydrate was added, and after reacting for 15 minutes, 1.02 g of zinc nitrate hexahydrate was added, and the reaction was continued for 20 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water, respectively. After freeze-drying, the product was calcined at 600 °C for 5 hours in air at a rate of 1 °C / min to obtain ZnBGN, which was then stored at room temperature for later use.

[0079] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:5. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 15 minutes to ensure complete dispersion. Then, 8.33 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 48 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0080] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 750℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0081] Example 7

[0082] 1) Preparation of ZnBGN: First, 0.8 g of urea and 1 g of hexadecylpyridine bromide were dissolved in 30 mL of deionized water. 27 mL of cyclohexane and 1 mL of isopropanol were slowly added, and the mixture was stirred vigorously at 25 °C for 2 hours to form a homogeneous emulsion. Then, 2.7 mL of tetraethyl orthosilicate was added dropwise, and after hydrolysis for 30 minutes, the temperature was raised to 70 °C and the reaction was continued for 8 hours. Then, 0.402 g of calcium nitrate tetrahydrate was added, and after reacting for 15 minutes, 1.02 g of zinc nitrate hexahydrate was added, and the reaction was continued for 14 hours. After the reaction was completed, the product was washed three times each with acetone, ethanol and water, respectively. After freeze-drying, the product was calcined in air at a rate of 1 °C / min to 600 °C for 5 hours to finally obtain ZnBGN, which was then stored at room temperature for later use.

[0083] 2) Preparation of ZnBGN@4-ICA: The mass ratio of ZnBGN to 4-ICA imidazole ligand molecules was 3:3.2. First, 5 mg of ZnBGN was dispersed in 500 μL of deionized water and sonicated for 8 minutes to ensure complete dispersion. Then, 5.37 mg of 4-ICA was dissolved in 1 mL of deionized water to prepare a solution, which was slowly added dropwise to the ZnBGN dispersion. The reaction system was continuously and vigorously stirred at room temperature for 30 hours to ensure complete coordination reaction. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was washed twice with deionized water to remove uncoordinated 4-ICA molecules. Finally, the ZnBGN@4-ICA composite material with Zn-N coordination active centers was obtained by freeze-drying.

[0084] 3) Preparation of IM-ZnBGN: The prepared ZnBGN@4-ICA composite material was placed in an argon protective atmosphere and heated to 650℃ at a constant heating rate of 5℃ / min. It was then calcined at this temperature for 2 hours and cooled to room temperature at a cooling rate of 5℃ / min to obtain IM-ZnBGN nanozyme material with lactone catalytic performance.

[0085] Comparative Example 1

[0086] Same as Example 1, except that in step 2), the imidazole ligand molecule used is 2-imidazolium formaldehyde (2-ICA).

[0087] Comparative Example 2

[0088] Same as Example 1, except that in step 2), the imidazole ligand molecule used is 1-methyl-2-imidazolium carboxaldehyde (MCA).

[0089] Comparative Example 3

[0090] Same as Example 1, except that in step 3), the calcination temperature is 800°C.

[0091] The ZnBGN nanozyme material with lactone enzyme catalytic properties obtained in the preparation process of this invention is a brownish-brown powder after freeze-drying.

[0092] Figure 1 These are scanning electron microscope (SEM) images of ZnBGN with different metal ion doping ratios used in this invention. The materials used were prepared in Examples 1-3. Figure 1 The original feed molar ratios of ZnBGN corresponding to (a), (b), and (c) are 70Si:20Ca:10Zn, 70Si:10Ca:20Zn, and 70Si:0Ca:30Zn, respectively. As can be seen from the figure, the ZnBGN prepared using the metal ion doping ratios described in this invention are all well-monodispersed, uniformly sized spherical nanoparticles with a wrinkled surface and a radial mesoporous structure. The particle sizes of ZnBGN with the original feed molar ratios of 70Si:20Ca:10Zn, 70Si:10Ca:30Zn, and 70Si:30Zn are 440 nm, 290 nm, and 590 nm, respectively.

[0093] Figure 2 This is a transmission electron microscope (TEM) image and elemental distribution of IM-ZnBGN used in this invention. The material used was prepared in Example 2. Figure 2 Image (a) is a transmission electron microscope (TEM) image of IM-ZnBGN. Figure 2 (b) shows the energy spectrum scanning region of IM-ZnBGN. Figure 2 (c), (d), (e), (f), and (g) are the distribution diagrams of Si, O, Ca, Zn, and N elements, respectively. It can be seen that the IM-ZnBGN prepared in Example 2 maintains a complete mesoporous structure and good monodispersity, with a particle size of 290 nm. Furthermore, the distribution of Zn and N elements indicates that the Zn-N catalytic active centers are uniformly distributed within the BGN.

[0094] Figure 3 Image (a) shows the characterization results of the catalytic activity of IM-ZnBGN lactone-like enzymes synthesized using different imidazole ligand molecules in this invention. Figure 3(b) shows fluorescence images of different reaction systems, using materials prepared in Example 2, Comparative Example 1, and Comparative Example 2. The 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe can be specifically hydrolyzed by lactonease to generate the hydrolysis product 2',7'-dichlorodihydrofluorescein (DCFH). This process effectively simulates the reaction characteristics of lactonease hydrolysis of AHL signal molecules. The generated DCFH is oxidized under the action of reactive oxygen species (ROS, such as O2-· / H2O2) to form the 2',7'-dichlorofluorescein (DCF) product, which exhibits specific fluorescence emission at 525 nm under 488 nm excitation light. Based on this reaction mechanism, quantitative analysis of the lactonease-like catalytic performance of IM-ZnBGN nanozymes can be achieved by monitoring the fluorescence intensity of DCF. In the figure, control group 1 represents the fluorescence intensity of the system after complete hydrolysis and oxidation of DCFH-DA with NaOH, and control group 2 represents the fluorescence intensity of the system without any hydrolysis treatment of DCFH-DA. Figure 3 As shown in (a) and (b), using the DCFH-DA fluorescent probe as a substrate, after co-incubation of the material with DCFH-DA, analysis of the fluorescence signal of the reaction system revealed that among the three selected imidazole organic ligand molecules, only IM-ZnBGN obtained by coordination-calcination with ZnBGN using 4-ICA as the ligand could efficiently catalyze the hydrolysis of DCFH-DA, exhibiting significant lactonease catalytic performance. This result indicates that the selection of 4-ICA as the functional ligand is key to obtaining highly efficient lactonease-like catalytic materials in the preparation method of the bioactive glass nanozyme with lactonease catalytic performance in this invention.

[0095] Figure 4 Image (a) shows the catalytic activity characterization results of the IM-ZnBGN-type lactoneases synthesized in this invention and obtained by calcining ZnBGN@4-ICA at 600℃, 700℃, and 800℃, respectively. Figure 4 (b) shows fluorescence images of different reaction systems, using materials prepared in Examples 2, 4, and Comparative Example 3. Figure 4As shown in (a) and (b), IM-ZnBGN prepared using the DCFH-DA fluorescent probe as a substrate and calcined at 600℃ and 700℃ exhibits significantly higher DCFH-DA hydrolysis performance compared to ZnBGN@4-ICA. Specifically, the IM-ZnBGN prepared in Example 4 achieved a DCFH-DA hydrolysis rate of 96.02% compared to control group 1, indicating superior lactone-like catalytic performance. However, the product obtained from ZnBGN@4-ICA calcined at 800℃ did not show significant lactone bond hydrolysis. This result indicates that during the preparation of IM-ZnBGN from ZnBGN@4-ICA using this invention, the calcination temperature must be strictly controlled within the specific range (550-750℃) described in this invention to obtain a material with highly efficient lactone-like catalytic performance.

[0096] The representative AHL molecule 3-oxo-C12-HSL secreted by Pseudomonas aeruginosa was selected as a substrate for analysis by liquid chromatography-mass spectrometry (LC-MS). Figure 5 This is the characterization result of the hydrolysis of AHL molecules by IM-ZnBGN synthesized in this invention. The material used was prepared in Example 2. Figure 5 (a) shows a comparison of the total ion chromatograms of AHL standards before and after treatment with IM-ZnBGN. Figure 5 (b) shows the mass spectrum of the AHL standard 3-oxo-C12-HSL, corresponding to... Figure 5 Peak 1 in (a) Figure 5 (c) shows the mass spectrum of its hydrolysis product 3-oxo-C12-HS, corresponding to Figure 5 Peak 2 in (a). As shown in the figure, in positive ion mode, the substrate molecular ion peaks appear at m / z 298.2 ([M+H)). + ) and 320.18([M+Na] + After catalysis by the material, the characteristic peak of the hydrolysis product 3-oxo-C12-HS shifted to m / z 316.21 ([M+H)). + ) and 338.19([M+Na] + The molecular weight increased by 18 Da (corresponding to the addition of one water molecule), which confirms that IM-ZnBGN can efficiently catalyze the hydrolysis of the lactone bond in the 3-oxo-C12-HSL molecule.

[0097] Figure 6 This illustrates the inhibitory effect of the synthesized IM-ZnBGN on biofilm formation in this invention. The illustration shows a representative photograph of the biofilm on a glass slide after crystal violet staining. The *E. coli* suspension (5 × 10⁻⁶) was used. 6The control group (CFU / mL) was co-incubated with ZnBGN and IM-ZnBGN (100, 250, 500 μg / mL) at 37℃ for 48 hours. After crystal violet staining and elution with 70% ethanol, the absorbance at 595 nm was measured and statistically analyzed. The results showed that the control group formed a mature biofilm with a dense structure and uniform surface coverage. Compared with the control group, both ZnBGN and IM-ZnBGN treatment groups showed significant inhibitory effects on biofilm formation, with looser biofilm structure, decreased coverage, and reduced thickness. The inhibition rate of the 500 μg / mL IM-ZnBGN group was as high as 60.44%, which was better than that of the ZnBGN group at the same concentration, indicating that IM-ZnBGN can stably exert an anti-biofilm effect.

[0098] Unlike traditional ion-doped, antibiotic-loaded antibacterial bioactive glass nanoparticles, this invention precisely constructs imidazole and Zn within BGN. 2+ With the coordination structure serving as the active site of the catalytic center, a novel antibacterial nanomaterial with highly efficient inhibition of bacterial biofilm formation has been developed, providing a new method to overcome the bottleneck problems of single function and insufficient antibacterial activity in the application of traditional bioactive glass in the treatment of periodontitis and other diseases and in regenerative medicine.

[0099] This invention involves reacting imidazole organic ligands with ZnBGN prepared via a microemulsion method in a specific ratio. After centrifugation purification, the mixture is lyophilized and calcined under nitrogen protection to prepare ZnBGN lactonease, which is then stored at room temperature for later use. This technique functionalizes BGN by in-situ constructing a Zn-N catalytic active center, not only preserving the excellent bone tissue regeneration properties of bioactive glass nanoparticles but also endowing the material with significant anti-infection properties through a lactonease-like catalytic mechanism.

[0100] Compared with traditional synthesis strategies for antibacterial bioactive glasses (such as ion doping or antibiotic loading), the preparation method of this invention features mild reaction conditions, simple operation, and no need for complex equipment. The prepared bioactive glass nanocomposite material exhibits good biocompatibility, enzyme-like catalytic activity, and anti-infection properties. In particular, this method effectively avoids the potential toxicity of ion doping and uncontrollable antibiotic release problems in traditional antibacterial BGNs, breaking through the limitations of antibacterial bioactive glasses in the field of regenerative medicine.

[0101] The above description is only of the preferred embodiment of the present invention and should not be construed as limiting the scope of the claims. The present invention is not limited to the above embodiments, and variations in its specific structure are permitted. All variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing a bioactive glass nanozyme with lactonease catalytic properties, characterized in that, Includes the following steps: An aqueous solution containing hexadecyl pyridine bromide and urea is subjected to a micelle self-assembly reaction with one of cyclohexane and n-hexane, and one of isopropanol and n-butanol, to obtain a micelle self-assembled microemulsion. Tetraethyl orthosilicate was added to a micelle self-assembled microemulsion, followed by hydrolysis and polycondensation. Then, calcium nitrate tetrahydrate and zinc nitrate hexahydrate were added to carry out a sol-gel conversion reaction, followed by drying and calcination to obtain zinc-doped bioactive glass. 4-Imidazole formaldehyde and zinc-doped bioactive glass were mixed in a reaction solvent and stirred to obtain an imidazole-coordinated bioactive glass composite material. By heat-treating imidazole-coordinated bioactive glass composite materials, bioactive glass nanozymes with lactone enzyme catalytic properties were obtained.

2. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The mass ratio of urea to hexadecylpyridine bromide is 0.3:1-0.8:1; The volume ratio of cyclohexane to water is 0.8:1 to 1.2:1; The volume ratio of n-hexane to water is 0.8:1 to 1.2:1; The volume ratio of isopropanol to water is 1:20-1:30; The volume ratio of n-butanol to water is 1:20-1:

30.

3. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The molar ratio of tetraethyl orthosilicate to the total molar ratio of calcium nitrate tetrahydrate and zinc nitrate hexahydrate is 7:3, and the molar ratio of calcium nitrate tetrahydrate to zinc nitrate hexahydrate is (3-x):x, 1≤x≤3.

4. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The self-assembly reaction temperature of micelles is 20-30℃, and the reaction time is 1-3 hours; the hydrolysis-condensation reaction temperature is 65-75℃, and the reaction time is 6-12 hours; the sol-gel conversion reaction temperature is 65-75℃, and the reaction time is 12-20 hours.

5. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The mass ratio of zinc-doped bioactive glass to 4-imidazolium formaldehyde is 3:0.1-3:

5.

6. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The reaction solvent is water. 4-Imidazole formaldehyde and zinc-doped bioactive glass are mixed in the reaction solvent and stirred. The reaction temperature is 20-30℃ and the reaction time is 24-48 hours.

7. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 1, characterized in that, The heat treatment temperature is 550-750℃, and the time is 2 hours.

8. The method for preparing bioactive glass nanozymes with lactonease catalytic properties according to claim 7, characterized in that, The heat treatment atmosphere is argon, and the temperature is increased to 550-750℃ at a heating rate of 5℃ / min. After the reaction, the temperature is reduced to room temperature at a cooling rate of 5℃ / min.

9. A bioactive glass nanozyme with lactonease catalytic properties prepared by the method according to any one of claims 1-8.

10. The application of a bioactive glass nanozyme with lactonease catalytic properties prepared by the method according to any one of claims 1-8 in the preparation of antibacterial drugs.

Images