Ionic liquid anti-caries coating with dual functions of inhibiting bacteria and promoting mineralization and preparation method thereof

Abstract

The invention discloses an ionic liquid anti-caries coating with dual functions of bacteriostasis and mineralization promotion and a preparation method thereof, belongs to the technical field of dental caries prevention in stomatology, and aims to overcome the defects that an existing anti-caries material fluoride is insufficient in bacteriostasis effect and relatively short in residence time on a tooth surface. And the risk of oral flora imbalance caused by the generation of drug resistance possibly caused by antibacterial mouthwash is also caused. According to the invention, alkyl imidazole is taken as a main body, is paired with ions such as strontium and fluorine with a mineralization promoting function, is modified by a silane coupling agent, and is properly added with an excipient, so that the constructed ionic liquid coating can be adhered to the surfaces of teeth for a long time, the dual effects of bacteriostasis and mineralization promoting can be durably exerted, and a more excellent anti-caries effect can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an ionic liquid anti-caries coating with dual functions of bacteriostasis and mineralization promotion and a preparation method thereof. Background Art

[0002] Dental caries is an infectious disease mediated by plaque biofilm, affected by various factors, and with a dynamic imbalance between demineralization and remineralization of dental hard tissues. The results of the fourth national oral health epidemiological survey show that the caries prevalence rate in people over 35 years old is more than 90%. Under special conditions such as military training and exercises, due to high-intensity workloads and poor living conditions, oral hygiene cannot be effectively maintained, and the risk of dental caries in special populations is further increased; in special environments such as high altitude with low oxygen and high cold, the gene expression or protein metabolism of Streptococcus mutans changes, its cariogenic ability is enhanced, and the risk of dental caries is increased.

[0003] Clinically, fluoride is the most commonly used remineralization material, and its application forms can be divided into two types: systemic fluoride use and topical fluoride use. Among them, topical fluoride use is one of the most widely used anti-caries measures at present, and the main routes include fluoride toothpaste, fluoride gel, fluoride foam, and fluoride coating, etc. The anti-caries mechanism of fluoride mainly includes three aspects: reducing the solubility of enamel, promoting the remineralization of demineralized enamel, and inhibiting bacterial metabolism and acid production. Combining fluoride with antibacterial materials such as chlorhexidine, traditional Chinese medicine extracts, and antibacterial peptides can further improve the anti-caries effect and reduce the incidence of dental caries. Although the significant effect of the combined use of fluoride and antibacterial materials in the prevention and treatment of dental caries has been confirmed, there are still the following problems: 1. The long-term use of chlorhexidine will lead to an imbalance in the oral microecology and even cause serious bacterial drug resistance problems; 2. There are a wide variety of traditional Chinese medicine extracts, and their action mechanisms are not clear, which to a certain extent limits their development and application; 3. Antibacterial peptides have unstable structures and high manufacturing costs, and related research is still in the in vitro experiment stage, and their safety and effectiveness remain to be further explored.

[0004] The main mechanism of fluoride in preventing dental caries is to replace the hydroxyl groups in hydroxyapatite crystals to form more stable fluorohydroxyapatite or fluorapatite, reduce the solubility of enamel, and form fluorapatite deposition on the enamel surface to promote the remineralization of demineralized enamel. However, fluoride still has certain defects in preventing dental caries. On the one hand, it does not have good antibacterial function and has limited effect on cariogenic bacteria. On the other hand, clinically used fluoride only prolongs the residence time of the material on the tooth surface through fluoride varnish and can only remain for 12 - 24 hours under saliva flushing, which weakens the caries prevention effect of fluoride to a certain extent. In addition, mouthwash is also a commonly used method for preventing dental caries. However, the active ingredients in antibacterial mouthwash can cause the generation of bacterial drug resistance and bring the risk of oral flora imbalance, so it cannot be used for a long time. Therefore, finding a safe and highly effective antibacterial material and integrating it with remineralization materials is the key to effectively preventing and managing dental caries. Summary of the Invention

[0005] In view of the above problems, the present application provides an ionic liquid anti-caries coating with dual functions of antibacterial and mineralization promotion and a preparation method thereof. The antibacterial function is exerted by alkylimidazole cations, fluoride ions and strontium ions are introduced to play a role in promoting mineralization, and it is modified by a silane coupling agent to form a strong chemical bond with the tooth surface, effectively prolonging the residence time of the coating on the tooth surface and exerting a long-term "antibacterial - mineralization promotion" anti-caries effect.

[0006] First, the present invention provides a preparation method of an ionic liquid anti-caries coating with dual functions of antibacterial and mineralization promotion, and the preparation method includes:

[0007] Step 1: Synthesize alkylimidazole cations with different chain lengths and screen the alkylimidazole cations;

[0008] Step 2: Introduce strontium ions or fluoride ions into the alkylimidazole cation liquid system screened in Step 1 to synthesize a composite ionic liquid;

[0009] Step 3: Blend rosin with the composite ionic liquid to prepare an ionic liquid anti-caries coating.

[0010] In some preferred examples of this aspect, the specific process of Step 1 includes:

[0011] Step 101: Weigh 3-chloropropyltriethoxysilane and 1-hexylimidazole bromide in a molar ratio of 1:1 and slowly add them to a single-neck flask, add ethyl acetate, and under nitrogen protection, stir magnetically at 70 °C for 48 h to obtain a yellowish-brown liquid product;

[0012] Step 102: Wash several times with ethyl acetate and vacuum dry to a viscous state to obtain 1-hexyl-3-propyltriethoxysilaneimidazole chloride;

[0013] Step 103: Using 1-octylimidazole bromide and 1-decylimidazole bromide as reactants, prepare 1-octyl-3-propyltriethoxysilane imidazole chloride and 1-decyl-3-propyltriethoxysilane imidazole chloride respectively according to Steps 101-102;

[0014] Step 104: Analyze the antibacterial properties and cytotoxicity of 1-hexyl-3-propyltriethoxysilane imidazole chloride, 1-octyl-3-propyltriethoxysilane imidazole chloride, and 1-decyl-3-propyltriethoxysilane imidazole chloride, and screen 1-hexyl-3-propyltriethoxysilane imidazole chloride for subsequent reactions.

[0015] In some preferred examples in this regard, the specific process of Step 2:

[0016] Weigh 1-hexyl-3-propyltriethoxysilane imidazole chloride and potassium hexafluorophosphate according to a mass ratio of 5:2.3 and add them to a flask. Then add acetone, and magnetically stir the reaction at room temperature for 6 hours. After that, add chloromethane. After observing the formation of a white precipitate, centrifuge at 12000 rpm for 5 min, collect the precipitate, and vacuum dry it to a viscous state to obtain 1-hexyl-3-propyltriethoxysilane imidazole hexafluorophosphate.

[0017] In some preferred examples in this regard, the specific process of Step 2:

[0018] Weigh 1-hexyl-3-propyltriethoxysilane imidazole chloride and sodium fluoride according to a mass ratio of 10:1 and add them to a flask. Then add acetone, and magnetically stir the reaction at room temperature for 6 hours. After that, add chloromethane. After observing the formation of a white precipitate, centrifuge at 12000 rpm for 5 min, collect the precipitate, and vacuum dry it to a viscous state to obtain 1-hexyl-3-propyltriethoxysilane imidazole fluoride.

[0019] In some preferred examples in this regard, the specific process of Step 2:

[0020] Weigh 1-hexyl-3-propyltriethoxysilane imidazole chloride and strontium chloride according to a mass ratio of 5:2 and add them to a flask. Then add acetone, and magnetically stir the reaction at room temperature for 6 hours. After that, add absolute ethanol, and carry out a condensation reflux reaction at 50 °C overnight. Centrifuge at 8000 rpm for 5 min to remove the precipitate, collect the supernatant, first evaporate to remove the solvent, and then place it in a vacuum and dry it to a viscous state to obtain 1-hexyl-3-propyltriethoxysilane imidazole strontium chloride complex.

[0021] In a second aspect, the present invention provides an ionic liquid anti-caries coating with dual functions of antibacterial and promoting mineralization, which is prepared by using a preparation method of an ionic liquid anti-caries coating with dual functions of antibacterial and promoting mineralization.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention utilizes the strong designability of ionic liquids. With alkyl imidazole as the main body, it is paired with strontium, fluorine and other ions with the function of promoting mineralization, modified with silane coupling agent, and appropriately added with excipients, so that the constructed ionic liquid coating can adhere to the tooth surface for a long time and play the dual functions of "bacteriostasis - promoting mineralization" persistently, achieving a more excellent anti - caries effect.

[0024] Compared with the anti - caries materials that only play the function of bacteriostasis or promoting mineralization alone, the anti - caries material of the present invention has the dual functions of bacteriostasis and promoting mineralization. At the same time, through effective means, the action time of the anti - caries material is extended, and the material does not produce bacterial drug resistance, and has good biological safety. Brief Description of the Drawings

[0025] Figure 1 It is the synthesis process of three kinds of alkyl imidazoles with different chain lengths.

[0026] Figure 2 It is the bacteriostasis of alkyl imidazole cations; A - Streptococcus mutans; B - Streptococcus sanguinis; C - Actinomyces viscosus.

[0027] Figure 3 It is the cytotoxicity of alkyl imidazole cations; A - 24h; B - 48h; C - 72h.

[0028] Figure 4 It is the anion replacement and balancing diagram.

[0029] Figure 5 It is the mineralization - promoting performance of the ionic liquid.

[0030] Figure 6 It is the mineralization - promoting performance of the ionic liquid.

[0031] Figure 7 It is the cytotoxicity of the ionic liquid.

[0032] Figure 8 It is the colony - counting result after drug treatment.

[0033] Figure 9 It is the transmission electron microscope image of bacteria after treatment with ionic liquid.

[0034] Figure 10 It is the scanning electron microscope image of bacteria after treatment with ionic liquid.

[0035] Figure 11 It is the scanning electron microscope image of bacterial biofilm after treatment with ionic liquid.

[0036] Figure 12 It is the laser confocal microscope image of live / dead staining of bacterial biofilm after treatment with ionic liquid.

[0037] Figure 13It is the mixing effect diagram of rosin and ionic liquid.

[0038] Figure 14 It is the laser confocal microscopy image of live / dead staining of bacterial biofilm on the surface of the ionic liquid anti-caries coating.

[0039] Figure 15 It is the scanning electron microscopy image of the enamel block after being immersed in physiological saline for 12 h after being treated with ionic liquid.

[0040] Figure 16 It is the release curves of F / Sr in Duraphat and ionic liquid coatings.

[0041] Figure 17 It is the Keyes score result after applying anti-caries materials to rat molars.

[0042] Figure 18 It is the QLF detection result after applying anti-caries materials to rat molars.

[0043] Figure 19 It is the HE staining result of the buccal mucosa and important organs after applying anti-caries materials to rat molars. Detailed implementation manners

[0044] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0045] Silane coupling agents are a class of compounds with dual characteristics. Their molecular structure contains two different functional groups. One can chemically react with organic materials, and the other can form strong adsorption or chemical bond binding with the surface of inorganic substances. This unique property makes silane coupling agents show excellent effects in improving the bonding strength between different materials and are widely used in improving the performance of adhesives. Therefore, ionic liquids can be modified with silane coupling agents to form covalent bonds with the tooth surface, thereby extending the effective action time of anti-caries materials.

[0046] Based on the above research background, the present invention utilizes the strong designability of ionic liquids. Taking alkylimidazole ionic liquids as the main body, they are modified with silane coupling agents to synthesize alkylsilylimidazole cations, and then paired with anions with remineralization functions such as strontium and fluorine. Finally, excipients with the function of delaying drug release are added. By using the bacteriostatic property of cations, the mineralization-promoting effect of anions, the chemical bond binding of silane coupling agents, and the drug slow-release function of excipients, an ionic liquid coating with dual "bacteriostatic - mineralization-promoting" effects is prepared, and an excellent anti-caries effect is exerted by extending the residence time of the material on the tooth surface.

[0047] The preparation method of the ionic liquid coating with dual "bacteriostatic - mineralization-promoting" effects is as follows:

[0048] Step 1: Synthesis and screening of cationic materials

[0049] As Figure 1 shown, the synthesis processes of three different chain-length alkyl imidazoles include:

[0050] 3-chloropropyltriethoxysilane (Aladdin) and 1-hexylimidazole bromide (Aladdin) with a molar ratio of 1:1, and 30 mL of ethyl acetate (Aladdin) were slowly added to a single-necked flask. Under nitrogen protection, the reaction was magnetically stirred at 70 °C for 48 h to obtain a yellowish-brown liquid product. The product was washed several times with ethyl acetate and dried under vacuum until it became viscous to obtain 1-hexyl-3-propyltriethoxysilaneimidazole chloride.

[0051] 1-octyl-3-propyltriethoxysilaneimidazole chloride and 1-decyl-3-propyltriethoxysilaneimidazole chloride were prepared in the same way.

[0052] The antibacterial properties (Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus) and cytotoxicity of the three alkyl imidazole cations were investigated:

[0053] Investigation on the antibacterial effects of the three imidazole cations against different cariogenic bacteria: The microbroth dilution method was used to determine the antibacterial properties of the synthesized imidazole cations against three common cariogenic bacteria, namely Streptococcus mutans (S. mutans), Streptococcus sanguis (S. sanguis), and Actinomyces viscosus (A. viscosus). The positive control group selected sodium fluoride with the same molar concentration. The bacterial suspension was diluted to 2×10 6 CFU / mL with BHI liquid medium. 100 μL of the diluted bacterial solution was added to a 96-well plate. Through preliminary experiments, a suitable concentration gradient was explored. 100 μL of the ionic liquid composed of various components was added to each well. Sodium fluoride with the same concentration was used as the positive control, DDW + bacterial solution as the negative control, and DDW + BHI medium as the blank control. Each group was set with 5 sub-wells. The 96-well plate was placed in an incubator at 37 °C with 5% CO2 for 24 h. The OD600 values of each group were measured with an enzyme-linked immunosorbent assay (ELISA) reader. The bacterial solution in the wells was spread on plates for counting, and the MIC and MBC values of the imidazole cations were calculated.

[0054] Investigation on the effects of the three imidazole cations on inhibiting the formation of plaque biofilms: Streptococcus mutans was diluted to 2×10 6 CFU / mL with BHI liquid medium containing 1% sucrose (BHIS). 100 μL of the bacterial suspension was inoculated onto a 96-well plate, and then 100 μL of 0.5 MIC, MIC, and 2 MIC imidazole cations were added. The mixture was cultured at 37 °C for 24 h. The crystal violet staining method was used to detect the OD595 value of the biofilm to evaluate the ability of the three imidazole cations to inhibit the formation of single-species plaque biofilms.

[0055] CCK-8 cytotoxicity assay: L929 cells were selected for cytotoxicity assay. According to the MIC and MBC values, concentration gradients were set, and the CCK-8 assay method was used to investigate the toxic effects of imidazole-based cations on L929 cells. L929 cells were seeded in 96-well plates and incubated overnight; ionic liquids were added and co-incubated for 24, 48, and 72 h; after the incubation time was reached, the medium was replaced with FBS-free medium containing 10% CCK-8 and incubated for 2 - 4 h; the absorbance of each well in the culture plate at 450 nm was read using a microplate reader and the relative cell viability was calculated.

[0056] The results are as Figure 2 and Figure 3 shown. The results indicate that the antibacterial activity of hexylimidazole cation: octylimidazole cation > hexylimidazole cation > decylimidazole cation; cytotoxicity: decylimidazole cation > octylimidazole cation > hexylimidazole cation. Considering the antibacterial activity and safety of the materials, hexyl and octylimidazole cations were selected for subsequent experiments.

[0057] Step 2: Introduce strontium ions and fluoride ions into the ionic liquid system to synthesize composite ionic liquids

[0058] Taking hexylimidazole cation as an example:

[0059] IL6(PF6): Weigh 1 g of 1-hexyl-3-propyltriethoxysilaneimidazolium chloride and 0.46 g of potassium hexafluorophosphate (Aladdin) into a flask, add 20 mL of acetone and stir magnetically at room temperature for 6 hours, then add 2 mL of chloromethane (Aladdin). After observing the formation of white precipitate, centrifuge at 12000 rpm for 5 min, collect the precipitate, and vacuum dry it to a viscous state to obtain 1-hexyl-3-propyltriethoxysilaneimidazolium hexafluorophosphate.

[0060] IL6(F): Weigh 1 g of 1-hexyl-3-propyltriethoxysilaneimidazolium chloride and 0.1 g of sodium fluoride (Aladdin) into a flask, add 20 mL of acetone (Aladdin) and stir magnetically at room temperature for 6 hours, then add 2 mL of chloromethane. After observing the formation of white precipitate, centrifuge at 12000 rpm for 5 min, collect the precipitate, and vacuum dry it to a viscous state to obtain 1-hexyl-3-propyltriethoxysilaneimidazolium fluoride.

[0061] IL6(Sr): Weigh 1 g of 1-hexyl-3-propyltriethoxysilaneimidazolium chloride and 0.4 g of strontium chloride (Aladdin) into a flask, add 30 mL of absolute ethanol (Aladdin), reflux with condensation at 50 °C overnight, centrifuge at 8000 rpm for 5 min to remove the precipitate, collect the supernatant, evaporate the solvent, and then place it in a vacuum dryer until it becomes viscous to obtain 1-hexyl-3-propyltriethoxysilaneimidazolium strontium chloride complex.

[0062] Replacing the above-mentioned process with octylimidazole cations can obtain 1-octyl-3-propyltriethoxysilane imidazole hexafluorophosphate (IL8(PF6)), 1-octyl-3-propyltriethoxysilane imidazole fluoride (IL8(F)), and 1-octyl-3-propyltriethoxysilane imidazole strontium chloride complex (IL8(Sr)).

[0063] The antibacterial, cytotoxic, and mineralization-promoting properties of the prepared ionic liquid materials were investigated:

[0064] The detection methods for antibacterial and cytotoxic properties were the same as above.

[0065] Detection method for mineralization-promoting property: Maxillary third molars newly extracted without obvious caries and defects were collected from the outpatient department of oral and maxillofacial surgery and prepared into enamel blocks of 5mm * 5mm * 2mm, which were polished, cleaned, and dried. After acid-etching the surface with 37% phosphoric acid for 30s, rinsed and dried, they were respectively immersed in ionic liquid solutions composed of three anions at 250 mM for 5 min, once in the morning and once in the evening. 1% sodium fluoride was used as the positive control, and DDW was used as the negative control. The rest of the time, they were immersed in artificial saliva, and the artificial saliva was changed every day. Before each change of the liquid, the surface of the sample was thoroughly rinsed with DDW for 1 min. After 7 days, the changes in the surface structure and mechanical properties of human tooth enamel before and after demineralization and after remineralization treatment were detected by scanning electron microscopy and microhardness tester to compare the differences in the effects of ionic liquids with three anion components on the remineralization of demineralized enamel.

[0066] Table 5 Antibacterial properties of ionic liquids

[0067]

[0068] The results showed that the antibacterial properties of IL6(Sr), IL6(F), IL6(PF6), IL8(Sr), and IL8(F) were basically the same, all slightly weaker than IL8, and the antibacterial effect of IL8(PF6) was relatively the worst.

[0069] The results of the mineralization-promoting function are as Figure 5 shown: IL(Sr) and IL(F) have a certain mineralization-promoting function, while the mineralization-promoting effect of IL(PF6) is not obvious.

[0070] Figure 6 What is shown is the recovery rate of the surface hardness of enamel, which can reflect the mineralization of enamel. The results showed that after treatment with 1% sodium fluoride and IL(F), the surface hardness of enamel increased significantly.

[0071] Figure 7 What is shown is the toxicity of ionic liquid materials with various combinations of cations and anions to cells.

[0072] Combined with the antibacterial, mineralization-promoting and cytotoxicity results of the materials, the most suitable cation-anion composition was screened. Using hexamethylimidazole as the cation, Sr and F were introduced through anion balancing and anion substitution to prepare two ionic liquids, IL6(Sr) and IL6(F), as the anti-caries coatings of the present invention for subsequent experiments and functional investigations.

[0073] Antibacterial effects of IL6(Sr) and IL6(F) against Streptococcus mutans, Streptococcus sanguinis and Actinomyces viscosus:

[0074] After Streptococcus mutans was cultured to the logarithmic growth phase, it was diluted with sterile BHI liquid medium to a bacterial suspension of 2×10 6 CFU / mL. The diluted bacterial suspension was mixed with 50 μM IL6, IL6(Sr) and IL6(F) (final concentration was MIC) in equal volumes and placed in a 37°C constant temperature water bath for 60 min. Sodium fluoride and SrCl2 at the same concentration were used as controls. After incubation for the specified time, drop plate counting was performed. The time-kill curve experimental method for Streptococcus sanguinis and Actinomyces viscosus was the same as above. Figure 8 It shows that the ionic liquid has good antibacterial effects.

[0075] Streptococcus mutans was cultured to the logarithmic growth phase at 37°C and 5% CO2. Take 0.5 mL of the bacterial suspension and 0.5 mL of 50 μM ionic liquid and place them in a centrifuge tube. DDW was used as the blank control and continued to be cultured at 37°C and 5% CO2 for 4 h. Centrifuge at 3000 rpm / min for 5 min, discard the supernatant, fix the bacterial precipitate with 2.5% glutaraldehyde, embed it in resin, make ultra-thin sections, and observe the morphology and cell integrity of Streptococcus mutans by transmission electron microscopy after staining.

[0076] Place a cell slide at the bottom of a 24-well plate, dilute Streptococcus mutans with BHI liquid medium to 1×10 8 CFU / mL, add 1 mL of the bacterial suspension and 1 mL of 50 μM ionic liquid to each well, and DDW was used as the blank control. Place the 24-well plate in an environment of 37°C and 5% CO2 and culture for 4 h. Fix with 2.5% glutaraldehyde, dehydrate with gradients of 50%, 60%, 70%, 80%, 90%, 95% and 100% ethanol, and observe the morphology and cell integrity of Streptococcus mutans by scanning electron microscopy.

[0077] Place a cell slide at the bottom of a 24-well plate, add 1 mL of each well diluted to 1×10 with BHIS liquid medium containing 1% sucrose 6A suspension of Streptococcus mutans at a concentration of CFU / mL was cultured in an environment of 37°C and 5% CO2 for 15 h. The medium was changed, and 1 mL of 25 μM ionic liquid was added to each well. BHI liquid medium served as the negative control, and the culture continued for 4 h. It was fixed with 2.5% glutaraldehyde and dehydrated step by step with 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol. The morphology of the Streptococcus mutans biofilm was observed by scanning electron microscopy.

[0078] Through Figure 9 , 10 , 11, it can be seen that after treatment with the ionic liquid, the bacterial structure was damaged and lysis and death occurred.

[0079] A cell slide was placed at the bottom of a 24-well plate. 1 mL of a suspension of Streptococcus mutans diluted to 1x10 6 CFU / mL with BHIS liquid medium containing 1% sucrose was added to each well. It was cultured in an environment of 37°C and 5% CO2 for 15 h. The medium was changed, and 1 mL of 25 μM ionic liquid was added to each well. BHI liquid medium served as the negative control, and the culture continued for 4 h. It was stained with a LIVE / DEAD detection kit, and the biofilm was observed by a laser confocal scanning microscope.

[0080] Through Figure 12 , it can be seen that after treatment with the ionic liquid, the proportion of live bacteria decreased and the number of dead bacteria increased.

[0081] Step 3: Using rosin as a shaping agent, rosin and the composite ionic liquid were blended to prepare an ionic liquid anti-caries coating with a concentration of 0.5 M (where rosin was 1 g / mL and absolute ethanol was 1 g / ml).

[0082] The appearance of the prepared ionic liquid anti-caries coating was as Figure 13 shown.

[0083] A cell slide was placed at the bottom of a 24-well plate. 0.2 mL of the coating was added with a syringe, artificial saliva was added, and it was incubated at 37°C for 2 h. Then, 1 mL of a suspension of Streptococcus mutans diluted to 1x10 7 CFU / mL with BHIS liquid medium containing 1% sucrose was added to each well. It was cultured in an environment of 37°C and 5% CO2 for 24 h. BHI liquid medium served as the negative control. It was stained with a LIVE / DEAD detection kit, and the ability of the material to inhibit biofilm formation was observed by a laser confocal scanning microscope. Through Figure 14 , it can be seen that the proportion of live bacteria on the surface of the ionic liquid anti-caries coating decreased, and the number of dead bacteria increased significantly.

[0084] A layer of anti-caries coating was applied to one side surface of the cell smear slide, immersed in 10 mL of DDW, with the material side facing down, placed at 37 °C for 12 h, and the surface morphology of the coating film was observed by scanning electron microscopy. It was found that the pores of Duraphat were fewer and scattered, while the rosin ionic liquid coating could form densely distributed pores after hardening into a film in water, as Figure 15 shown.

[0085] A small amount of ionic liquid material was aspirated with a 1 mL syringe and applied to one side surface of the slide, immersed in 10 mL of DDW, with the material side facing down, placed at 37 °C. The leaching solutions were taken out at 3 h, 6 h, 12 h, and 24 h respectively, and the concentrations of F - and Sr 2+ were measured by ion chromatography and inductively coupled plasma spectrometry respectively. The slide without the coated material was used as the blank group.

[0086] The calculation method and formula for the fluoride release rate are as follows:

[0087] Fluoride release amount (mg) = (fluoride ion concentration in the soaking solution - average value of fluoride ion concentration in the blank control) * volume of the soaking solution

[0088] Fluoride dissolution rate (%) = (fluoride release amount / fluoride mass in the coating film) * 100%

[0089] The method for measuring the strontium ion release rate is the same as above.

[0090] The results are as Figure 16 shown. At 12 h, the fluoride release rate of the fluoride-containing ionic liquid coating reached about 40%, while that of Duraphat was only about 20%. The strontium release rate of the strontium-containing ionic liquid coating was relatively low, which might be related to the formation of coordination bonds by strontium ions.

[0091] Aiming at the disadvantages of existing anti-caries materials such as insufficient antibacterial effect of fluorides and short residence time on the tooth surface, as well as the risk of drug resistance and oral flora imbalance caused by antibacterial mouthwashes, the anti-caries coating prepared by the present invention has the dual functions of "antibacterial-promoting remineralization", is not easy to produce drug resistance, and has good biological safety. This anti-caries coating can adhere to the tooth surface for a long time and continuously exert the dual functions of "antibacterial-promoting remineralization" by utilizing the antibacterial property of cations, the remineralization effect of anions, the chemical bond combination of silane coupling agents, and the drug slow-release function of excipients, and has a more excellent anti-caries effect.

[0092] Next, animal experiments were carried out using the prepared anti-caries materials:

[0093] This study has passed the review of the Animal Ethics Committee of the Air Force Medical University.

[0094] Rat caries model

[0095] SPF-SD male rats at 21 days of age were selected to establish a caries model. They were fed with feed uniformly mixed with sodium ampicillin (1000 g of ordinary feed mixed with 2 g of sodium ampicillin) and drinking water containing potassium penicillin (800,000 units, 1 bottle of potassium penicillin mixed with 200 mL of glucose water) for 5 days to remove the influence of endogenous bacteria. One day later, Streptococcus mutans was inoculated on the occlusal surfaces of the molars of the rats, with a bacterial concentration of 1×10 8 CFU / mL, 600 μL each time, once a day for 5 consecutive days, and high-sugar feeding was given during this period. To determine the inoculation effect of Streptococcus mutans, the molars of the rats were cleaned 1 day later and the materials were coated according to the grouping, and high-sugar feeding was continued for 3 weeks.

[0096] Experimental grouping: The self-control method was adopted. Twelve molars of the upper and lower jaws of the rats were selected for the experiment. The experimental group of rats was coated with ionic liquid anti-caries coatings (three kinds); the positive control group of rats was coated with Duraphat; the blank group of rats was given PBS. There were 5 rats in each group, with a total of 25 rats. The molars of the rats in the blank group were taken X-ray films, and the successful establishment of the model was confirmed by the presence of low-density images.

[0097] Evaluation of anti-caries effect

[0098] At the end of the experiment, the dental plaque on the surface of the molars of the animals was scraped for colony counting; subsequently, the animals were sacrificed and samples were taken to detect the caries lesions of the molars of the experimental animals. The detection indexes included Keyes score and QLF detection.

[0099] Keyes score:

[0100] The jaw bone specimens of the animals were stained in 0.4% murexide solution for 12 h. Using a diamond saw blade with a thickness of 0.1 mm and a diameter of 25 mm, the dentition was cut along the mesial-distal sagittal plane of the jaw bone. Under a stereomicroscope, the caries lesions of the molars were evaluated according to the Keyes caries lesion scoring method. The range and depth of murexide infiltration into the teeth represented the range and severity of the caries lesions, which were divided into four grades:

[0101] Pure enamel caries (E): The caries only exists in the enamel;

[0102] Mild dentin caries (Ds): 1 / 4 of the dentin between the enamel and the pulp chamber roof is invaded by caries;

[0103] Moderate dentin caries (Dm): The caries lesion progresses to 1 / 4 to 3 / 4 of the dentin between the enamel and the pulp chamber roof;

[0104] Extensive dentin caries (Dx): The caries lesion range reaches 3 / 4 of the dentin.

[0105] The scores of all Class E carious lesions on the smooth surfaces of the molars were added up to obtain the total score of Class E carious lesions on the smooth surfaces of this tooth. The same method was used to score the carious lesions of Class E, Ds, Dm, and Dx in the pits and fissures. The scoring process ensured single blinding. All scores were double-evaluated by two persons, and the average of the scores of the two persons was taken as the final score for statistical analysis.

[0106] Figure 17 The results showed that the depth and scope of carious lesion staining on the smooth surfaces and in the pits and fissures of the molars of the rats in the blank group were deeper and more extensive than those in other groups. There were carious lesion manifestations such as discontinuous enamel and low-density transmission areas, while there were fewer carious lesion manifestations in the Duraphat group and the group treated with the ionic liquid anti-caries coating. The Keyes scoring method was used to statistically analyze the degree of carious lesions. The results showed that there were significant differences in the degree of carious lesions between the rats in the anti-caries coating group and the blank group. The IL6(F)+rosin treatment group had the best effect, effectively reducing the occurrence frequency and severity of carious lesions on the smooth surfaces and in the pits and fissures.

[0107] QLF detection: The quantitative light-induced fluorescence system was used to take fluorescence images of the animal molars before and after the experiment to observe the caries situation.

[0108] Figure 18 These are the QLF detection results. The dark areas with reduced fluorescence brightness in the QLF fluorescence pictures represent the carious lesion sites of the rat molars. The percentage of fluorescence loss (ΔF, %) and the lesion volume (ΔQ, %px) of each specimen were measured through the system analysis software to determine the severity of carious lesions. The results showed that the ionic liquid anti-caries coating could effectively reduce the severity of carious lesions in rat molars. Among them, the IL6(F)+rosin treatment group had the least carious lesion area and the lightest fluorescence loss, and the anti-caries effect was the best.

[0109] Biological safety

[0110] Macroscopically observe whether there is inflammation or injury on the buccal mucosa of the experimental animals. Take the buccal mucosa (about 2 mm×4 mm) corresponding to the molars of the rats in each group and the tissues of the important organs such as the heart, liver, spleen, lungs, and kidneys for HE staining to observe whether there are pathological changes.

[0111] Figure 19 It was shown that there were no pathological changes in the buccal mucosa, heart, liver, spleen, lungs, and kidneys of the rats in each group.

[0112] The above are only several embodiments of the present application, and do not impose any form of limitation on the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any person skilled in the relevant art can make some changes or modifications within the scope of the technical solution of the present application by using the disclosed technical content, which are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. A method for preparing an ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization, characterized in that: The preparation method comprises: Step 1: synthesizing alkyl imidazolium cations with different chain lengths and screening the alkyl imidazolium cations; Step 2: introducing strontium ions or fluoride ions into the alkyl imidazolium cation liquid system screened in step 1 to synthesize a composite ionic liquid; Step 3: Blending rosin with the composite ionic liquid to prepare an ionic liquid anti-caries coating.

2. The method for preparing an ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization according to claim 1, characterized in that: The specific process of step 1 includes: Step 101: 3-chloropropyltriethoxysilane and 1-hexyl imidazole bromide were weighed in a molar ratio of 1:1 and slowly added into a single-necked flask, and ethyl acetate was added. Under nitrogen protection, the mixture was reacted at 70° C. with magnetic stirring for 48 hours to obtain a yellow-brown liquid product; Step 102: Wash with ethyl acetate several times and vacuum dry until viscous to obtain 1-hexyl-3-propyltriethoxysilane imidazole chloride; Step 103: using 1-octyl imidazole bromide and 1-decyl imidazole bromide as reactants, preparing 1-octyl-3-propyl triethoxysilyl imidazole chloride and 1-decyl-3-propyl triethoxysilyl imidazole chloride respectively according to steps 101-102; Step 104: Analyze the antibacterial activity and cytotoxicity of 1-hexyl-3-propyltriethoxysilyl imidazole chloride, 1-octyl-3-propyltriethoxysilyl imidazole chloride and 1-decyl-3-propyltriethoxysilyl imidazole chloride, and select 1-hexyl-3-propyltriethoxysilyl imidazole chloride for subsequent reactions.

3. The method for preparing an ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization according to claim 2, characterized in that: The specific process of step 2: 1-Hexyl-3-propyltriethoxysilyl imidazole chloride and potassium hexafluorophosphate were weighed in a mass ratio of 5:2.3 and added to a flask, and acetone was added. After reacting for 6 hours under magnetic stirring at room temperature, methyl chloride was added. After observing the generation of white precipitate, the mixture was centrifuged at 12000 rpm for 5 minutes, the precipitate was collected, and vacuum dried to a viscous state to obtain 1-hexyl-3-propyltriethoxysilyl imidazole hexafluorophosphate.

4. The method for preparing an ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization according to claim 2, characterized in that: The specific process of step 2: 1-Hexyl-3-propyltriethoxysilyl imidazole chloride and sodium fluoride were weighed in a mass ratio of 10:1 and added to a flask, and acetone was added. After reacting for 6 hours under magnetic stirring at room temperature, methyl chloride was added. After observing the generation of white precipitate, the mixture was centrifuged at 12000 rpm for 5 minutes, the precipitate was collected, and vacuum dried to a viscous state to obtain 1-hexyl-3-propyltriethoxysilyl imidazole fluoride.

5. The method for preparing an ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization according to claim 2, characterized in that: The specific process of step 2: 1-Hexyl-3-propyltriethoxysilane imidazole chloride and strontium chloride were weighed in a mass ratio of 5:2 and added to a flask, and acetone was added. After reacting under magnetic stirring at room temperature for 6 hours, anhydrous ethanol was added, and the reaction was carried out at 50°C for condensation reflux overnight. The mixture was centrifuged at 8000 rpm for 5 minutes to remove the precipitate, and the supernatant was collected. After evaporation to remove the solvent, the mixture was placed in a vacuum dryer until it became viscous to obtain a 1-hexyl-3-propyltriethoxysilane imidazole strontium chloride complex.

6. An ionic liquid anti-caries coating having dual functions of antibacterial and promoting mineralization, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 5.

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