Improved mesoporous silica material as well as preparation method and application thereof in prevention of decayed teeth

Abstract

The invention relates to the field of biomedical materials, and particularly discloses an improved mesoporous silica material as well as a preparation method and application thereof in prevention of decayed teeth. The improved mesoporous silica material is prepared from the following raw materials in parts by weight: 10 to 12 parts of alkannin, 20 to 24 parts of mesoporous silica, 6 to 8 parts of dopamine, 10 to 12 parts of polyethylene glycol, 3 to 5 parts of compatibilizer and 0.5 to 1.5 parts of silane coupling agent. The preparation method comprises the following steps: modifying PDA-PEG on the surfaces of mesoporous silica particles, and loading alkannin into PDA-PEG modified MSNs mesopores to form the MSPP mesoporous silica nano preparation. The improved mesoporous silica material disclosed by the invention has the characteristics of natural components, good antibacterial property and mineralization induction, and plays an important role in preventing decayed teeth.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and more specifically, to an improved mesoporous silica material, a preparation method thereof, and an application thereof in preventing dental caries. Background Art

[0002] Dental caries is a common oral disease caused by the combined effects of bacteria, diet, host and time. The initial symptoms of dental caries are usually white patches or yellow-brown spots on the tooth surface, which will form cavities in the later stages. Dental caries can cause severe pain when it damages the dental pulp. If not treated in time, it can also cause sepsis or bacteremia, which seriously endangers human life and health.

[0003] Caries prevention and treatment has been widely studied, but has not yet been fully resolved. Finding natural anti-caries active ingredients has always been an important research direction for caries prevention and treatment. The prior art has found that a variety of Xinjiang traditional Chinese medicines have excellent anti-caries efficacy, but most of them do not have the properties of specific elimination of bacterial biofilms, pH response, and promotion of enamel regeneration and repair, and are limited in clinical application. Currently, the treatment of caries mostly adopts drug therapy, surgical treatment and other methods. These methods not only have poor prevention and treatment effects, but also may cause other complications, such as accidental pulp perforation, pulpal pain, periodontal pain, secondary caries, etc. Therefore, based on the deficiencies of the prior art, the present application provides a modified mesoporous silica material, a preparation method thereof, and its application in the prevention of dental caries. Summary of the invention

[0004] In order to solve the problem in the related art that most traditional Chinese medicines do not have the performance of specifically eliminating bacterial biofilms, targeting enamel and responding to pH, and the treatment process is prone to other complications, the present application provides a modified mesoporous silica material, a preparation method thereof, and its application in preventing dental caries.

[0005] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10-12 parts of shikonin, 20-24 parts of mesoporous silica (MSNs), 6-8 parts of dopamine, 10-12 parts of polyethylene glycol, 3-5 parts of a compatibilizer, and 0.5-1.5 parts of a silane coupling agent.

[0006] Preferably, the preparation method of shikonin comprises the following steps:

[0007] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it; stirring and mixing the crushed sample with water to obtain a mixed solution;

[0008] (2) adding a composite enzyme to the mixed solution for enzymolysis to obtain a primary extract;

[0009] (3) Add 1-ethyl-3-methylimidazole diamine nitrile and lecithin to the primary extract, stir and mix for 1-2 hours, and centrifuge to obtain shikonin.

[0010] Preferably, in step (2), the complex enzyme is composed of xylanase and β-glucanase mixed in a mass ratio of 1-3:0.5-2.5.

[0011] Preferably, in step (3), the amount of 1-ethyl-3-methylimidazole dicyanamide and lecithin added is 4-6% of the mass of the initial extract, and the mass ratio of 1-ethyl-3-methylimidazole dicyanamide to lecithin is 1-3:1.

[0012] Preferably, the volume expander consists of collagen, calcium alginate and deionized water in a mass ratio of 4-6:2-4:100.

[0013] Preferably, the silane coupling agent is silane coupling agent KH-570.

[0014] The present application also proposes a method for preparing an improved mesoporous silica material, comprising the following steps:

[0015] S1, mixing the mesoporous silica solution with the silane coupling agent KH-570, and performing ultrasonic dispersion reaction to obtain modified mesoporous silica (KH-570-MSNs);

[0016] S2, dissolving dopamine in Tris-HCl, adding polyethylene glycol and stirring evenly, carrying out grafting reaction, and washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0017] S3, adding KH-570-MSNs, PDA-PEG and a compatibilizer into Tris-HCl, performing ultrasonic dispersion at room temperature for 1-3 hours, and then reacting for 1-2 hours under nitrogen protection, centrifuging and freeze-drying to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PDA-PEG);

[0018] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS buffer, stir to mix, then centrifuge at room temperature and dry. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0019] Preferably, the mesoporous silica solution in step S1 is prepared by the following method: dispersing mesoporous silica into an ethanol solution with a mass concentration of 70-80% to obtain a mesoporous silica solution, wherein the amount of the ethanol solution is 8-12 times the amount of the mesoporous silica.

[0020] Preferably, the mesoporous silica solution in step S1 is prepared by the following method: dispersing MSNs in an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution.

[0021] Preferably, in step S3, the amount of Tris-HCl used is 45-55 times the amount of the compatibilizer used.

[0022] Preferably, in step S4, the amount of PBS buffer used is 90-110 times the amount of shikonin used.

[0023] The present application also proposes an application of a modified mesoporous silica material, wherein the modified mesoporous silica material is applied to prevent dental caries.

[0024] In summary, this application has the following beneficial effects:

[0025] 1. This application uses mesoporous silica, dopamine, polyethylene glycol, a compatibilizer, a silane coupling agent and other raw materials to prepare an improved mesoporous silica material, and loads the natural active ingredient shikonin onto the surface of the mesoporous silica. After PDA-PEG modification, the pharmacological effects of shikonin such as anti-inflammatory and antibacterial can be effectively enhanced, and the ability of shikonin to promote the regeneration and repair of oral soft and hard tissues and enamel is improved, thereby obtaining a nano drug delivery system with targeted enamel, good antibacterial properties, and induced mineralization.

[0026] 2. This application uses mesoporous silica material as a drug carrier, which has a high specific surface area, high mechanical strength and thermal stability. The biocompatibility and stability of nanoparticles can be improved by PDA-PEG modification on the surface of mesoporous silica; adding a compatibilizer consisting of collagen, calcium alginate and deionized water during the PDA-PEG modification process synergistically enhances biocompatibility; in addition, collagen is rich in protein, which provides a suitable microenvironment for cell proliferation and growth, can accelerate the regeneration of tooth tissue, and improve the prevention and treatment efficiency of caries.

[0027] 3. In the process of extracting shikonin, the present application adds a complex enzyme for enzymatic hydrolysis, which can accelerate the destruction of the cell wall structure, promote the decomposition of the cell wall, and make it easier to decompose substances such as polysaccharides, thereby improving the product purity and extraction efficiency. Then, 1-ethyl-3-methylimidazole diamine nitrile and lecithin are added to perform secondary extraction of shikonin. Multiple extractions fully release the active ingredients of shikonin, significantly enhancing the antioxidant and antibacterial effects.

[0028] 4. In the improved mesoporous silica preparation process of the present application, the mesoporous silica is first modified with a silane coupling agent KH-570 to improve the reactivity of the nanoparticles and enhance their adhesion, thereby facilitating the subsequent attachment of drug molecules; the mesoporous silica is then modified with PDA-PEG to improve the biocompatibility and stability of the nanoparticles, thereby creating a stable environment for the delivery of drug molecules; finally, shikonin is loaded onto the surface of the PDA-PEG-modified modified mesoporous silica to fully exert the activity of the drug molecules, synergistically enhance the antibacterial ability of the drugs, and improve the efficiency of drug delivery. DETAILED DESCRIPTION

[0029] The present application is further described in detail below with reference to the embodiments.

[0030] Examples 1-3 provide a modified mesoporous silica material and a preparation method thereof.

[0031] Mesoporous silica (MSNs) (item number: 101016, purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.), methacryloxypropyltrimethoxysilane (model: KH-570, purchased from Hangzhou Jessica Chemical Co., Ltd.), xylanase (model: RCMJT10, purchased from Weifang Ruichen Biotechnology Co., Ltd.), β-glucanase (item number: S23089-25g, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), and lecithin (item number: YB22253, purchased from Hunan Yunbang Biotechnology Co., Ltd.) used in the examples and comparative examples of the present application.

[0032] Example 1

[0033] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0034] The preparation method of shikonin comprises the following steps:

[0035] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, crushing it and passing it through an 80-mesh sieve, stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0036] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0037] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0038] A method for preparing an improved mesoporous silica material comprises the following steps:

[0039] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0040] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0041] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / LT ris-HCl in an amount 45 times that of the compatibilizer, and ultrasonically dispersed at room temperature for 1 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 kHz, and then reacted at room temperature for 1 h under nitrogen protection, centrifuged at 10000 r / min for 10 min, and freeze-dried at -20°C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PD A-PEG);

[0042] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 90 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0043] Example 2

[0044] An improved mesoporous silica material comprises the following raw materials in parts by weight: 11 parts of shikonin, 22 parts of mesoporous silica (MSNs), 7 parts of dopamine, 11 parts of polyethylene glycol, 4 parts of a compatibilizer, and 1 part of a silane coupling agent.

[0045] The preparation method of shikonin comprises the following steps:

[0046] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 55° C. and the drying time to be 35 min, crushing it and passing it through a 90-mesh sieve, stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0047] (2) adding a composite enzyme (0.3% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 2:1.5, and performing enzymolysis at a temperature of 45°C for 2.5 h to obtain a primary extract;

[0048] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 5% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 2:1), stir and mix at 500 r / min for 1.5 h, and centrifuge at 11000 r / min for 40 min to obtain shikonin.

[0049] A method for preparing an improved mesoporous silica material comprises the following steps:

[0050] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 20 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0051] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0052] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 5:3:100) were added to 0.1 mol / LT ris-HCl with a volume of 50 times the volumetric agent, and ultrasonic dispersion was performed at room temperature for 2 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 KHz. Then, under nitrogen protection, the mixture was reacted at room temperature for 1.5 h, centrifuged at 10,000 r / min for 15 min, and freeze-dried at -20°C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MS Ns-PDA-PEG);

[0053] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 2 h, then centrifuge at 9000 r / min at room temperature for 30 min, and dry at 55°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0054] Example 3

[0055] An improved mesoporous silica material comprises the following raw materials in parts by weight: 12 parts of shikonin, 24 parts of mesoporous silica (MSNs), 8 parts of dopamine, 12 parts of polyethylene glycol, 5 parts of a compatibilizer, and 1.5 parts of a silane coupling agent.

[0056] The preparation method of shikonin comprises the following steps:

[0057] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 60° C. and the drying time to be 40 min, passing the crushed sample through a 100-mesh sieve, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0058] (2) adding a composite enzyme (0.4% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 3:2.5, and performing enzymolysis at a temperature of 50°C for 3 h to obtain a primary extract;

[0059] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 6% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 3:1), stir and mix at 600 r / min for 2 h, and centrifuge at 12000 r / min for 50 min to obtain shikonin.

[0060] A method for preparing an improved mesoporous silica material comprises the following steps:

[0061] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times that of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 h. Control the ultrasonic power to 500 W and the ultrasonic frequency to 25 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0062] S2, dissolving dopamine in 0.1 mol / L Tris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400 r / min for 1 h for grafting reaction, and washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0063] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 6:4:100) were added to 0.1 mol / LT ris-HCl with a volume of 55 times the volumetric agent, and ultrasonic dispersion was performed at room temperature for 3 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 KHz, and then reacted at room temperature for 2 h under nitrogen protection, centrifuged at 10000 r / min for 20 min, and freeze-dried at -20 ° C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PDA-PEG);

[0064] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 110 times the amount of shikonin, stir at 1000 r / min for 3 h, then centrifuge at 10000 r / min for 30 min at room temperature, and dry at 60°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0065] Comparative Example 1

[0066] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0067] The preparation method of shikonin comprises the following steps:

[0068] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0069] (2) adding xylanase (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution for enzymatic hydrolysis at 40° C. for 2 h to obtain a primary extract;

[0070] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0071] A method for preparing an improved mesoporous silica material comprises the following steps:

[0072] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0073] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0074] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / LT ris-HCl in an amount 50 times that of the compatibilizer, and ultrasonically dispersed at room temperature for 1 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 kHz, and then reacted at room temperature for 1 h under nitrogen protection, centrifuged at 10000 r / min for 10 min, and freeze-dried at -20°C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PD A-PEG);

[0075] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0076] Comparative Example 2

[0077] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0078] The preparation method of shikonin comprises the following steps:

[0079] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0080] (2) Adding complex enzyme (the amount added is 0.2% of the mass of the mixed solution), 1-ethyl-3-methylimidazole diamine nitrile and lecithin (the amount of 1-ethyl-3-methylimidazole diamine nitrile and lecithin added is 4% of the mass of the mixed solution, and the mass ratio of 1-ethyl-3-methylimidazole diamine nitrile and lecithin is 1:1) to the above mixed solution, stirring and mixing at 400 r / min at 40°C for 1 hour, and then centrifuging at 10000 r / min for 30 minutes to obtain shikonin.

[0081] Wherein, the composite enzyme in step (2) is composed of xylanase and β-glucanase mixed in a mass ratio of 1:0.5.

[0082] A method for preparing an improved mesoporous silica material comprises the following steps:

[0083] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0084] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0085] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / LT ris-HCl in an amount 50 times that of the compatibilizer, and ultrasonically dispersed at room temperature for 1 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 kHz, and then reacted at room temperature for 1 h under nitrogen protection, centrifuged at 10000 r / min for 10 min, and freeze-dried at -20°C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PD A-PEG);

[0086] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0087] Comparative Example 3

[0088] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0089] The preparation method of shikonin comprises the following steps:

[0090] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0091] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0092] (3) Add 1-ethyl-3-methylimidazole diamine cyanamide to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine cyanamide added is 4% of the mass of the primary extract), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0093] A method for preparing an improved mesoporous silica material comprises the following steps:

[0094] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0095] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0096] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / LT ris-HCl in an amount 45 times that of the compatibilizer, and ultrasonically dispersed at room temperature for 1 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 kHz, and then reacted at room temperature for 1 h under nitrogen protection, centrifuged at 10000 r / min for 10 min, and freeze-dried at -20°C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PD A-PEG);

[0097] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 90 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0098] Comparative Example 4

[0099] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0100] The preparation method of shikonin comprises the following steps:

[0101] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0102] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0103] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0104] A method for preparing an improved mesoporous silica material comprises the following steps:

[0105] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0106] S2, dissolving dopamine in 0.1mol / LTris-HCl (the amount used is 20 times that of dopamine), stirring at 400r / min for 1h, centrifuging at 10000r / min for 10min, discarding the supernatant, washing with deionized water for 3 times to remove free dopamine, vacuum drying, controlling the drying temperature to 55°C, and the drying time to 1h, to obtain polydopamine-modified modified mesoporous silica (KH-570-MSNs-PD A);

[0107] S3, KH-570-MSN-PDA, polyethylene glycol, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / L Tris-HCl with a volume of 50 times the volumetric agent, and ultrasonically dispersed at room temperature for 1 hour, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 kHz. Then, the mixture was reacted at room temperature for 1 hour under nitrogen protection, centrifuged at 10,000 r / min for 10 minutes, and freeze-dried at -20°C for 3 hours to obtain PDA-PEG-modified KH-570-modified mesoporous silica (KH-570-MSNs-PDA-PEG);

[0108] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0109] Comparative Example 5

[0110] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0111] The preparation method of shikonin comprises the following steps:

[0112] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0113] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0114] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0115] A method for preparing an improved mesoporous silica material comprises the following steps:

[0116] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times that of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 h. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0117] S2, dissolving dopamine in 0.1 mol / L Tris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400 r / min for 1 h, carrying out grafting reaction, and washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0118] S3. KH-570-MSNs and shikonin were dissolved in PBS buffer (10 mM, pH 7.4) buffer with a 100-fold amount of shikonin, stirred at 1000 r / min for 1 h, then centrifuged at 8000 r / min for 30 min at room temperature, and dried at 50°C for 30 min. The collected precipitate was modified mesoporous silica (KH-570-MSNs-Shikonin) loaded with shikonin in response to pH.

[0119] S4. KH-570-MSN-Shikonin, PDA-PEG, and a compatibilizer (composed of collagen, calcium alginate, and deionized water in a mass ratio of 4:2:100) were added to 0.1 mol / L Tris-HCl (50 times the amount of the compatibilizer), and ultrasonically dispersed at room temperature for 1 h. The ultrasonic power was controlled to be 400 W and the ultrasonic frequency to be 20 kHz. Then, under nitrogen protection, the mixture was reacted at room temperature for 1 h, centrifuged at 10,000 r / min for 10 min, and freeze-dried at -20°C for 3 h to obtain a pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0120] Comparative Example 6

[0121] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0122] The preparation method of shikonin comprises the following steps:

[0123] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0124] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0125] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0126] A method for preparing an improved mesoporous silica material comprises the following steps:

[0127] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0128] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0129] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of collagen and deionized water in a mass ratio of 6:100) were added to 0.1 mol / L Tris-HCl with a volume of 50 times the volumetric agent, and ultrasonically dispersed at room temperature for 1 hour, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 KHz, and then reacted at room temperature for 1 hour under nitrogen protection, centrifuged at 10000 r / min for 10 minutes, and freeze-dried at -20°C for 3 hours to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PDA-PEG);

[0130] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0131] Comparative Example 7

[0132] An improved mesoporous silica material comprises the following raw materials in parts by weight: 10 parts of shikonin, 20 parts of mesoporous silica (MSNs), 6 parts of dopamine, 10 parts of polyethylene glycol, 3 parts of a compatibilizer, and 0.5 parts of a silane coupling agent.

[0133] The preparation method of shikonin comprises the following steps:

[0134] (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it, controlling the drying temperature to be 50° C. and the drying time to be 30 min, passing through an 80-mesh sieve after crushing, and stirring and mixing the crushed sample with water (the amount used is 10 times that of the crushed sample) to obtain a mixed solution;

[0135] (2) adding a composite enzyme (the amount added is 0.2% of the mass of the mixed solution) to the mixed solution in a mass ratio of xylanase to β-glucanase of 1:0.5, and performing enzymolysis at a temperature of 40°C for 2 h to obtain a primary extract;

[0136] (3) Add 1-ethyl-3-methylimidazole diamine and lecithin to the primary extract (the amount of 1-ethyl-3-methylimidazole diamine and lecithin added is 4% of the mass of the primary extract, and the mass ratio of 1-ethyl-3-methylimidazole diamine and lecithin is 1:1), stir and mix at 400 r / min for 1 h, and centrifuge at 10000 r / min for 30 min to obtain shikonin.

[0137] A method for preparing an improved mesoporous silica material comprises the following steps:

[0138] S1. Disperse MSNs into an ethanol solution with a mass concentration of 75% (the amount used is 10 times the amount of MSNs) to obtain an MSNs solution, add KH-570 and mix evenly, and perform ultrasonic dispersion reaction for 1 hour. Control the ultrasonic power to 500 W and the ultrasonic frequency to 15 KHz to obtain modified mesoporous silica (KH-570-MSNs);

[0139] S2, dissolving dopamine in 0.1mol / LTris-HCl (the dosage is 20 times that of dopamine), adding polyethylene glycol, stirring at 400r / min for 1h, carrying out grafting reaction, washing with deionized water to obtain polyethylene glycol grafted polydopamine (PDA-PEG);

[0140] S3, KH-570-MSNs, PDA-PEG, and a compatibilizer (composed of calcium alginate and deionized water in a mass ratio of 6:100) were added to 0.1 mol / L Tris-HCl with a volume of 50 times the volumetric agent, and ultrasonically dispersed at room temperature for 1 h, with the ultrasonic power controlled at 400 W and the ultrasonic frequency at 20 KHz, and then reacted for 1 h under nitrogen protection, centrifuged at 10000 r / min for 10 min, and freeze-dried at -20 ° C for 3 h to obtain PDA-PEG-modified modified mesoporous silica (KH-570-MSNs-PDA-PEG);

[0141] S4. Dissolve KH-570-MSNs-PDA-PEG and shikonin in PBS (10 mM, pH 7.4) buffer solution 100 times the amount of shikonin, stir at 1000 r / min for 1 h, then centrifuge at 8000 r / min at room temperature for 30 min, and dry at 50°C for 30 min. The collected precipitate is the pH-responsive shikonin@PDA-PEG mesoporous silica (KH-570-MSNs-Shikonin@PDA-PEG, MSPP) nanoformulation.

[0142] Performance Testing

[0143] The anti-caries effects of the improved mesoporous silica materials prepared in Examples 1-3 of the present application and Comparative Examples 1-7 were tested respectively.

[0144] 1. Determination of anti-caries performance

[0145] 1. Establishment of rat dental caries model:

[0146] (1) 60 17-day-old male SD rats of SPF grade were selected and randomly divided into cages for feeding, 5 rats per cage, a total of 12 cages, and randomly divided into 12 groups, namely, Example 1-3 groups, Comparative Example 1-7 groups, positive control group and negative control group;

[0147] (2) First, rats were fed a diet containing 0.1% ampicillin, 0.1% chloramphenicol, and 0.1% carbenicillin and water containing 4000 U / ml penicillin for 3 days to inhibit the original flora in the rats' mouths and promote the colonization of mutans streptococci;

[0148] (3) Starting from the fourth day, the rats were smeared with the bacterial solution of Streptococcus mutans UA159 in the logarithmic phase in the oral cavity for 3 consecutive days, and were given the cariogenic diet Keyes 2000 and 5% sucrose solution as drinking water;

[0149] (4) On the 7th day, oral swabs were used to collect samples, which were spread on MSB plates and incubated at 37°C for 2 days. If frosty blue rough colonies of typical mutans Streptococcus were visible on the MSB plates after incubation, it indicated that mutans Streptococcus had successfully colonized in the rats’ mouths and the rat caries model was established.

[0150] (5) After the rat caries model was established, the Example 1-3 group and the Comparative Example 1-7 group were coated with the improved mesoporous silica products prepared in Example 1-3 and Comparative Example 1-7, respectively, and the positive control group was coated with 1000 ppm F - The negative control group was treated with 0.5% DMSO on the bilateral mandibular teeth in the oral cavity of rats for 5 minutes, three times a day, for three consecutive weeks.

[0151] (II) Keyes Index Analysis

[0152] Smooth surface and pit and fissure caries lesions were scored according to the Keyes diagnosis and scoring method. In the pit and fissure caries scoring, the lesion depth was divided into enamel only (E), slightly involved in dentin (Ds), moderately involved in dentin (Dm) and extensively involved in dentin (Dx). The measurement results are shown in Table 1.

[0153] Table 1:

[0154]

[0155]

[0156] Note: Mean±SD, different letters in each column indicate that the index has significant difference among the groups (P<0.05), and the same letters indicate that the index has no statistically significant difference among the groups (P>0.05) Micro-CT analysis.

[0157] It can be seen from Table 1 that compared with the negative control group, the improved mesoporous silica materials prepared in Examples 1-3 and Comparative Examples 1-7 significantly reduced the incidence and severity of pit caries, the incidence and severity of pit caries in Examples 1-3 were significantly lower than those in Comparative Examples 1-7, and the therapeutic effects of the improved mesoporous silica materials prepared in Examples 1-3 and Comparative Examples 1-7 were also significantly better than those in the positive control group, indicating that the improved mesoporous silica materials of the present application can effectively prevent and treat caries and have important application value in preventing dental caries.

[0158] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A modified mesoporous silica material, characterized in that: The invention comprises the following raw materials in parts by weight: 10-12 parts of shikonin, 20-24 parts of mesoporous silica, 6-8 parts of dopamine, 10-12 parts of polyethylene glycol, 3-5 parts of a compatibilizer and 0.5-1.5 parts of a silane coupling agent.

2. The improved mesoporous silica material according to claim 1, characterized in that: The preparation method of shikonin comprises the following steps: (1) cutting fresh lithospermum root into slices, drying the sliced ​​lithospermum root and then crushing it; stirring and mixing the crushed sample with water to obtain a mixed solution; (2) adding a composite enzyme to the mixed solution for enzymolysis to obtain a primary extract; (3) Add 1-ethyl-3-methylimidazole diamine nitrile and lecithin to the primary extract, stir and mix for 1-2 hours, and centrifuge to obtain shikonin.

3. The improved mesoporous silica material according to claim 2, characterized in that: In the step (2), the complex enzyme is composed of xylanase and β-glucanase mixed in a mass ratio of 1-3:0.5-2.

5.

4. The improved mesoporous silica material according to claim 2, characterized in that: In the step (3), the amount of 1-ethyl-3-methylimidazole dicyanamide and lecithin added is 4-6% of the mass of the initial extract, and the mass ratio of 1-ethyl-3-methylimidazole dicyanamide to lecithin is 1-3:

1.

5. The improved mesoporous silica material according to claim 1, characterized in that: The volume expander consists of collagen, calcium alginate and deionized water in a mass ratio of 4-6:2-4:

100.

6. The improved mesoporous silica material according to claim 1, characterized in that: The silane coupling agent is silane coupling agent KH-570.

7. A method for preparing the improved mesoporous silica material according to any one of claims 1 to 6, characterized in that: The method comprises the following preparation steps: S1, mixing the mesoporous silica solution with a silane coupling agent, and performing ultrasonic dispersion reaction to obtain modified mesoporous silica; S2, dissolving dopamine in Tris-HCl, adding polyethylene glycol and stirring evenly, carrying out grafting reaction, and washing with deionized water to obtain polyethylene glycol-grafted polydopamine; S3, adding modified mesoporous silica, polyethylene glycol grafted polydopamine, and a compatibilizer to Tris-HCl, and ultrasonically dispersing for 1-3 hours at room temperature, and then reacting for 1-2 hours under nitrogen protection, centrifuging, and freeze-drying to obtain modified mesoporous silica modified with polyethylene glycol grafted polydopamine; S4. Dissolve the modified mesoporous silica modified with polyethylene glycol grafted polydopamine and shikonin in PBS buffer, stir and mix, then centrifuge at room temperature and dry. The collected precipitate is the pH-responsive shikonin@polyethylene glycol grafted polydopamine modified mesoporous silica nanoformulation.

8. The method for preparing a modified mesoporous silica material according to claim 7, characterized in that: The mesoporous silica solution in step S1 is prepared by the following method: dispersing mesoporous silica into an ethanol solution with a mass concentration of 70-80% to obtain a mesoporous silica solution, wherein the amount of the ethanol solution is 8-12 times the amount of the mesoporous silica.

9. A use of the improved mesoporous silica material according to any one of claims 1 to 6, characterized in that: The improved mesoporous silica material is used for preventing dental caries.

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