Auxiliary pith capping material containing zinc ionic liquid modified chitosan as well as preparation method and application of auxiliary pith capping material

Abstract

The invention discloses an auxiliary pulp capping material containing zinc ionic liquid modified chitosan as well as a preparation method and application of the auxiliary pulp capping material, and relates to the technical field of dental medical materials. The material comprises a solid phase and a liquid phase, the solid phase is carboxymethyl chitosan; the liquid phase is 1-vinyl-3-dodecylbrominated imidazole zinc chloride, and the pH value of the liquid phase is 7-8; the structural formula of the 1-vinyl-3-dodecyl brominated imidazole zinc chloride is as shown in the formula 1 in the specification. The material comprises solid-phase carboxymethyl chitosan and liquid-phase 1-vinyl-3-dodecyl brominated imidazole zinc chloride, through the synergistic effect of the antibacterial activity of zinc ions and the biocompatibility of chitosan, various oral pathogenic bacteria are effectively inhibited, dental pulp repair is promoted, and the material has the advantages of broad-spectrum antibacterial property, good biocompatibility and convenient operation.

Description

Technical Field

[0001] This invention relates to the field of dental medical materials technology, specifically to an auxiliary pulp capping material of zinc ion liquid modified chitosan, its preparation method, and its application. Background Technology

[0002] Caries-related pulpitis is a common and prevalent type of pulpitis in clinical practice. The traditional treatment for pulpitis is to directly remove the infected pulp through root canal treatment. However, root canal treatment can damage the integrity of the tooth, causing the pulp to lose its vitality and important functions, making it prone to reinfection and tooth fracture. Therefore, pulp preservation is an effective way to improve the long-term survival rate of affected teeth.

[0003] Currently, the most commonly used pulp capping agents in clinical practice are novel bioceramic pulp capping agents such as mineral trioxide aggregates (MTA) and iRootBPplus. These pulp capping agents release OH... - and Ca 2+ OH - This increases the local pH value of the dental pulp tissue cross-section, creating an alkaline environment that has antibacterial properties and promotes mineralization. 2+ It participates in intracellular signal transduction pathways and maintains and regulates normal biological processes, induces undifferentiated mesenchymal cells in the dental pulp to differentiate into odontoblast-like cells, and forms reparative dentin at the pulp fracture surface, thereby preserving the root pulp and maintaining its physiological function.

[0004] The strongly alkaline environment created by the novel bioceramic capping agent has a strong antibacterial effect against common pathogens such as Enterococcus faecalis and Candida albicans, but a poor antibacterial effect against facultative anaerobes such as Streptococcus and Lactobacillus. Summary of the Invention

[0005] To comprehensively address the above-mentioned problems, this invention aims to provide an auxiliary medullary capping material modified with zinc ionic liquid, its preparation method, and its application, thereby improving the antibacterial and bacteriostatic effects of the auxiliary medullary capping material.

[0006] The first aspect of this invention provides an auxiliary medullary capping material of chitosan modified with zinc ionic liquid, comprising a solid phase and a liquid phase;

[0007] The solid phase is carboxymethyl chitosan;

[0008] The liquid phase is 1-vinyl-3-dodecylimidazolium bromide zinc chloride;

[0009] The structural formula of the 1-vinyl-3-dodecylimidazolium bromide zinc chloride is shown in Formula 1:

[0010]

[0011] Through the above technical solution, carboxymethyl chitosan serves as the solid phase, providing a biocompatible framework. Its carboxyl groups combine with the cations in the ionic liquid through electrostatic interactions to form a stable complex. 1-Vinyl-3-dodecylimidazolium bromide zinc chloride in the liquid phase gradually releases zinc ions upon contact with dental pulp tissue, inhibiting the growth of various pathogenic bacteria. The long-chain alkyl structure of the ionic liquid modulates the material's hydrophobicity, preventing premature degradation. This composite system forms a protective layer on the dental pulp fracture surface, maintaining local microenvironmental stability and promoting reparative dentin formation; simultaneously, the composite system forms a stable physical barrier, reducing the risk of secondary bacterial infection. The continuous release of zinc ions prolongs the antibacterial period, while the synergistic effect of carboxymethyl chitosan and the ionic liquid ensures the structural integrity of the material in the moist dental pulp environment.

[0012] Preferably, the weight-to-volume ratio of the solid phase to the liquid phase is in the range of 1%-6%.

[0013] Through the above technical solution, when the weight-to-volume ratio of the solid phase to the liquid phase is controlled at 1%-6%, the three-dimensional network structure formed by carboxymethyl chitosan can effectively coat the ionic liquid, avoiding excessive material fluidity due to excessive liquid phase, which could affect the sealing of the pulp cross-section. Simultaneously, this ratio ensures the continuous release of zinc ions in the pulp microenvironment. By adjusting the ratio to 6%, the material can still maintain sufficient flexibility to adapt to the pulp cavity morphology, while the lower limit of 1% avoids structural loosening due to insufficient solid phase. This application can enhance the antibacterial effect against Streptococcus and Lactobacillus species, while maintaining the activity of pulp cells, promoting odontoblast differentiation and the formation of reparative dentin, thereby improving the success rate of vital pulp preservation.

[0014] The second aspect of this invention provides a method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material, comprising the following steps: S2, adding 1-vinylimidazolium to 1-bromododecane and mixing evenly to react and generate 1-vinyl-3-dodecyl imidazolium bromide; S3, adding ZnCl2 to the 1-vinyl-3-dodecyl imidazolium bromide and reacting to obtain 1-vinyl-3-dodecyl imidazolium bromide zinc chloride liquid; S4, adding the carboxymethyl chitosan to the 1-vinyl-3-dodecyl imidazolium bromide zinc chloride liquid and mixing evenly to obtain the auxiliary medullary capping material.

[0015] Through the above technical solution, during the preparation process, 1-vinylimidazole undergoes an alkylation reaction with 1-bromododecane to generate a quaternary ammonium salt intermediate. Subsequently, zinc ions are introduced into the imidazole backbone through an ion exchange reaction with ZnCl2, forming an ionic liquid with antibacterial activity. Carboxymethyl chitosan, as a solid matrix, is mixed with the ionic liquid to form a pulp capping material with antibacterial and biocompatibility. This preparation method controls the synthesis process of the ionic liquid through stepwise reactions, ensuring effective loading of zinc ions. Simultaneously, it utilizes the film-forming and biodegradable properties of carboxymethyl chitosan to achieve stable coverage of the pulp cross-section and efficient preparation of the pulp capping material, solving the problem of insufficient antibacterial effect of traditional pulp capping agents against facultative anaerobes. The zinc ionic liquid exerts its antibacterial effect by disrupting bacterial cell membranes and inhibiting biofilm formation, while carboxymethyl chitosan provides a suitable microenvironment to promote pulp cell migration and differentiation, thereby effectively controlling infection while preserving pulp activity.

[0016] Preferably, in step S1, the preparation of 1-vinyl-3-dodecyl imidazole bromide includes the following steps: adding 1-vinylimidazolium and 1-bromododecane to ethyl acetate and mixing them evenly, then washing and drying the product with ethyl acetate to obtain 1-vinyl-3-dodecyl imidazole bromide.

[0017] The above technical solution removes unreacted monomers and low-boiling-point byproducts dissolved in the solvent by centrifugation. The 1-vinyl-3-dodecyl imidazole bromide obtained after drying provides a high-purity precursor for the subsequent zinc ion coordination reaction, ensuring the integrity of the final ionic liquid structure. This solves the problem of unstable antibacterial properties of the capping material due to insufficient purity of intermediates in the traditional preparation process. The final zinc-containing ionic liquid can more effectively inhibit the growth of Streptococcus and Lactobacillus, while maintaining the biocompatibility of the carboxymethyl chitosan matrix.

[0018] Preferably, the 1-vinyl-3-dodecyl imidazole bromide comprises the following raw materials in parts by weight: 4-5 parts of 1-vinylimidazolium; 10-15 parts of 1-bromododecane.

[0019] By precisely controlling the proportion of raw materials, the reaction byproducts are reduced, and the yield and stability of intermediates are improved, laying the foundation for the subsequent zinc ion loading step. This improves the synthesis efficiency of 1-vinyl-3-dodecyl imidazole bromide and provides a reliable guarantee for the subsequent preparation of modified chitosan materials with stable zinc ion loading capacity.

[0020] Preferably, in step S1, the reaction temperature is 40℃-50℃ and the reaction time is 40h-60h.

[0021] Through the above technical solution, the temperature is controlled within the range of 40-50℃ during the preparation of 1-vinyl-3-dodecyl imidazole bromide. At this temperature, the active group of 1-vinylimidazolium and the long-chain alkyl group of 1-bromododecane can effectively combine to form a stable quaternary ammonium salt structure, avoiding carbon chain breakage or imidazole ring opening caused by excessively high temperature or reaction time. This ensures the stable binding of zinc ions and chitosan in the final pulp capping material, enhancing its targeted antibacterial effect against facultative anaerobic bacteria in pulp infections. When the reaction time is set to 40-60 hours, the content of unreacted free 1-bromododecane in the reaction system can be reduced to below the detection limit, while the product viscosity reaches a threshold suitable for subsequent processing steps. After the reaction, unreacted small molecule impurities are removed by washing with ethyl acetate, finally obtaining a high-purity intermediate.

[0022] Preferably, in step S2, the 1-vinyl-3-dodecyl imidazole zinc chloride comprises the following raw materials in parts by weight: 8-12 parts of 1-vinyl-3-dodecyl imidazole; 3-5 parts of ZnCl2.

[0023] Through the above technical solution, this ratio ensures that the cation sites of imidazolium salt fully bind with zinc ions, avoiding the residue of unreacted metal salts due to excessive ZnCl2, while maintaining the stability of the ionic liquid. During the reaction, Cl- in ZnCl2 reacts with Br- in imidazolium salt. - An exchange occurs, forming a zinc-containing ionic liquid structure. The zinc ion loading directly affects the antibacterial properties of the material. This application enables uniform loading of zinc ions in the ionic liquid, enhancing the material's antibacterial effect against Streptococcus and Lactobacillus.

[0024] Preferably, in step S2, the reaction temperature is room temperature and the reaction time is 20-30 hours.

[0025] Through the above technical solution, 1-vinyl-3-dodecyl imidazole bromide is mixed with zinc chloride at room temperature. Mechanical stirring ensures uniform dispersion of the reactants, allowing zinc ions to gradually replace bromide ions and form a stable coordination structure with the imidazole groups. This ensures that the resulting capping material, after mixing with carboxymethyl chitosan, has a uniform distribution of antibacterial components, effectively inhibiting the activity of various pathogenic bacteria during the capping process. Simultaneously, it avoids the negative impact of high-temperature treatment on the biocompatibility of chitosan. The reaction time is controlled within the range of 20-30 hours, ensuring complete reaction while avoiding the accumulation of byproducts or damage to the ionic liquid structure that may occur with prolonged reactions. During this process, no heating device is required to maintain reaction activity, thus simplifying the operation process and reducing energy consumption.

[0026] The third aspect of this invention provides the application of a zinc-containing ionic liquid-modified chitosan auxiliary capping material in the preparation of antibacterial products.

[0027] The beneficial effects of this invention are: the material comprises solid-phase carboxymethyl chitosan and liquid-phase 1-vinyl-3-dodecyl imidazole zinc chloride bromide. Through the synergistic effect of the antibacterial activity of zinc ions and the biocompatibility of chitosan, it effectively inhibits a variety of oral pathogens and promotes pulp repair. It has the advantages of broad-spectrum antibacterial activity, good biocompatibility and convenient operation. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation method of the auxiliary medullary capping material of the present invention;

[0029] Figure 2 This is a schematic diagram illustrating the synthesis of 1-vinyl-3-dodecylimidazolium bromide zinc chloride according to the present invention;

[0030] Figure 3 This is a diagram showing the room temperature state of 1-vinyl-3-dodecylimidazolium bromide zinc chloride according to the present invention;

[0031] Figure 4 This is the Fourier transform infrared spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride according to the present invention;

[0032] Figure 5 This is the 1H NMR spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride according to the present invention;

[0033] Figure 6 This is the X-ray energy dispersive spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride according to the present invention;

[0034] Figure 7 The ball-and-stick model of the zinc coordination anion and the electrospray ionization mass spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride are shown in the present invention.

[0035] Figure 8 The time-killing curves of three antibacterial agents, NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride, against S. mutans are shown in the figure.

[0036] Figure 9 This is a time-killing curve of three antibacterial agents, NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride, against L. casei.

[0037] Figure 10 This diagram illustrates the minimum inhibitory concentrations for biofilm formation of the three antibacterial agents NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride of the present invention.

[0038] Figure 11This diagram illustrates the minimum biofilm removal concentrations of the three antibacterial agents NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride of the present invention.

[0039] Figure 12 This is a staining image of live and dead bacteria in a biofilm after 1 hour of treatment with the three antibacterial agents NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride, which are the lowest biofilm removal concentrations of the present invention.

[0040] Figure 13 This is a graph showing the ratio of live to dead bacteria in the biofilm after 1 hour of action of the three antibacterial agents NaClO, CHX, and 1-vinyl-3-dodecyl imidazole zinc chloride at the lowest biofilm removal concentration of the present invention.

[0041] Figure 14 This is a graph showing the MIC changes of S. mutans under continuous exposure and passage at 1 / 2 MIC of the drug according to the present invention;

[0042] Figure 15 This is a graph showing the MIC changes of L. casei under continuous exposure and passage at 1 / 2 MIC of the drug according to the present invention;

[0043] Figure 16 This is a scanning electron microscope image showing the morphological changes of S. mutans and L. casei after 12 h of treatment with 1-vinyl-3-dodecyl imidazole zinc chloride under MIC.

[0044] Figure 17 This is a schematic diagram showing the reactive oxygen species levels of S. mutans and L. casei after 12 hours of reaction with 1-vinyl-3-dodecyl imidazole zinc chloride at the MIC.

[0045] Figure 18 This is a cytotoxicity test diagram of 1-vinyl-3-dodecyl imidazole zinc chloride according to the present invention;

[0046] Figure 19 This is a hemolytic activity test diagram of 1-vinyl-3-dodecyl imidazole zinc chloride according to the present invention;

[0047] Figure 20 The diagram shows the room temperature properties of IL-Zn (1-vinyl-3-dodecylimidazolium chloride) / CMCS (carboxymethyl chitosan) prepared in Examples 1-6 of this invention.

[0048] Figure 21 The ΔOD of IL-Zn / CMCS prepared in Examples 1-6 of this invention on S. mutans and L. casei bacterial cultures after 24, 48, and 72 hours of treatment. 600 Schematic diagram;

[0049] Figure 22This is a microscopic illustration of the adhesion of IL-Zn / CMCS to L929 cells in Examples 2 and 3 of this application of the present invention.

[0050] Figure 23 This is a statistical graph showing the effect of IL-Zn / CMCS on L929 cell adhesion rate in Examples 2 and 3 of the present invention;

[0051] Figure 24 This is a scanning electron microscope schematic diagram showing the effect of IL-Zn / CMCS on the adhesion morphology of L929 cells in this invention;

[0052] Figure 25 This is a schematic diagram of the animal experimental groupings for Example 2 of the present invention, the blank control group, and the control group;

[0053] Figure 26 This is a schematic diagram showing the typical HE staining results of dental pulp in Example 2, the blank control group, and the control group 4 weeks post-operation.

[0054] Figure 27 This is a schematic diagram showing the HE staining results of the main organs of Example 2, the blank control group, and the control group at 4 weeks post-operation. Detailed Implementation

[0055] The following combination Figures 1-27 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0056] The raw materials and reagents involved in this invention are as follows:

[0057] All solvents used in this invention are commercially available analytical grade reagents. Among them, 1-vinylimidazole, 1-bromododecane, carboxymethyl chitosan, methanol, and ZnCl2 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and ethyl acetate was purchased from Sinopharm Chemical Reagent Co., Ltd. Streptococcus mutans (UA159) and Lactobacillus casei (ATCC393) were provided by the Oral Microbiology Laboratory of the National Key Laboratory of Oral and Maxillofacial System Reconstruction and Regeneration, Air Force Medical University.

[0058] A zinc ionic liquid-modified chitosan auxiliary medullary capping material comprises a solid phase and a liquid phase. The solid phase is carboxymethyl chitosan, and the liquid phase is 1-vinyl-3-dodecyl imidazole zinc chloride. The structural formula of 1-vinyl-3-dodecyl imidazole zinc chloride is shown in Formula 1.

[0059]

[0060] Figure 1This is a flowchart of a method for preparing a zinc-containing ionic liquid-modified chitosan auxiliary medullary capping material according to an embodiment of the present invention. The preparation method includes the following steps: S1, adding 1-vinylimidazolium to 1-bromododecane and mixing evenly to generate 1-vinyl-3-dodecyl imidazolium bromide; S2, adding ZnCl2 to the 1-vinyl-3-dodecyl imidazolium bromide and reacting to obtain 1-vinyl-3-dodecyl imidazolium bromide zinc chloride liquid; S3, adding the carboxymethyl chitosan to the 1-vinyl-3-dodecyl imidazolium bromide zinc chloride liquid and mixing evenly to obtain the auxiliary medullary capping material; wherein, the synthesis schematic diagram of 1-vinyl-3-dodecyl imidazolium bromide zinc chloride is shown in Figure 1. Figure 2 As shown.

[0061] Example

[0062] Example 1

[0063] A method for preparing a zinc ionic liquid-modified chitosan auxiliary operculum trophic material includes the following steps:

[0064] S1. Add 50 mL of ethyl acetate to a 100 mL round-bottom flask, then add 1-vinylimidazolium (0.05 mol, 4.7 g) and 1-bromododecane (0.05 mol, 12.2 g). Purge the round-bottom flask with argon gas to remove oxygen for 15 min, and stir magnetically at 45 °C for 48 h. Wash the reaction product three times with ethyl acetate, centrifuging at 1000 rpm for 5 min each time. After the viscous liquid turns into a white solid, dry it in a vacuum drying oven for 12 h to obtain 1-vinyl-3-dodecylimidazolium bromide.

[0065] S2. Add (0.03 mol, 4.1 g) ZnCl2 to (0.03 mol, 9.9 g) 1-vinyl-3-dodecyl imidazolium bromide and react at room temperature for 24 h to obtain 1-vinyl-3-dodecyl imidazolium bromide zinc chloride liquid.

[0066] S3. Add 0.1g of carboxymethyl chitosan to 10ml of 1-vinyl-3-dodecyl imidazole zinc chloride liquid and mix well to obtain the auxiliary medullary capping material.

[0067] The 1-vinyl-3-dodecylimidazolium chloride zinc chloride prepared in Example 1 was as follows at room temperature: Figure 3 As shown.

[0068] Example 2

[0069] A method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material differs from Example 1 in that the weight-volume ratio of the solid phase to the liquid phase is in the range of 2%.

[0070] Example 3

[0071] A method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material differs from Example 1 in that the weight-volume ratio concentration of the solid phase to the liquid phase is in the range of 3%.

[0072] Example 4

[0073] A method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material differs from Example 1 in that the weight-volume ratio of the solid phase to the liquid phase is in the range of 4%.

[0074] Example 5

[0075] A method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material differs from Example 1 in that the weight-volume ratio of the solid phase to the liquid phase is in the range of 5%.

[0076] Example 6

[0077] A method for preparing a zinc ionic liquid-modified chitosan auxiliary medullary capping material differs from Example 1 in that the weight-volume ratio of the solid phase to the liquid phase is in the range of 6%.

[0078] Experimental Analysis:

[0079] 1. Fourier transform infrared spectroscopy analysis of 1-vinyl-3-dodecylimidazolium bromide zinc chloride (IL-Zn) prepared in Example 1:

[0080] The Fourier transform infrared spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride in Example 1 was measured using a Fourier transform infrared spectrometer - IRTracer 100 (Tsushima, Japan). The test results are as follows: Figure 4 As shown, NH, CH, CN, C=C and C=N tensile vibration peaks can be observed. These characteristic peaks are basically consistent with the main functional groups of 1-vinyl-3-dodecylimidazolium bromide zinc chloride, indicating the presence of long-chain alkyl and imidazole rings, which proves the successful synthesis of the material.

[0081] 2. NMR characterization analysis of 1-vinyl-3-dodecylimidazolium bromide zinc chloride prepared in Example 1:

[0082] The 1H NMR spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride in Example 1 was measured using a Bruker Anavce NEO 400M NMR spectrometer (Bruker, Germany). The results are as follows: Figure 5 As shown, the obtained chemical shift and peak shape are consistent with the theoretical structure of H on the target compound 1-vinyl-3-dodecylimidazolium bromide zinc chloride, and no obvious impurity signal was observed.

[0083] 3. The X-ray energy dispersive spectroscopy (EDS) spectrum of 1-vinyl-3-dodecylimidazolium bromide zinc chloride from Example 1 is shown below. Figure 6 As shown, the main elemental peaks of the sample include C, N, Br, Cl, and Zn, which are consistent with the elemental composition of the target compound 1-vinyl-3-dodecylimidazolium bromide zinc chloride. In terms of elemental content, the mass fractions of C, N, Br, Cl, and Zn are 42.09%, 7.9%, 17.94%, 17.22%, and 14.85%, respectively, with corresponding atomic fractions of 70.01%, 11.26%, 4.48%, 9.7%, and 4.54%. The ratio of Zn, Cl, and Br is approximately 1:2:1, consistent with the expected structure of the target compound. Figure 7 Ball-and-stick model of zinc coordination anion and electrospray ionization mass spectrum of IL-Zn.

[0084] 4. Antibacterial performance test of 1-vinyl-3-dodecyl imidazole zinc chloride from Example 1:

[0085] Using *Streptococcus mutans* and *Lactobacillus casei*, which are closely related to the occurrence and development of carious pulpitis, as experimental strains, IL-Zn was cultured in BHI and MRS media at 37°C under facultative anaerobic conditions (90% N2, 5% CO2, 5% H2). IL-Zn showed good antibacterial effects against planktonic bacteria and dual-species biofilms. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the classic clinical bactericide sodium hypochlorite (NaClO), the bacteriostatic agent chlorhexidine (CHX), and IL-Zn in this example were tested. The comparison of the MIC and MBC results of the three antibacterial agents against *S. mutans* and *L. casei* is shown in Table 1.

[0086] Table 1. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MCCs) of three antimicrobial agents against *S. mutans* and *L. casei*.

[0087]

[0088] Figure 8 and Figure 9 The figures show the time-killing curves of three antibacterial agents against *S. mutans* and *L. casei* at MBC. At MBC, the bactericidal rate against airborne bacteria was faster than that of CHX, but significantly slower than that of NaClO. The minimum inhibitory concentration of IL-Zn for biofilm formation was 2.61 μg / mL. Figure 10 This is a schematic diagram showing the minimum inhibitory concentrations (MICs) for three antibacterial agents to inhibit biofilm formation. Figure 11 Table 2 shows the minimum biofilm removal concentrations of the three antimicrobial agents. The minimum biofilm inhibition concentrations and minimum biofilm removal concentrations of the three antimicrobial agents for bimicrobial biofilm formation are shown in Table 2.

[0089] Table 2. Minimum inhibitory concentrations (MICs) and minimum biofilm removal concentrations (MRCs) of three antimicrobial agents against dual-species biofilm formation.

[0090]

[0091] S. mutans and L. casei bacterial suspensions were diluted to 1×10⁻⁶ with 1% BHIS. 7 CFU / mL, then mixed homogeneously in 1% BHIS at a 1:1 ratio. Place cell smears into 24-well plates and add 1 mL of a 1×10⁻⁶ CFU / mL solution. 7 CFU / mL mixed bacterial culture was inoculated into 24-well plates and cultured at 37°C under facultative anaerobic conditions for 24 h. After biofilm formation, the supernatant was discarded, and the cells were gently washed with sterile PBS. The minimum bactericidal concentrations of IL-Zn, CHX, and NaClO were added, and the cells were cultured at 37°C for 1 h. Afterward, the culture medium was aspirated from each well, and the cell smears containing the dual-species biofilm were gently washed twice with PBS. Following the bacterial live / dead staining kit instructions, green fluorescent dye DMAO, red fluorescent dye PI, and detection buffer were mixed in a 1:1:8 ratio to prepare the working solution for live / dead staining. 100 μL of the working solution was added, and staining was performed in the dark for 15 min. The working solution was discarded, and excess stain was removed by washing with PBS. The biofilm morphology was observed using a confocal laser scanning microscope. One hour after drug intervention on the biofilm, the biofilm in the blank control group (Control) was dense and concentrated, with a large area occupied by live bacteria. In the NaClO group, dead bacteria accounted for approximately 70.6% of the total bacterial count, in the CHX group approximately 50%, and in the IL-Zn group approximately 64.4%, which was close to that of the NaClO group (P>0.05), but significantly higher than that of the CHX group and the blank control group (Control) (P<0.05). The results are as follows. Figure 12 , 13 As shown, Figure 12 This image shows staining of live and dead bacteria in a biofilm after 1 hour of treatment with three antimicrobial agents at the lowest biofilm clearance concentration. Figure 13 This is a graph showing the ratio of live to dead bacteria in the biofilm after 1 hour of treatment with three antimicrobial agents at the lowest biofilm removal concentration.

[0092] After 15 generations of exposure to CHX at 1 / 2 MIC concentration, the final MIC of *S. mutans* became 4 times the initial MIC. However, after 15 generations of exposure to IL-Zn at 1 / 2 MIC concentration, the MIC remained unchanged. Figure 14 As shown; L. casei showed no change in MIC after 15 generations of exposure to both drugs at 1 / 2 MIC concentrations, as... Figure 15 As shown, this suggests that the risk of bacterial resistance is low when IL-Zn is applied at low concentrations.

[0093] Figure 16The image shows the morphological changes of S. mutans and L. casei after 12 hours of treatment with IL-Zn minimum inhibitory concentration under a scanning electron microscope. After 12 hours of treatment, some Streptococcus mutans showed significant morphological changes, exhibiting shrinkage and deformation. Lactobacillus casei showed less obvious morphological changes than Streptococcus mutans, but changes in bacterial surface roughness were visible.

[0094] Figure 17 This diagram illustrates the reactive oxygen species (ROS) levels of *S. mutans* and *L. casei* after 12 hours of treatment with IL-Zn at the minimum inhibitory concentration (MIC). For *S. mutans*, compared to the control group, the ZnCl2 group showed a slight increase in ROS-positive cells, while the IL-Zn group showed a more significant increase. For *L. casei*, the IL-Zn group showed over 10% ROS-positive cells, while the ZnCl2 group showed little change. These results demonstrate that IL-Zn exerts its antibacterial effect against *S. mutans* and *L. casei* by generating more ROS. Excessive ROS production can induce bacterial DNA oxidative damage and lipid peroxidation, leading to bacterial death.

[0095] 5. Biocompatibility analysis of 1-vinyl-3-dodecylimidazolium bromide zinc chloride from Example 1:

[0096] The cytotoxicity of the drug was detected by the CCK-8 assay, and the results were as follows: Figure 18 As shown, Figure 18 The graph shows the cytotoxicity test results of IL-Zn. When the concentration of the ionic liquid is not higher than 2.61 μg / mL, the cell survival rate is above 80%, indicating that the ionic liquid has good biocompatibility within the effective antibacterial concentration range.

[0097] The biocompatibility of the material was further verified through a hemolysis test, and the results were as follows: Figure 19 As shown, Figure 19 The graph shows the hemolytic activity test results of three antibacterial drugs. At the MIC concentration, the hemolysis rate of NaClO reached over 80%, while the hemolysis rates of CHX and IL-Zn were both below 5%, indicating that zinc-containing ion liquids have good blood compatibility.

[0098] 6. Analysis of the antibacterial properties of the auxiliary capping material:

[0099] (1) Figure 20 The diagram shows the room temperature properties of IL-Zn (1-vinyl-3-dodecyl imidazole zinc chloride) / CMCS (carboxymethyl chitosan) in Examples 1-6. The rheological characteristics of the auxiliary medullary capping materials in Examples 1-6 were evaluated and are shown in Table 3. According to the rheological characteristics, 2% IL-Zn / CMCS and 3% IL-Zn / CMCS have good operability and are suitable for local application.

[0100] Table 3 Rheological characteristics of IL-Zn / CMCS with different mass volume fractions

[0101] IL-Zn / CMCS(w / v) Viscosity (mPa·s) Flow velocity 1％ 12.05±0.15 Faster 2％ 70.48±0.12 Moderate 3％ 299.2±1.6 Slower 4％ 1027.3±3.2 slow 5％ 2830±8 slow 6％ 6158±12 slow

[0102] (2) The combined antibacterial effects of IL-Zn and CMCS on S. mutans in Examples 1-6 and the combined antibacterial effects of IL-Zn and CMCS on L. casei are shown in Tables 4-5.

[0103] Table 4. Combined antibacterial effect of IL-Zn and CMCS against S. mutans

[0104]

[0105] Table 5. Combined antibacterial effect of IL-Zn and CMCS against L. casei

[0106]

[0107]

[0108] The bacterial suspensions of S. mutans and L. casei were diluted to 2 × 10⁻⁶ with the corresponding liquid culture medium. 6 CFU / mL was inoculated into 96-well plates, 100 μL per well. Subsequently, 100 μL of different drugs were added to each well according to the grouping criteria. The final concentration of the bacterial culture in each well was 1 × 10⁻⁶. 6 CFU / mL. After incubation at 37℃ under facultative anaerobic conditions for 24, 48, and 72 hours, the absorbance at OD600 was measured, and the results are as follows: Figure 21 As shown, concentrations of 2% IL-Zn / CMCS and above can maintain antibacterial effects against S. mutans and L. casei for up to 72 hours.

[0109] (3) The checkerboard method showed that the FICI of IL-Zn and CMCS in combination was 0.83, indicating that the antibacterial properties of the two in combination had an additive effect. Table 6 shows the FICI of IL-Zn and CMCS against S. mutans and L. casei.

[0110] Table 6. FICI of IL-Zn and CMCS on S. mutans and L. casei

[0111]

[0112] 7. Biocompatibility analysis of 1-vinyl-3-dodecyl imidazole zinc chloride combined with carboxymethyl chitosan:

[0113] L929 cells were seeded into 24-well plates containing cell spreaders at a density of 5 × 10⁶ cells / well.4 Cells were incubated in wells at 37°C with 5% CO2 for 6 hours before seeding, with different concentrations of IL-Zn / CMCS added to each well. The cells were then removed from the slides, rinsed three times with PBS buffer, and fixed with 2.5% glutaraldehyde solution for 12 hours. Cells were then dehydrated sequentially with ethanol at gradient concentrations of 30%, 50%, 70%, 90%, 100%, and 100%. Cell morphology was observed using scanning electron microscopy. The cell morphology of the blank control group, the 2% IL-Zn / CMCS group, and the 3% IL-Zn / CMCS group was observed under a microscope, and quantitative analysis was performed. 3% IL-Zn / CMCS significantly affected the adhesion of L929 cells, as shown in the results below. Figure 22 and Figure 23 As shown in the figure. Further observation of L929 cell adhesion using scanning electron microscopy indicated that 2% IL-Zn / CMCS had no significant effect on L929 cell adhesion; the cells spread well and exhibited good biocompatibility. The results are as follows. Figure 24 As shown.

[0114] 8. In vivo efficacy verification analysis of auxiliary medullary capping materials:

[0115] A bacterial pulp infection model was established in SD rats as follows: Eighteen 10-week-old male SPF-grade SD rats with intact dentition, weighing 300-380g, were used. Thirty-six maxillary first molars were randomly divided into three groups: a blank control group, a control group, and an experimental group (IL-Zn / CMCS from Example 2), with 12 teeth in each group for subsequent experimental studies. 1×10⁻⁶ teeth were prepared in advance. 5 A suspension of *Streptococcus mutans* and *Lactobacillus casei* at CFU / mL was prepared. Rats were weighed and anesthetized. After anesthesia took effect, the rats were fixed in a supine position. The oral cavity and target tooth were disinfected with iodine-soaked cotton balls. Using an FG 1 / 4 ball bur, intermittent drilling was performed on the occlusal surface of the maxillary first molar in the central fossa until the bottom of the fossa turned pink. A 40# K-file was used to apply pressure to create a pulp foramen, opening and exposing the pulp cavity. The pulp cavity was cleaned with small cotton balls of physiological saline and then dried with dry cotton balls. 5 μL of 1×10⁻⁶ CFU / mL solution was added. 5 A bacterial pulp injury model was established using a CFU / mL suspension of *Streptococcus mutans* and *Lactobacillus casei*. In the blank control group, no bacteria were added; the pulp was capped directly with iRoot BP Plus after drilling, followed by light-cured glass ionomer filling. In the experimental group (IL-Zn / CMCS), bacteria were added, 2% IL-Zn / CMCS was applied, followed by iRoot BP Plus pulp capping and light-cured glass ionomer filling. Figure 25 This is a schematic diagram of an animal experiment.

[0116] Four weeks post-surgery, SD rats were euthanized, and their maxillary dentition along with the maxilla was removed, fixed, decalcified, embedded, sectioned, and stained with hematoxylin and eosin (HE) for observation. Figure 26 As shown, in the blank control group, calcified bridges formed under pulp capping agent, with no obvious inflammatory cell infiltration and good root pulp condition; in the control group, the coronal pulp showed extensive necrosis, reaching the root pulp, with abundant inflammatory cell infiltration in the root pulp; in the IL-Zn / CMCS group, a small amount of inflammatory cell infiltration and calcified bridge formation were observed in the pulp wound after treatment, but no obvious tissue destruction was observed in the coronal pulp and root pulp. The detailed criteria for pulp histological scoring are shown in Table 7.

[0117] Table 7. Detailed Scoring Rules for Pulp Histology

[0118]

[0119] Table 8 shows the statistical results of pulp histology scores for Example 2, Control Group 1, and Control Group 2.

[0120] Table 8. Statistical analysis of pulp histological scores for each group

[0121]

[0122]

[0123] Note: Same letters indicate no statistically significant difference (p>0.05), different letters indicate a statistically significant difference (p<0.05).

[0124] The above results demonstrate that bacterial infection control is crucial in pulp preservation, and that 2% IL-Zn / CMCS can exert an antibacterial effect in vivo to reduce pulp damage caused by bacteria.

[0125] Figure 27 This is a schematic diagram showing the HE staining results of major organs in each group 4 weeks post-surgery. No significant abnormalities were observed in the heart, liver, spleen, lungs, and kidneys of rats in each group, suggesting that the IL-Zn / CMCS in Example 2 has good biocompatibility for local application.

[0126] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A zinc-containing ionic liquid-modified chitosan auxiliary medullary capping material, characterized in that, Including solid and liquid phases; The solid phase is carboxymethyl chitosan; The liquid phase is 1-vinyl-3-dodecylimidazolium bromide zinc chloride; The structural formula of the 1-vinyl-3-dodecylimidazolium bromide zinc chloride is shown in Formula 1:

2. The auxiliary medullary capping material of chitosan modified with zinc ionic liquid according to claim 1, characterized in that, The weight-to-volume ratio concentration of the solid phase to the liquid phase ranges from 1% to 6%.

3. A method for preparing an auxiliary medullary capping material of chitosan modified with zinc ionic liquid, characterized in that, Includes the following steps: S1. Add 1-vinylimidazol to 1-bromododecane and mix thoroughly to produce 1-vinyl-3-dodecylimidazolium bromide. S2. Add ZnCl2 to the 1-vinyl-3-dodecyl imidazole bromide and react to obtain 1-vinyl-3-dodecyl imidazole zinc chloride liquid; S3. The carboxymethyl chitosan is added to 1-vinyl-3-dodecyl imidazole zinc chloride liquid and mixed evenly to obtain the auxiliary medullary capping material.

4. The method for preparing the auxiliary medullary capping material of chitosan modified with zinc ionic liquid according to claim 3, characterized in that, In step S1, the preparation of 1-vinyl-3-dodecyl imidazole bromide includes the following steps: The product obtained by mixing 1-vinylimidazolium and 1-bromododecane in ethyl acetate, washing with ethyl acetate and drying, yields 1-vinyl-3-dodecylimidazolium bromide.

5. The method for preparing the auxiliary medullary capping material of chitosan modified with zinc ionic liquid according to claim 3, characterized in that, The 1-vinyl-3-dodecyl imidazole bromide comprises the following raw material components in parts by weight: 4-5 parts of 1-vinylimidazole; 10-15 parts of 1-bromododecane.

6. The method for preparing the auxiliary medullary capping material of zinc-containing ionic liquid modified chitosan according to claim 3, characterized in that, In step S1, the reaction temperature is 40℃-50℃ and the reaction time is 40h-60h.

7. The method for preparing the auxiliary endothelial material of chitosan modified with zinc ionic liquid according to claim 3, characterized in that, In step S2, the 1-vinyl-3-dodecylimidazolium bromide zinc chloride comprises the following raw materials in parts by weight: 8-12 parts of 1-vinyl-3-dodecyl imidazole bromide; ZnCl2 3 parts - 5 parts.

8. The method for preparing the auxiliary medullary capping material of chitosan modified with zinc ionic liquid according to claim 3, characterized in that, In step S2, the reaction temperature is room temperature and the reaction time is 20-30 hours.

9. The application of the zinc ionic liquid-modified chitosan auxiliary capping material according to any one of claims 1-2 in the preparation of antibacterial products.

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