A dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, and a preparation method and application thereof

Abstract

The present application relates to the technical field of dental medical materials and its preparation technology, disclose a kind of 2-amino imidazole derivative PMMA microsphere-containing dental adhesive and its preparation method and application, comprising the following steps: S1, preparation 2A4: combination raw material preparation 4-pentadecyl-1H-imidazole-2-amine hydrochloride (2A4);S2, preparation 2A4@PMMA microsphere: emulsion polymerization is used, 2A4 obtained in S1 is dissolved in dichloromethane, then MMA and TEGDMA and emulsifier OP-10 are added and uniformly obtained oil phase solution;Mixed deionized water and sodium persulfate obtain aqueous solution;The oil phase solution is added dropwise to the aqueous solution, and stirred to form a stable emulsion;After heating reaction, the supernatant is discarded by centrifugation, and the precipitate is vacuum dried to obtain 2A4@PMMA microsphere;S3, preparation dental adhesive: Bis-GMA, UDMA, TEGDMA are mixed to prepare adhesive resin monomer;Adhesive resin monomer, functional monomer, photoinitiator, anhydrous ethanol are mixed, then uniformly stirred by magnetic stirring, to obtain dental adhesive.

Description

Technical Field

[0001] This invention relates to the field of dental medical materials and their preparation technology, specifically to a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, its preparation method, and its application. Background Technology

[0002] Dental adhesives are core auxiliary materials in dental restorative procedures such as resin fillings for tooth defects and crown restorations. They are primarily used to achieve a strong bond between the resin filling / restoration and the tooth structure (enamel and dentin). Their performance directly determines the stability, durability, and treatment outcome of the restoration. Located between the restoration and the tooth structure, dental adhesives enhance the overall mechanical properties of the tooth structure and, through optimized formulation, improve aesthetics, allowing the restoration to match the color of natural teeth. They play an irreplaceable role in restoring tooth defects caused by caries, trauma, etc. However, secondary caries is a major cause of resin filling failure, closely related to the action of cariogenic bacteria, and currently used adhesives in clinical practice do not possess antibacterial properties. Therefore, the development of high-performance dental adhesives has significant clinical and industrial value in improving the efficacy of dental restorations, preventing secondary caries, and improving patients' oral health.

[0003] In existing technologies, to improve the overall performance of dental adhesives and address the clinical pain point of secondary caries after restoration, the focus is mainly on enhancing the antibacterial properties of adhesives, resulting in several technical approaches: First, the direct contact antibacterial strategy utilizes chemical covalent bonds to anchor antibacterial molecules to the polymer backbone. The special molecular chains of the antibacterial agent exert their antibacterial effect upon contact with bacteria. Broad-spectrum antibacterial agents, represented by quaternary ammonium compounds, attract bacteria to the resin material surface through electrostatic adsorption. The hydrophobic groups in their structure can directly insert into the phospholipid bilayer of the bacterial cell membrane, causing cell membrane rupture and ultimately bacterial death. Second, the release-type antibacterial strategy releases pre-loaded antibacterial agents into the surrounding environment to exert their antibacterial effect, typically through physical mixing. Represented by metals and their oxides, the released metal ions enter the cell and, by disrupting bacterial enzyme systems, interfere with metabolism, damage DNA, and inhibit bacterial division and proliferation.

[0004] However, existing technologies for improving the antibacterial properties of dental adhesives still have many significant drawbacks and are difficult to meet the actual needs of clinical restoration: (1) It is difficult to balance antibacterial properties with oral microecological compatibility. Although traditional broad-spectrum antibacterial agents can inhibit cariogenic bacteria, they cannot distinguish between pathogenic bacteria and oral symbiotic bacteria, which can easily lead to bacterial resistance and oral flora imbalance, disrupt the balance of oral microecology, and long-term use is not conducive to oral health; (2) The antibacterial activity of direct contact antibacterial agents depends on the degree of exposure of antibacterial agent molecules in the polymer network. If the molecular chains become entangled, the antibacterial activity is significantly reduced and is weaker than that of free antibacterial agents; (3) Release-type antibacterial agents are prone to initial burst release, resulting in excessively high local drug concentrations and cytotoxicity; and rapid consumption of drugs will lead to insufficient antibacterial activity and decreased durability in the later stage, making it difficult to meet the needs of long-term restoration. In summary, there is an urgent need for a new type of dental adhesive that can break through the existing technical bottlenecks and achieve a synergistic improvement in targeted antibacterial, long-term sustained release and strong bonding, effectively prevent secondary caries and improve the long-term efficacy of dental restoration. Summary of the Invention

[0005] The present invention aims to provide a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, its preparation method and application, in order to solve the technical problem that dental adhesives prepared by existing technologies cannot simultaneously achieve both targeted selectivity and long-lasting antibacterial ability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, comprising the following steps: S1. Preparation of 2A4: 4-pentadecanyl-1H-imidazol-2-amine hydrochloride (2A4) was prepared by combining raw materials. S2. Preparation of 2A4@PMMA microspheres: Using emulsion polymerization, the 2A4 obtained in S1 was dissolved in dichloromethane, and then methyl methacrylate (MMA), triethylene glycol dimethacrylate (TEGDMA), and emulsifier octylphenyl ether (OP-10) were added and mixed to obtain an oil phase solution. Deionized water and sodium persulfate were mixed to obtain an aqueous phase solution. The above oil phase solution was added dropwise to the aqueous phase solution, and the mixture was stirred to form a stable emulsion. After heating the emulsion to react, the supernatant was discarded by centrifugation, and the precipitate was dried under vacuum to obtain 2A4@PMMA microspheres. S3. Preparation of dental adhesive: Bisphenol A glycidyldimethacrylate (Bis-GMA), urethane dimethacrylate (UDMA), and TEGDMA are mixed to prepare an adhesive resin monomer system; the adhesive resin monomer system, functional monomers, photoinitiator, and anhydrous ethanol are mixed and magnetically stirred until homogeneous to obtain the dental adhesive.

[0007] Preferably, as an improvement, in S1, the steps for preparing 2A4 are as follows: S1-1. Palmitoyl chloride, trimethylsilyldiazomethane and acetonitrile / tetrahydrofuran mixed solvent were added to the reaction flask; hydrobromic acid solution was added until the reaction was complete; the reaction solution was extracted with ethyl acetate, dried and concentrated to obtain white solid intermediate 1. S1-2. Intermediate 1, Boc-guanidine, sodium iodide and N,N-dimethylformamide are mixed and reacted at room temperature; the reactants are extracted with ethyl acetate, dried and concentrated to obtain intermediate 2. S1-3. Intermediate 2, dichloromethane, and trifluoroacetic acid are mixed and stirred to react. The reactants are dissolved in ethyl acetate and then slowly added dropwise to hydrochloric acid / ethyl acetate solution. A white solid precipitates out. The mixture is stirred in an ice bath and allowed to crystallize overnight. The mixture is then filtered to obtain the crude product. The crude product is dissolved in dichloromethane and purified by column chromatography to obtain 2A4.

[0008] Preferably, as an improvement, in S1, the mixing mass ratio of palmitoyl chloride, trimethylsilyldiazomethane, and acetonitrile / tetrahydrofuran mixed solvent is (40~50g):(15~25g):(250~350ml); the mixing mass ratio of intermediate 1, Boc-guanidine, sodium iodide, and N,N-dimethylformamide is (30~40g):(20~30g):(200~250ml); and the mixing mass ratio of intermediate 2, dichloromethane, and trifluoroacetic acid is (25~35g):(100~150ml):(35~45ml).

[0009] Preferably, as an improvement, in S1, the ethyl acetate extraction is performed by liquid-liquid extraction with 150-200 ml of ethyl acetate three times; the drying is performed by drying with 30-40 g of anhydrous sodium sulfate for 15-30 min; and the concentration is performed by rotary evaporation at 35-40°C for 30-45 min.

[0010] Preferably, as an improvement, in S2, the volume ratio of the oil phase solution to the aqueous phase solution in the emulsion is 30~60ml:100~200ml; the dropping rate of the oil phase solution is 1~2 drops / s.

[0011] Preferably, as an improvement, in S2, the ratio of 2A4, dissolved in dichloromethane, MMA, and TEGDMA in the oil phase solution is 0.5~2g:15~30ml:12~24ml:3~9ml; after mixing the raw materials in the oil phase solution, ultrasonic-assisted dissolution at 50~150W is further performed for 5~15min; the ratio of deionized water and sodium persulfate in the aqueous phase solution is 100~200ml:0.05~0.20g.

[0012] Preferably, as an improvement, in S2, the heating reaction of the emulsion is carried out at 70~80℃ for 3~6h; the centrifugation is carried out at 8000~12000rpm for 10~15min; and the drying is carried out at 60~70℃ for 24~48h.

[0013] Preferably, as an improvement, in S3, the mixing mass ratio of the adhesive resin monomer, functional monomer, photoinitiator, and anhydrous ethanol is 80~85:5~10:0.1~0.2:10~20.

[0014] Preferably, as an improvement, the photoinitiator comprises CQ and EDB, and the functional monomer comprises 10-MDP.

[0015] Preferably, as an improvement, this solution also provides a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, including the dental adhesive prepared by the above method.

[0016] Preferably, as an improvement, the application of a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres in selective antibacterial activity against Streptococcus mutans.

[0017] The principles and advantages of this scheme are: This preparation method achieves efficient integration of antibacterial components and adhesive substrates through a standardized process of "precise synthesis of 2-aminoimidazole derivative (2A4) → loading of 2A4@PMMA microspheres → preparation of dental adhesive resin system". This results in a dental adhesive with excellent antibacterial properties and stable adhesive performance, overcoming the pain point of insufficient antibacterial properties of traditional dental adhesives, and taking into account both pharmaceutical safety and clinical suitability, thus providing high-quality material support for dental clinical bonding.

[0018] 1. This scheme employs a multi-step precise synthesis and purification process to prepare high-purity, high-activity 2A4, laying the core material foundation for subsequent microsphere loading and adhesive antibacterial properties, meeting the purity requirements of pharmaceutical-grade materials. Specifically, the synthesis process is clearly divided into steps, using suitable raw materials such as palmitoyl chloride and Boc-guanidine, combined with specialized solvents such as acetonitrile / tetrahydrofuran and dichloromethane, to precisely control reaction conditions, ensuring the efficient synthesis of intermediates 1 and 2 and reducing side reactions. After each reaction, purification treatments such as ethyl acetate extraction, drying, and concentration are performed, and finally, precise purification is achieved through column chromatography (EA:PE gradient elution) to effectively remove unreacted raw materials and impurities, obtaining high-purity white solid 2A4, ensuring its antibacterial activity and avoiding impurities affecting subsequent microsphere loading and adhesive performance. The synthesis process is highly controllable, with standardized steps, enabling the large-scale preparation of 2A4, meeting the batch production needs of subsequent dental adhesives.

[0019] 2. This solution uses emulsion polymerization to achieve efficient and stable loading of 2A4 and PMMA microspheres, taking into account the dispersibility of microspheres and the compatibility of antibacterial components, giving the adhesive long-lasting antibacterial properties while ensuring the structural stability of microspheres. Specifically, emulsion polymerization was used, where 2A4 was dissolved in dichloromethane and then ultrasonically dissolved with MMA, TEGDMA, and OP-10 to form a homogeneous oil phase. This oil phase was then combined with deionized water and sodium persulfate to ensure uniform dispersion of the system, preventing 2A4 agglomeration and ensuring its uniform loading on the surface and interior of PMMA microspheres. Precise control of the dropping rate (1-2 drops / second), stirring speed (450 rpm), reaction temperature (80℃), and reaction time (3 h) ensured emulsion stability, resulting in microspheres with uniform particle size and regular structure. High-speed centrifugation (8000 rpm, 10 min) followed by vacuum drying (60℃, 24 h) efficiently separated the microspheres from the reaction system, removing residual solvents and impurities while ensuring thorough drying and structural stability of the microspheres, preventing uneven dispersion during subsequent fusion with the adhesive resin system. The PMMA microspheres, as a carrier, exhibit excellent biocompatibility and mechanical strength suitable for dental bonding requirements, enabling long-term sustained release of 2A4 and enhancing the antibacterial durability of the adhesive.

[0020] 3. This solution constructs a resin system adapted to the needs of dental clinical practice. The components are scientifically formulated, and the preparation process is simple, ensuring the adhesive's bonding performance, light-curing efficiency, and safety, thus meeting practical clinical requirements. Specifically, Bis-GMA, UDMA, and TEGDMA are used in synergistic preparation of the adhesive resin. The component combination matches the performance requirements of dental adhesives, possessing both good bonding strength and biocompatibility, suitable for hard tissue bonding scenarios. Precise addition of a photoinitiator (0.7 wt% CQ + 0.7 wt% EDB) ensures high light-curing efficiency, enabling rapid adhesive curing and shortening clinical operation time. The addition of 10 wt% anhydrous ethanol as a solvent promotes complete dissolution of all components. 8 hours of magnetic stirring ensures uniform mixing of the system, preventing component stratification and ensuring stable adhesive performance. The preparation process is simple, requiring no complex equipment, and the reaction conditions are mild, enabling large-scale production. Furthermore, no harmful byproducts are generated, meeting the safety requirements of pharmaceutical materials and reducing the risks of clinical application. Attached Figure Description

[0021] Figure 1 This is a synthetic route diagram of compound 2A4 in Example 1 of the present invention.

[0022] Figure 2 The FTIR (A), TAG (B), and structural formula (C) of 4-pentadecanyl-1H-imidazol-2-amine hydrochloride (2A4) in Experimental Example 1 of this invention are shown.

[0023] Figure 3 The images shown are (A) and (B) of the 2A4@PMMA microspheres in Experimental Example 2 of this invention, obtained by scanning electron microscopy (SEM).

[0024] Figure 4 The images shown are FTIR, TGA, and XPS images of the 2A4@PMMA microspheres in Experimental Example 2 of this invention (A, FTIR spectrum of the microspheres; B, TG-DTG curve of the microspheres; C, XPS spectrum of the microspheres: (C1) XPS full spectrum scan; (C2) C1s spectrum; (C3) N1s spectrum).

[0025] Figure 5 The images are scanning electron microscope (SEM) images of the Streptococcus mutans biofilm at 24h and 48h in Experimental Example 4 of this invention (A1-A6, 1000x magnification for observing bacterial quantity; B1-B6, 5000x magnification for observing biofilm formation indicated by orange arrows; C1-C6, 30000x magnification for observing bacterial morphology indicated by yellow arrows).

[0026] Figure 6The survival ability of Streptococcus mutans in three groups of dental adhesives in Experiment Example 4 of this invention within 24 hours is shown in Figure A. Agar plate images with different contents of 2A4@PMMA microspheres; Figure B. Colony-forming unit (CFU) count of 2A4@PMMA microspheres, showing a concentration-dependent antibacterial effect; ns: no significant difference. : p <0.05; : p <0.01; : p <0.001).

[0027] Figure 7 The results of the cytotoxicity of the extract in Experiment Example 5 of this invention are as follows: (A) Cell viability staining results; (B) CCK-8 results: B1 shows no significant difference in cell growth among all groups at the same time point. p >0.05), B2 indicates that all groups showed an increasing trend in cell count over time; ns: no significant difference; : p <0.05; : p <0.01; : p <0.001).

[0028] Figure 8 The results show the micro-tensile strength of the adhesive material containing 2A4@PMMA microspheres in Experimental Example 6 of this invention. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0030] Example 1 This solution provides a method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, comprising the following steps: S1. Prepare 2A4, the synthetic route is as follows: Figure 1As shown, palmitoyl chloride (Pal-Cl), trimethylsilyldiazomethane, and a 1:1 mixture of acetonitrile (CH3CN) / tetrahydrofuran (THF) solvent were added to a reaction flask (the specific mass ratio of the palmitoyl chloride, trimethylsilyldiazomethane, and acetonitrile / tetrahydrofuran mixture was 40-50 g : 15-25 g : 250-350 ml). A 48% hydrobromic acid (HBr) solution was added dropwise until the reaction was complete. The mixture was extracted with ethyl acetate (EA), dried, and concentrated to obtain a white solid intermediate 1.

[0031] Boc-guanidine, sodium iodide (NaI), and N,N-dimethylformamide (DMF) were added to a reaction flask (the specific mass ratio of intermediate 1, Boc-guanidine, sodium iodide, and N,N-dimethylformamide was 30-40 g : 20-30 g : 200-250 ml), and the reaction was carried out at room temperature. The mixture was extracted with ethyl acetate (EA), dried, and concentrated to obtain intermediate 2.

[0032] Dichloromethane (DCM) and trifluoroacetic acid (CF3COOH) were added to a reaction flask (the specific mass ratio of intermediate 2, dichloromethane, and trifluoroacetic acid was 25-35 g: 100-150 ml: 35-45 g), and the mixture was stirred. The solution was dissolved in ethyl acetate (EA) and then slowly added dropwise to an ethyl acetate hydrochloride (EA / HCl) solution, precipitating a white solid. The mixture was stirred in an ice bath and allowed to crystallize overnight. The crystals were then filtered to obtain the crude product. The crude product was dissolved in dichloromethane and purified by column chromatography (EA:PE = 1:20 to 1:1) to finally obtain 2A4.

[0033] S2. Preparation of 2A4@PMMA microspheres: Loading 2A4 onto PMMA microspheres (2A4@PMMA) is the second step in preparing the antibacterial dental adhesive. Using emulsion polymerization, 0.5 g of the 2-aminoimidazole derivative (2A4) was dissolved in 15 ml of dichloromethane (DCM). 12 ml of MMA, 3 ml of TEGDMA, and 0.1% OP-10 were added. The solution was sonicated for 5 min to obtain the oil phase solution. 100 ml of deionized water and sodium persulfate were added to a three-necked flask as initiators to obtain the aqueous phase solution. The prepared oil phase liquid was added dropwise to the aqueous phase at a rate of 1-2 drops per second using a dropping funnel, and the mixture was stirred at 450 rpm for 30 min until a stable emulsion was formed. The mixture was heated to 80°C and reacted for 3 h. After the reaction was completed, the microspheres were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried in a vacuum drying oven at 60℃ for 24 h. The dried microspheres were then collected.

[0034] S3. Preparation of dental adhesive: Bis-GMA, UDMA, and TEGDMA were mixed to prepare the adhesive resin monomer. 5 wt% 10-MDP was added as a functional monomer, 0.7 wt% CQ and 0.7 wt% EDB were added as photoinitiators, and 10 wt% anhydrous ethanol was added as a solvent. The above reagents were placed in a sample vial equipped with a magnetic stir bar and magnetically stirred for 8 hours until homogeneous, thus completing the synthesis of the resin monomer system.

[0035] Experimental Example 1: Chemical Structure Analysis of 2A4 The chemical structure of 2A4 was characterized by Fourier transform infrared spectroscopy (FTIR). Since the subsequent loading of the drug into the microspheres involves temperature changes, the thermal stability of 2A4 was evaluated by thermogravimetric analysis (TGA) to exclude the effect of temperature on the structure of 2A4 (the results are shown in Figure 2).

[0036] Figure 2 A shows the FTIR results for 2A4. At 3340 cm⁻¹ -1 and 3171 cm -1 The absorption peaks at 1696 cm⁻¹ correspond to the stretching vibrations of the NH₂ and NH bonds, respectively, indicating the presence of primary amine groups and their protonated forms in the sample. -1 The absorption peak at 1191 cm⁻¹ is attributed to the C=N stretching vibration, a characteristic vibrational mode of the imidazole ring skeleton; -1 and 1139 cm -1 The absorption peak at 847 cm⁻¹ likely originates from the CN stretching vibration, consistent with the characteristics of aromatic amines. -1 The absorption peak at 2920 cm⁻¹ likely corresponds to the out-of-plane bending vibration of the CH bond on the imidazole ring. These characteristic peaks indicate the presence of an imidazole ring structure in the compound (distinguished in green in the figure). Meanwhile, the peak at 2920 cm⁻¹... -1 and 2853 cm -1 The absorption peaks at 1472 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of CH₂, respectively. -1 The absorption peak at 721 cm⁻¹ is due to the shear bending vibration of CH₂. -1 The absorption peak corresponds to the planar rocking vibration of CH2, and the above characteristic peaks indicate the presence of a long-chain alkyl chain structure in the compound (distinguished in orange in the figure). Comprehensive analysis of all characteristic absorption peaks confirms that the synthesized 2A4 simultaneously possesses the characteristic structures of an imidazole ring and a long-chain alkyl chain, and the vibrational modes of each functional group are highly consistent with the target molecule structure.

[0037] Figure 2B shows the thermogravimetry (TG) and its derivative thermogravimetry (DTG) plots for 2A4. The entire thermal decomposition process is mainly divided into four stages. The first stage is the removal of physically adsorbed water, from room temperature to 75.1℃, with a mass loss of 2.40%. The second stage is the removal of water of crystallization, from 75.1℃ to 173.1℃, with the mass loss changing from 2.40% to 3.24%, a total mass loss of 0.84%. The third stage is the main decomposition stage, from 173.1℃ to 271.1℃, with the mass loss changing from 3.24% to 19.17%, a total mass loss of 15.93%. Meanwhile, the DTG plots show that the fastest weight loss rate (-4.29% / min) occurs at 243.1℃, indicating that the thermal decomposition reaction is most vigorous at this temperature. The fourth stage is the imidazole ring decomposition stage, which occurs between 271.1℃ and 359.1℃. The mass loss changes from 19.17% to 32.69%, with a total mass loss of 13.52% in this stage, indicating the continuous decomposition of the cyclic structure at high temperatures. In summary, the chemical structure of 2A4 maintains good thermal stability up to 173.1℃ and is not easily decomposed by heat.

[0038] Example 2: Structural Analysis of 2A4@PMMA Microspheres The second step in preparing the antibacterial dental adhesive is loading 2A4 onto PMMA microspheres (2A4@PMMA). Using emulsion polymerization, 0.5 g of the 2-aminoimidazole derivative (2A4) was dissolved in 15 ml of dichloromethane (DCM). 12 ml of MMA, 3 ml of TEGDMA, and 0.1% OP-10 were added to the solution. The solution was dissolved using ultrasound for 5 min to obtain the oil phase. 100 ml of deionized water and sodium persulfate were added to a three-necked flask as initiators to obtain the aqueous phase. The prepared oil phase was added dropwise to the aqueous phase at a rate of 1-2 drops per second using a dropping funnel, and the mixture was stirred at 450 rpm for 30 min until a stable emulsion was formed. The mixture was then heated to 80°C and reacted for 3 h. After the reaction, the microspheres were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried in a vacuum drying oven at 60℃ for 24 h. The dried microspheres were then collected, and their morphology was characterized by high-resolution scanning electron microscopy (SEM) (results are shown in the figure). Figure 3 (As shown). Figure 3 The image shows a SEM image of 2A4@PMMA drug-loaded microspheres synthesized by emulsion polymerization, which characterizes the typical morphology of the microspheres. The particles are arranged in aggregates with good dispersibility and an average particle size of 359.02 ± 3.15 nm.

[0039] To evaluate the loading status of drug 2A4, 2A4@PMMA microspheres were analyzed by Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), and X-ray photon spectroscopy (XPS). For FTIR analysis, the microsphere powder and potassium bromide powder were thoroughly mixed at a 1:100 ratio and ground. The mixture was then scanned using a Fourier transform infrared spectrometer (Bruker, Vertex 70) with a scanning range of 40 to 4000 cm⁻¹. -1 For TGA analysis, the microsphere powder was placed in an alumina pot and analyzed using a thermogravimetric analyzer (Netzsch, TG209F1Libra R) at a temperature range of 25 to 350 °C, a nitrogen flow rate of 20 mL / min, and a heating rate of 10 °C / min. For XPS analysis, the microsphere powder was ultrasonically dispersed and then drop-coated onto a silicon wafer, dried under vacuum at 60 °C for 2 h, and the elemental composition, chemical state, and relative content of C, O, and N on the surface of the PMMA microspheres were analyzed using X-ray electron spectroscopy (Thermo Fisher, K-ALPHA).

[0040] Figure 4 The results of FTIR, TGA, and XPS analyses of 2A4@PMMA are shown. Figure 4 A shows the Fourier transform infrared (FTIR) absorption spectra of 2A4, PMMA, and 2A4@PMMA microspheres. The 2A4@PMMA (red curve) shows the 1731 cm⁻¹ peak of the PMMA (purple curve). -1 The characteristic peak of C=O and the 3442 cm⁻¹ of 2A4 (green curve) -1 The characteristic peak of NH is also visible at 1638 cm⁻¹. -1 The shifts in position and intensity demonstrate an interaction between 2A4 and PMMA. In conclusion, 2A4 is effectively encapsulated within PMMA.

[0041] Figure 4 B shows the thermogravimetric analysis (TG-DGT) spectrum of 2A4@PMMA microspheres. The entire thermal decomposition process can be divided into two main stages. The first stage is the desorption and pyrolysis of the long alkyl chain of the 2A4 drug (distinguished in red in the figure), starting from room temperature to 225.1℃, with a mass loss of 8.4%. The second stage is the complete pyrolysis of the PMMA polymer backbone (distinguished in orange in the figure), from 225.1℃ to 445.1℃, with the mass loss increasing from 8.4% to 100%. Furthermore, the DTG curve shows that the fastest weight loss rate (-11.01% / min) occurs at 355.1℃, indicating that the thermal decomposition reaction is most vigorous at this temperature. In conclusion, 2A4 has been successfully incorporated into PMMA microspheres.

[0042] Figure 4C represents the high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of 2A4@PMMA microspheres. To clarify the chemical state of nitrogen in the PMMA microspheres and verify the successful loading of the target drug 2A4 onto the microspheres, XPS analysis was performed on the drug-loaded microsphere samples. Figure 4 In the C1 energy spectrum, a significant peak of N1s was detected in 2A4@PMMA microspheres. Figure 4 In C2, the peak values ​​of 288.61 eV, 286.339 eV, and 284.80 eV correspond to C=O, CO, and CC bonds, respectively. These bonds are the main chemical bonds in PMMA. Figure 4 In C3, the peaks at 401.30 eV, 399.73 eV, and 398.40 eV correspond to -NH2, NH-, and -N= bonds, respectively, which are the major chemical bonds in 2A4. These results further support the successful incorporation of 2A4 into PMMA.

[0043] Experimental Example 3: Effect of 2A4@PMMA microspheres on adhesive properties To evaluate the effect of 2A4@PMMA microspheres on adhesive performance, microspheres were added to dental adhesives in different proportions. Bis-GMA, UDMA, and TEGDMA were mixed to prepare the adhesive resin monomers. 5 wt% 10-MDP was added as a functional monomer, 0.7 wt% CQ and 0.7 wt% EDB were added as photoinitiators, and 10 wt% anhydrous ethanol was added as a solvent. The above reagents were placed in a sample vial equipped with a magnetic stir bar and magnetically stirred for 8 hours until homogeneous, thus completing the synthesis of the resin monomer system. The control group was identical to the experimental group except for the absence of 2A4@PMMA. The prepared 2A4@PMMA microspheres were incorporated into the adhesive resin at 5 wt% or 10 wt%, resulting in two experimental groups. The prepared dental adhesive samples were used to assess their inhibitory effect on cariogenic bacterial biofilms (Example 4), their effect on cell growth (Example 5), and their effect on adhesive performance (Example 6).

[0044] The circular samples used in Experiments 4 and 5 were prepared using a 1 mm thick, 10 mm diameter polyvinylsiloxane mold and cured using a UV curing lamp at 1200 mW / cm² per side. 2 Sterilization was performed by irradiation with ultraviolet light for 20 seconds. The sample preparation for Experiment 6 is described in detail in Experiment 6.

[0045] Experimental Example 4: Inhibitory effect of 2A4@PMMA adhesive on cariogenic bacterial biofilm To evaluate the inhibitory effect of 2A4@PMMA adhesive on cariogenic bacterial biofilms, the biofilm morphology of *Streptococcus mutans* on resin discs was observed by scanning electron microscopy (SEM), and the biofilm growth kinetics of the material were assessed by calculating CFU using the direct contact method. Samples were placed in sterile 24-well plates, six per group. Three wells were used for SEM observation, and three for CFU calculation. Each well contained a circular resin sample disc (10 mm in diameter), and 1.5 mL of 10% 2A4@PMMA adhesive was added. 6 CFU / mL BHIS medium. Biofilms cultured on resin slides for SEM observation at 24 h and 48 h were washed three times with PBS and then immersed in 2.5% glutaraldehyde at 4 °C for 12 h. Specimens were washed with PBS and subjected to a gradient ethanol dehydration (30%, 50%, 75%, 85%, 95%, and 100%). The sample surfaces were then sputter-coated with gold and observed using a scanning electron microscope (SEM, Gemini 500, CARL SEISS, Germany). Resin slides used for CFU calculation were removed from the wells after 24 h of culture and vortexed in 1 mL PBS solution for serial dilution to 10⁻⁶. 6 Inoculate 50 μL of each dilution onto BHI agar medium and incubate the plates at 37°C for 48 hours. Visually count the colonies and transform them using the following method at colony forming units per milliliter (CFU / mL): (average colony count × dilution factor) / plate volume. Count the colonies and convert them to CFU / mL as described above.

[0046] Figure 5 The results of the three adhesive groups after 24 hours and 48 hours are shown. S. mutans SEM images of the biofilm. The colonization density, biofilm structure, and morphological changes of individual bacteria were observed at different magnifications (1000×, 5000×, 30000×). Figure 5 (A1~A6) illustrates the changes in bacterial count. At 1000× magnification, at the same time point, the adhesive containing microspheres (5 wt% group and 10 wt% group) showed a higher bacterial count than the adhesive without microspheres (0 wt% group). S. mutans The growth is sparser, and the number of bacteria tends to decrease over time. Figure 5 (B1~B6) shows the changes in bacterial biofilm, at 5000× magnification, the 0 wt% group compared to the 5 wt% and 10 wt% groups. S. mutans The biofilm (indicated by orange arrows) grew more densely, and the biofilm in the 5 wt% and 10 wt% groups showed a decreasing trend over time. Figure 5(C1~C6) illustrates the changes in bacterial morphology. At 30,000× magnification, both the 5 wt% and 10 wt% groups showed morphological deformation such as shrinkage and breakage (indicated by yellow arrows). These results show that, compared with the control group, the biofilm on the adhesive surface containing 2A4@PMMA is sparser, and the bacterial morphology shows shrinkage and breakage.

[0047] Figure 6 This study demonstrates the ability of three groups of dental adhesive resins to survive 24 hours of Streptococcus mutans infection using a bacterial colony count (CFU) test. Figure 6 As shown in Figure A, the number of bacterial colonies gradually decreases as the content of 2A4@PMMA microspheres in the adhesive increases. Figure 6 B presents the results of quantitative statistical analysis. The log10 values ​​of CFU / mL for the 0 wt%, 5 wt%, and 10 wt% groups were 8.17±0.05, 7.62±0.10, and 7.33±0.02, respectively. Compared with the 0 wt% control group, the bacterial counts in the 5 wt% and 10 wt% 2A4@PMMA groups were significantly reduced. p The concentration-dependent antibacterial effect of 2A4@PMMA microspheres in dental adhesives is indicated by a concentration-dependent value (<0.05).

[0048] Experimental Example 5: Effects of 2A4@PMMA adhesive on cell growth To assess the effect of 2A4@PMMA adhesive on cell growth, gingival mesenchymal stem cells (GMSCs) were subjected to CCK-8 assays and cell viability / death staining using an indirect extract cytotoxicity assay to analyze the potential cytotoxic effects of the formulated adhesive. Extracts were prepared from six samples in each group.

[0049] First, qualitative analysis of cell viability and cytotoxicity was performed using cell viability and mortality staining. Each sample was immersed in 10 mL of α-MEM medium for 24 hours to prepare the extract. On the same day, 200 μL of cells containing GMSCs (concentration 1.25 x 10⁻⁶) was added. 5 α-MEM (cells / mL) was seeded in 24-well plates for 24 hours. 200 μL of the extract from each sample was seeded into wells containing GMSCs. A group of cells was treated with α-MEM medium without the extract as a negative control. Culture was terminated at 1, 3, and 5 days. 200 μL of 10% cell viability / deadness staining solution was added to each well, and the cells were incubated at 37°C for 30 min. Observation and imaging were performed using an inverted fluorescence microscope.

[0050] Next, quantitative analysis of CCK-8 cytotoxicity was performed. Each sample was immersed in 1 mL of α-MEM medium for 24 hours to prepare an extract. On the same day, 100 μL of GMSCs (concentration of 3 x 10⁻⁶ cells) was added.4 Cells were seeded with α-MEM (cells / mL) in 96-well plates for 24 hours. 100 μL of the extract from each sample was seeded into wells containing GMSCs. A cohort of cells was treated with α-MEM medium without the extract as a negative control. Culture was terminated at 1, 3, and 5 days. 100 μL of complete medium containing 10% CCK-8 was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance of the solution in each well was analyzed at 450 nm, and cell viability was normalized to that of untreated cells (negative control). Results are expressed as a percentage of viability (100% = viability of the negative control).

[0051] Figure 7 A shows the results of cell viability and death staining for the material extract. The CellCytotoxicity Assay Kit used contains dual fluorescent staining solutions of Calcein-AM and Propidium Iodide (PI). Calcein-AM stains live cells, showing green fluorescence, while PI stains dead cells, showing red fluorescence. Gingival mesenchymal stem cells (GMSCs) in all groups showed time-dependent proliferation, maintaining the typical fibroblast-like phenotype of mesenchymal stem cells with no obvious signs of apoptosis, indicating that the material extract is non-cytotoxic.

[0052] Figure 7 B shows the results of CCK-8 assay for the cytotoxicity of the material extract. The CCK-8 reagent used contains WST-8, which, in the presence of an electron-coupling reagent, can be reduced by mitochondrial dehydrogenases to generate a highly water-soluble orange-yellow Formazan dye. The amount of Formazan produced is directly proportional to the number of surviving cells. This characteristic makes it possible to directly analyze cell proliferation and cytotoxicity. Figure 7 B1 showed that on days 1, 3, and 5 of co-culture, the relative cell viability of the 5wt% and 10wt% groups was similar to that of the control group without microspheres, with no significant difference among the three groups. p >0.05). Figure 7 B2 showed that each group of cells exhibited an increasing trend over time, indicating that the material did not adversely affect the growth of GMSCs.

[0053] Experimental Example 6: Effect of 2A4@PMMA microspheres on the adhesive properties of adhesives To assess whether the addition of 2A4@PMMA microspheres affects the adhesive properties of the adhesive, its micro-tensile bond strength (μ-TBS) needs to be measured. The adhesive without 2A4@PMMA microspheres was used as a control group.

[0054] Immediate μ-TBS was evaluated using five third molars (eight sprites per group). Teeth were embedded in epoxy resin, and the occlusal surface of the crown was cut using a diamond saw to expose the dentin in the middle of the crown. Teeth were polished with 600-grit silicon carbide sandpaper under water cooling for 60 seconds. Three bonding materials were then bonded to Filtek™ Z350 resin according to clinical procedures, achieving a total height of 4–5 mm. A commercially available general-purpose dental adhesive (Singlebond Universal) served as a commercial control group. Prepared specimens were stored in distilled water at 37°C for 24 hours. A precision cutter was used to obtain specimens with an area of ​​approximately 1 × 1 mm. 2 Rectangular dentin-resin rod-shaped specimens were used. The area was calculated by averaging three equidistant measurement points on each side of the surface. Eight central rod-shaped specimens were selected from the same tooth, fixed to a micro-tensile fixture with cyanoacrylate adhesive, and the fixture was mounted on a universal testing machine. A tensile force (tensile rate 1 mm / min) was applied, and the μ-TBS value (MPa) of the sample was calculated according to the following formula:

[0055] F represents the magnitude of the applied force (N), and S represents the cross-sectional area of ​​the specimen (mm²). 2 All fracture surfaces were examined using a stereomicroscope at 10-40x magnification. Failure modes were categorized as adhesive failure, cohesive failure (including composite resin cohesive failure and dentin cohesive failure), and mixed failure. Cohesive failure could not be used for µ-TBS numerical statistics, but its proportion must be recorded.

[0056] Figure 8 The results show the microtensile bond strength of the materials. The microtensile bond strengths of the antibacterial dental adhesive with additions of 5 wt% and 10 wt% were 24.20 ± 5.9 MPa and 23.86 ± 5.3 MPa, respectively, while the 0 wt% group without microspheres had a strength of 25.02 ± 5.9 MPa. There was no statistically significant difference among the three groups. p >0.05), and its performance is comparable to that of the commonly used commercial adhesive Singlebond Universal (26.44 ± 6.28 MPa). p >0.05).

[0057] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, characterized in that: Includes the following steps: S1. Preparation of 2A4: 4-pentadecanyl-1H-imidazol-2-amine hydrochloride (2A4) was prepared by combining raw materials. S2. Preparation of 2A4@PMMA microspheres: Using emulsion polymerization, the 2A4 obtained in S1 was dissolved in dichloromethane, and then MMA, TEGDMA and emulsifier OP-10 were added and mixed to obtain an oil phase solution; deionized water and sodium persulfate were mixed to obtain an aqueous phase solution; the above oil phase solution was added dropwise to the aqueous phase solution and stirred to form a stable emulsion; after heating the emulsion to react, the supernatant was discarded by centrifugation, and the precipitate was dried under vacuum to obtain 2A4@PMMA microspheres; S3. Preparation of dental adhesive: Bis-GMA, UDMA and TEGDMA are mixed to prepare adhesive resin monomers; the adhesive resin monomers, functional monomers, photoinitiator and anhydrous ethanol are mixed and stirred evenly with magnetic force to obtain dental adhesive.

2. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 1, characterized in that: In S1, the steps for preparing 2A4 are as follows: S1-1. Palmitoyl chloride, trimethylsilyldiazomethane and acetonitrile / tetrahydrofuran mixed solvent were added to the reaction flask; hydrobromic acid solution was added until the reaction was complete; the reaction solution was extracted with ethyl acetate, dried and concentrated to obtain white solid intermediate 1. S1-2. Intermediate 1, Boc-guanidine, sodium iodide and N,N-dimethylformamide are mixed and reacted at room temperature; the reactants are extracted with ethyl acetate, dried and concentrated to obtain intermediate 2. S1-3. Intermediate 2, dichloromethane, and trifluoroacetic acid are mixed and stirred to react. The reactants are dissolved in ethyl acetate and then slowly added dropwise to hydrochloric acid / ethyl acetate solution. A white solid precipitates out. The mixture is stirred in an ice bath and allowed to crystallize overnight. The mixture is then filtered to obtain the crude product. The crude product is dissolved in dichloromethane and purified by column chromatography to obtain 2A4.

3. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 2, characterized in that: In S1, the mass ratio of palmitoyl chloride, trimethylsilyldiazomethane, and acetonitrile / tetrahydrofuran mixed solvent is (40~50g):(15~25g):(250~350ml); the mass ratio of intermediate 1, Boc-guanidine, sodium iodide, and N,N-dimethylformamide is (30~40g):(20~30g):(200~250ml); and the mass ratio of intermediate 2, dichloromethane, and trifluoroacetic acid is (25~35g):(100~150ml):(35~45ml).

4. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 3, characterized in that: In S2, the volume ratio of the oil phase solution to the aqueous phase solution in the emulsion is 30~60ml:100~200ml; the dropping rate of the oil phase solution is 1~2 drops / s.

5. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 4, characterized in that: In S2, the ratio of 2A4, dissolved in dichloromethane, MMA and TEGDMA, and emulsifier OP-10 in the oil phase solution is 0.5~2g: 15~30ml: 12~24ml: 3~9ml: 0.01~0.08g; after mixing the raw materials in the oil phase solution, ultrasonic-assisted dissolution at 50~150W is also performed for 5~15min; the ratio of deionized water and sodium persulfate in the aqueous phase solution is 100~200ml: 0.05~0.20g.

6. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 5, characterized in that: In S2, the heating reaction of the emulsion is carried out at 70-80°C for 3-6 hours; the centrifugation is carried out at 8000-12000 rpm for 10-15 minutes; and the drying is carried out at 60-70°C for 24-48 hours.

7. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 6, characterized in that: In S3, the mixing mass ratio of the adhesive resin monomer, functional monomer, photoinitiator, and anhydrous ethanol is 80~85:5~10:0.1~0.2:10~20.

8. The method for preparing a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 7, characterized in that: The photoinitiator includes CQ and EDB, and the functional monomer includes 10-MDP.

9. A dental adhesive containing 2-aminoimidazole derivative PMMA microspheres, characterized in that: The dental adhesive prepared by the method according to any one of claims 1 to 8.

10. The application of a dental adhesive containing 2-aminoimidazole derivative PMMA microspheres according to claim 9 in its selective antibacterial action against Streptococcus mutans.

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