WO2023138191A1 - 一种口腔护理组合物及其制备方法和应用 - Google Patents

一种口腔护理组合物及其制备方法和应用 Download PDF

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WO2023138191A1
WO2023138191A1 PCT/CN2022/131838 CN2022131838W WO2023138191A1 WO 2023138191 A1 WO2023138191 A1 WO 2023138191A1 CN 2022131838 W CN2022131838 W CN 2022131838W WO 2023138191 A1 WO2023138191 A1 WO 2023138191A1
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care composition
oral care
mal
peg
drug
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French (fr)
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黄翠
许玥
郭景梅
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武汉大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/16—Fluorine compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers

Definitions

  • the invention relates to the technical fields of biological materials and oral care, in particular to an oral care composition and its preparation method and application.
  • Dental caries is one of the most common diseases and high incidence in the oral cavity at present, and the resulting tooth defect can lead to the loss of the integrity of the tooth shape, thereby affecting the occlusal function.
  • the main treatment method is to reconstruct and repair the defective tissue through repair materials after removing the infected tissue mechanically and/or chemically, so as to restore the shape and function of the tooth.
  • this is a remedial measure after the damage is formed, and there may be a risk of secondary caries after the restoration of the tooth defect. Therefore, starting from the etiology, the development of methods for preventing and repairing early defects has great social benefits and application prospects.
  • the present invention provides an oral care composition that can adhere to the surface of teeth, have long-lasting drug release, and can be released rapidly in an acidic environment caused by dental caries, and its preparation method and application.
  • the present invention provides an oral care composition, comprising drug-loaded micelles, the drug-loaded micelles comprising polymer micelles and enamel repair medicines physically coated in polymer micelles, micelles particles formed by water-soluble polymers of the structure shown in polymer micelles formula I in an aqueous solution in which tannic acid exists, and the shells of which are linked to salivary protein polypeptides through chemical bonds:
  • x, y, z are natural numbers, representing the degree of polymerization, and the Mw weight average molecular weight of water-soluble polymer is 5000 ⁇ 6000g/mol;
  • the oral care composition provided by the present invention coats the enamel repairing drug with the micellar particles formed by the water-soluble polymer in the aqueous solution of tannic acid, and the saliva protein polypeptide stathelin connected to the surface of the micellar particles can make the drug-loaded micellar particles adhere to the surface of the tooth enamel, so as to achieve the purpose of long-term retention in the oral cavity; in the early stage of caries, the oral environment is acidified, the borate bond between the water-soluble polymer and tannic acid is broken, the gate is opened, and the tannic acid and sodium fluoride are released to play a highly effective antibacterial and antibacterial effect.
  • the remineralization and restoration effect realizes the intelligent and on-demand release of drugs, and achieves the purpose of preventing dental caries and early restoration.
  • the particle size of the micelles is 200-400 nm, more preferably 300 ⁇ 50 nm, so that the micelles can better penetrate into the biofilm formed by Streptococcus mutans.
  • the polymer micelles are connected to the salivary protein polypeptide through a thioether bond.
  • the enamel restoration drug is sodium fluoride.
  • the oral care composition is in the form of solid, paste, gel composition or liquid composition.
  • the oral care composition described above further comprises an anti-caries agent, a desensitizing agent, a viscosity regulator, a diluent, a surfactant, an emulsifier, a foam regulator, a pH regulator, an abrasive, a mouthfeel, a sweetener, a flavoring agent, a coloring agent, a preservative, an amino acid, an antioxidant, an anti-calculus agent, a fluoride ion source, a thickener, an active agent for preventing or treating a condition or disorder of hard or soft tissues of the oral cavity, an adhesive, a whitening agent, and combinations thereof.
  • the present invention provides the use of the above oral care composition in the preparation of a medicament for reducing or inhibiting bacteria in the oral cavity of a patient, and in the preparation of a medicament for promoting enamel remineralization and repair.
  • the present invention provides an ex vivo method of reducing or inhibiting bacteria or promoting enamel remineralization repair in a removable oral device of a patient comprising applying the above oral care composition to the surface of the removable oral device.
  • the present invention provides a method for preparing the above-mentioned oral care composition, comprising:
  • the drug-loaded micelles and salivary protein polypeptides are formed into chemical bonds in triethanolamine to obtain the oral care composition according to claim 1.
  • the method for preparing the water-soluble polymer of structure shown in formula I comprises:
  • MAL-PEG-b-PZLL is subjected to amidation reaction with 3-fluoro-4-carboxyphenanthrene boronic acid after debenzylation protection to obtain a water-soluble polymer with the structure shown in formula I.
  • the present invention firstly prepares water-soluble polymers containing boric acid groups, which can form borate bonds with tannic acid and conjugate with tannic acid to form micelles. During the formation of micelles, sodium fluoride is physically encapsulated. Finally, salivary protein polypeptides with tooth-adhesive functions are modified on the surface of drug-loaded micelles to form classic spherical micelles. The micelles can slowly release tannic acid and sodium fluoride under normal physiological conditions in the oral cavity, and play antibacterial and repairing functions. Nitric acid is released due to the breakage of the borate ester bond, and tannic acid and sodium fluoride present the effects of initial burst release and long-term sustained release.
  • the oral care composition prepared by the invention has tooth adhesion and pH responsiveness, can identify Streptococcus mutans early, can improve the bacterial targeting and local drug concentration of drugs, achieves intelligent and on-demand release, thereby reducing the toxic and side effects of drugs and the generation of bacterial drug resistance, and can prevent tooth hard tissue defects, and has great advantages and potentials.
  • Fig. 1 is the nuclear magnetic resonance spectrum of the water-soluble polymer MAL-PEG-b-PLL/PBA with the structure shown in formula I prepared by the present invention.
  • Fig. 2 is a transmission electron microscope image of MAL-PEG-b-PLL/PBA, CLM@NaF and CLM@NaF-Pep prepared in the present invention.
  • Figure 3 is the infrared spectrum detection results of MAL-PEG-b-PLL/PBA, CLM (MAL-PEG-b-PLL/PBA+TA), CLM@NaF, Pep and CLM@NaF-Pep prepared in the present invention.
  • Figure 4 shows the pH-controlled release effect of CLM@NaF-Pep prepared in the present invention on tannic acid and sodium fluoride.
  • Fig. 5 is the growth curve of CLM@NaF-Pep prepared in the present invention as a nanomaterial for caries prevention and early repair to inhibit Streptococcus mutans.
  • Fig. 6 is an effect diagram of the anti-Streptococcus mutans biofilm ability of the CLM@NaF-Pep prepared in the present invention and the control group.
  • Figure 7 is the quantitative detection and scanning electron microscope images of calcium and phosphorus ions for CLM@NaF-Pep prepared in the present invention and various control groups to inhibit enamel demineralization and promote its remineralization.
  • the selected raw materials are all commercially available.
  • micellar composites loaded with tannic acid and sodium fluoride and linked to sialoprotein polypeptides
  • Co-react polyethylene glycol diamine (CAS No.: 24991-53-5, NH 2 C 2 H 2 -(CH 2 O) n -C 2 H 2 NH 2 ) with 3-maleimidopropionic acid (MAL-) to modify MAL on the polymer to obtain the product MAL-PEG-NH 2 .
  • MAL- 3-maleimidopropionic acid
  • ⁇ -(benzyloxycarbonyl)-L-lysine N-carboxyanhydride ( ⁇ -benzyloxycarbonyl-L-lysine-N-carboxy-cyclic anhydride, Lys(Z)-NCA) (1.96g, 6.4mmol) was dissolved in 30mL N,N-dimethylformamide (DMF), by adding MAL-PEG-NH 2 (2.0g, 0.4mmol), in 3 Stir under dry argon at 5°C for 72h to allow polymerization to occur. Then the solvent was evaporated by rotary evaporation, the product was dissolved in 25 mL CHCl 3 , and precipitated in excess ether to obtain the product MAL-PEG-b-PZLL.
  • DMF N,N-dimethylformamide
  • the reaction solution was dialyzed with 0.01N NaOH solution, 0.01N HCl solution and deionized water using a dialysis bag (MWCO 3500), and the dialysis product was freeze-dried to obtain the product MAL-PEG-b-PLL/PBA.
  • the measured number average molecular weight Mn of MAL-PEG-b-PLL/PBA is 4458 g/mol, and the weight average molecular weight Mw is 5642 g/mol.
  • the MAL-PEG-b-PLL/PBA obtained by the above method was subjected to nuclear magnetic resonance, and the obtained spectrum is shown in FIG. 1 .
  • the peak at about 7.3ppm is the characteristic peak of FPBA phenylene, which confirms that FPBA is successfully connected to the polymer through conjugation with amine groups.
  • the transmission electron microscope results of MAL-PEG-b-PLL/PBA are shown in Figures 2a, 2d, and 2g. In the figure, it can be seen that there are a large number of small particles scattered around the larger particle size polymer. According to the scale in the figure, the particle size of the larger polymer is only about 100nm, and the structure of micelles has not yet formed.
  • micellar particles A 5 mg/mL aqueous solution of tannic acid (TA) was prepared, and 274 ⁇ L of the tannic acid aqueous solution was slowly and uniformly added dropwise to the sodium fluoride-carrier aqueous solution, stirred at room temperature for 4 hours, and the resulting solution was collected by ultrafiltration and centrifugation for 20 minutes to obtain drug-loaded micelle particles loaded with sodium fluoride and tannic acid (CLM@NaF).
  • CLM@NaF drug-loaded micelle particles loaded with sodium fluoride and tannic acid
  • the particle size of the synthesized micelles is about 300nm, and it has a classic spherical core-shell structure of micelles, with regular morphology and good dispersion.
  • the loading of tannic acid and sodium fluoride, and the linking of peptides will not have adverse effects on the morphology and dispersion of micellar particles.
  • CLM reference substance CLM
  • MAL-PEG-b-PLL/PBA+TA Add tannic acid aqueous solution dropwise to polymer MAL-PEG-b-PLL/PBA aqueous solution, stir and mix for self-assembly, and obtain CLM (MAL-PEG-b-PLL/PBA+TA) micellar particles after ultrafiltration and purification.
  • Embodiment 2 drug pH controlled release test
  • the pH-controlled release effects of CLM@NaF-Pep on tannic acid and sodium fluoride are shown in Figure 4: when the pHs are 7.4 and 5.0, respectively, the controlled-release effects of tannic acid are shown in Figure 4A; when the pHs are 7.4, 6.5, and 5.0, respectively, the controlled-release effects of sodium fluoride are shown in Figure 4B. It can be seen from Figure 4 that under normal oral physiological conditions (pH 7.4), the cumulative release rates of tannic acid and sodium fluoride within 24 hours are about 40% and 50%, respectively, which has a long-term sustained release effect and reflects the high stability of CLM@NaF-Pep.
  • Embodiment 3 Inhibition Streptococcus mutans growth effect test
  • the Streptococcus mutans cultured to the exponential phase was collected by centrifugation for 3 minutes (5000 rpm), and diluted to 10 6 CFU/mL with BHI medium to obtain a bacterial suspension.
  • CLM@NaF-Pep was added to the BHI medium with pH 7.4, 6.5 and 5.0, respectively, to prepare the dispersion solution of the experimental group; PBS was used as the blank control solution, and chlorhexidine (CHX) was used as the positive control solution, and distributed into 96-well plates (160 ⁇ L per well). Subsequently, 40 ⁇ L of the above bacterial suspension was added to the dispersion liquid of the experimental group and the solution of the control group and mixed evenly.
  • the 96-well plate was placed in a 37°C incubator, and cultured for 0.5, 1, 2, 4, 8, 12, and 24 h, respectively, and then 100 ⁇ L of culture solution was taken from each group, inoculated into a new 96-well plate, and the absorbance of each well was recorded at 600 nm using a microplate reader to evaluate the antibacterial activity. Three replicate wells were set up for all experiments, and the average values were calculated for comparison, and the experiments were repeated three times.
  • the growth curve of inhibiting Streptococcus mutans (Streptococcus mutans) is shown in Figure 5. From Figure 5, it can be seen that CLM@NaF-Pep exhibits high antibacterial activity against Streptococcus mutans, and as the pH decreases, its growth inhibition activity of Streptococcus mutans is stronger, and the bactericidal effect of Streptococcus mutans is even higher than that of chlorhexidine when the pH is 5.0.
  • HA slices (diameter 5 mm, thickness 2 mm) were prepared, and the HA slices were sterilized by high-pressure steam sterilization.
  • the HA tablets were treated with equal amounts of deionized water (ddH 2 O), CLM@NaF aqueous dispersion, Pep aqueous solution, and CLM@NaF-Pep aqueous dispersion, respectively, and washed three times with PBS to remove the non-adhered substances on the HA tablets, and four groups of pretreated HA tablets were obtained.
  • deionized water ddH 2 O
  • CLM@NaF aqueous dispersion CLM@NaF aqueous dispersion
  • Pep aqueous solution CLM@NaF-Pep aqueous dispersion
  • the Streptococcus mutans that had grown to the exponential phase was diluted with BHI medium to an OD 600 of 0.5, and then the bacterial suspension was inoculated onto the pretreated HA sheet, and placed in a 48-well plate, so that the pretreated HA sheet was completely immersed in the variable chain suspension, and anaerobically cultured at 37°C for 1.5h. Then aspirate the bacterial liquid, carefully rinse with PBS to remove the unadhered bacterial liquid, add an appropriate amount of sugar-containing medium, and incubate anaerobically at 37°C for 24-48h. Finally, the samples were rinsed with PBS, and the biofilm on the HA sheet was collected by ultrasound.
  • the biofilm suspension was serially diluted, spread on a BHI agar plate, and after 48 hours of incubation, the number of colonies was counted. Experiments were repeated three times.
  • the CFU (colony forming units) count chart of the anti-Streptococcus mutans biofilm ability is shown in Figure 6.
  • the HA sheet treated with CLM@NaF-Pep aqueous dispersion (represented by CLM@NaF-Pep in Figure 6) not only has antibacterial adhesion, but also CLM@NaF-Pep can penetrate into the biofilm and kill the bacteria in the biofilm, confirming that the constructed CLM@NaF-Pep has a high removal effect of cariogenic biofilm on the tooth surface.
  • Example 5 Inhibition of enamel demineralization and promotion of its remineralization detection
  • the caries-free third molars were collected, the roots were removed with a slow saw, and the crown was cut perpendicular to the long axis of the tooth from the mesial-distal direction, and then the sample was cut into 5*4*1.5mm 3 enamel slices. Polish with 600-grit and 1200-grit sandpaper, and then sonicate in deionized water (ddH 2 O) for 20 minutes. The enamel surface of the dental piece was etched with 37% phosphoric acid for 1 min, and then rinsed with ddH 2 O for 1 min.
  • the dental tablets were randomly divided into 4 groups (blank control group, CLM@NaF group, Pep group, CLM@NaF-Pep group), immersed in deionized water, CLM@NaF aqueous dispersion, Pep aqueous solution, CLM@NaF-Pep aqueous dispersion, and then rinsed with PBS to remove unadhered substances.
  • the enamel side of each group of tooth slices was facing up, and the other sides were covered with acid-resistant nail polish.

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Abstract

本发明公开了一种口腔护理组合物及其制备方法和应用，包括载药胶束，载药胶束包括聚合物胶束和物理包覆于聚合物胶束中的釉质修复药物，聚合物胶束式I所示结构的水溶性聚合物在单宁酸存在的水溶液中形成的胶束粒子，其外壳通过化学键与唾液蛋白多肽连接。本发明提供的口腔护理组合物通过唾液蛋白多肽粘附在牙齿表面，在龋齿造成的口腔酸性环境中打开单宁酸与水溶性聚合物之间的硼酸酯键，释放单宁酸和氟化钠，能够实现药物的智能、按需释放，从而达到预防龋齿和修复牙齿缺损的目的。

Description

一种口腔护理组合物及其制备方法和应用 技术领域
本发明涉及生物材料和口腔护理技术领域，尤其涉及一种口腔护理组合物及其制备方法和应用。
背景技术
龋病是目前口腔的最主要的常见病和高发病之一，随之导致的牙体缺损可致使牙体形态完整性丧失，从而影响咬合功能。当全口多颗牙齿缺损时甚至导致咬合关系丧失，患者口腔功能及美观受到严重损害。目前主要的治疗手段为机械和/或化学去除感染组织后，通过修复材料对缺损组织进行重建和修复，达到恢复牙齿形态和功能的目的。然而从整个牙体缺损疾病发展的过程来看，这是一种损害形成后的补救措施，并且牙体缺损修复后还可能面临继发龋的风险。因此，从病因出发，开发具有预防和修复早期缺损的方法具有极大的社会效益和应用前景。
目前的研究认为，细菌感染是龋病的主要病因，其主要致病菌变异链球菌(S.mutans)聚集、定植在牙齿表面并产酸、脱矿牙齿硬组织，最终造成牙体硬组织缺损。目前龋病的药物治疗多为使用涂剂、漱口水等或是将抗菌药物掺入充填材料提高修复界面的抗菌性能，但仍然存在药物释放不持久、特异性不强等缺陷。如何在龋损早期识别、介入并中断其进一步发展，同时修复缺损组织具有重 要的临床意义。
发明内容
针对现有口腔护理产品在口腔内停留时间短、药物释放不持久、特异性不强等缺陷，本发明提供一种可粘附于牙齿表面、药物释放持久且可在龋齿造成的酸性环境中迅速释放的口腔护理组合物及其制备方法和应用。
本发明提供的技术方案具体如下：
第一方面，本发明提供一种口腔护理组合物，包括载药胶束，所述载药胶束包括聚合物胶束和物理包覆于聚合物胶束中的釉质修复药物，聚合物胶束式I所示结构的水溶性聚合物在单宁酸存在的水溶液中形成的胶束粒子，其外壳通过化学键与唾液蛋白多肽连接：
Figure PCTCN2022131838-appb-000001
其中，x、y、z为自然数，代表聚合度，水溶性聚合物的Mw重均分子量为5000～6000g/mol；优选地，x＝80～100、y＝2～10、z＝2～8，式I所示结构的水溶性聚合物MAL-PEG-b-PLL/PBA的数均分子量Mn为4400±100g/mol，重均分子量Mw为5500±100g/mol。
基于以上技术方案，本发明提供的口腔护理组合物通过水溶性聚 合物在单宁酸存在的水溶液中形成的胶束粒子将釉质修复药物包覆其中，胶束粒子表面连接的唾液蛋白多肽stathelin可使载药胶束粒子能黏附牙釉质表面，达到长期留存口腔中的目的；龋损发生早期，口腔环境酸化，水溶性聚合物与单宁酸之间的硼酸酯键断裂，打开门控，释放单宁酸与氟化钠，发挥高效抗菌与促再矿化修复效用，实现药物的智能、按需释放，达到预防龋齿和早期修复的目的。
作为上述技术方案的优选，胶束粒子的粒径为200～400nm，进一步优选为300±50nm，便于胶束粒子可以更好地渗透入变异链球菌形成的生物膜。
作为上述技术方案的优选，聚合物胶束通过硫醚键与唾液蛋白多肽连接。
作为上述技术方案的优选，釉质修复药物为氟化钠。
作为上述技术方案的优选，口腔护理组合物呈固体、糊剂、凝胶组合物或液体组合物形式。
上述口腔护理组合物进一步包含抗龋齿剂、脱敏剂、粘度调节剂、稀释剂、表面活性剂、乳化剂、泡沫调节剂、pH调节剂、研磨剂、口感剂、甜味剂、调味剂、着色剂、防腐剂、氨基酸、抗氧化剂、抗牙结石剂、氟离子源、增稠剂、用于预防或治疗口腔的硬或软组织的病状或病症的活性剂、粘合剂、增白剂及其组合。
第二方面，本发明提供上述口腔护理组合物在制备用于减少或抑制患者口腔中的细菌的药物中的用途，以及在制备用于促进釉质再矿化修复的药物中的用途。
第三方面，本发明提供一种在患者可拆卸的口腔装置中减少或抑制细菌或促进釉质再矿化修复的离体方法，包括向可拆卸的口腔装置的表面施用上述口腔护理组合物。
第四方面，本发明提供一种制备上述口腔护理组合物的方法，包括：
制备如式I所示结构的水溶性聚合物；
使式I所示结构的水溶性聚合物与釉质修复药物在水溶液中混合，滴加单宁酸，得到分散在水溶液中的载药胶束；
使载药胶束与唾液蛋白多肽在三乙醇胺中生成化学键，得到权利要求1所述的口腔护理组合物。
作为上述技术方案的优选，制备如式I所示结构的水溶性聚合物的方法包括：
使3-马来酰亚胺基丙酸通过酰胺键连接在聚乙二醇二胺一端，得到MAL-PEG-NH 2；
使MAL-PEG-NH 2与ε-苄氧羰基-L-赖氨酸-N-羧基-环内酸酐发生聚合反应，得到MAL-PEG-b-PZLL；
使MAL-PEG-b-PZLL脱苄基保护后与3-氟-4-羧基菲硼酸进行酰胺化反应，得到如式I所示结构的水溶性聚合物。
本发明首先制备含硼酸基团的水溶性聚合物，其能与单宁酸形成硼酸酯键，并与单宁酸共轭形成胶束，在形成胶束粒子过程中物理包封氟化钠，最后在载药胶束粒子表面修饰具有牙齿黏附功能的唾液蛋白多肽，形成经典的球形胶束粒子，该胶束粒子在口腔正常生理条件 下可缓释单宁酸和氟化钠，发挥抗菌和修复作用，而当龋损发生口腔pH微环境酸化时，单宁酸因硼酸酯键断裂而释放，单宁酸和氟化钠呈现初期突释与长期缓释的效果。
本发明具有以下优点：
本发明制备的口腔护理组合物具有牙齿粘附性和pH响应性，能早期识别变异链球菌，能提高药物的细菌靶向性和局部药物浓度，做到智能、按需释放，从而降低药物的毒副作用及细菌耐药性的产生，且能预防牙体硬组织缺损，具有极大的优势和潜力。
附图说明
图1为本发明制备的式I所示结构的水溶性聚合物MAL-PEG-b-PLL/PBA的核磁共振谱图。
图2为本发明制备的MAL-PEG-b-PLL/PBA、CLM@NaF和CLM@NaF-Pep的透射电镜图。
图3为本发明制备的MAL-PEG-b-PLL/PBA、CLM(MAL-PEG-b-PLL/PBA+TA)、CLM@NaF、Pep和CLM@NaF-Pep的红外光谱检测结果。
图4为本发明制备的CLM@NaF-Pep对单宁酸和氟化钠的pH控释效果。
图5为本发明制备的CLM@NaF-Pep作为预防龋病并早期修复的纳米材料抑制变异链球菌(Streptococcus mutans)的生长曲线。
图6为本发明制备的CLM@NaF-Pep与对照组的抗变异链球菌生物膜能力的效果图。
图7为本发明制备的CLM@NaF-Pep与各对照组抑制釉质脱矿、促进其再矿化的钙磷离子定量检测与扫描电镜图。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰明白，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并不是为了限定本发明。
以下是本发明具体实验实施例，仅代表本发明优选的实施例，不代表对本发明具体方案的限定，部分材料的选择及手段为本领域人员可选，但整体覆盖原理的替代方案均在本发明保护的范围之内。
特别地，本发明描述的实施例中，选用的原料均为市售。
实施例1：负载单宁酸和氟化钠、连接唾液蛋白多肽的胶束复合材料的制备
(1)水溶性聚合物的制备：
将聚乙二醇二胺(CAS No.：24991-53-5，NH 2C 2H 2-(CH 2O) n-C 2H 2NH 2)与3-马来酰亚胺基丙酸(MAL-)共反应，将MAL修饰于聚合物上，得到产物MAL-PEG-NH 2。
将ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride(ε-苄氧羰基-L-赖氨酸-N-羧基-环内酸酐，Lys(Z)-NCA)(1.96g，6.4mmol)溶于30mL N,N-二甲基甲酰胺(DMF)中，通过加入MAL-PEG-NH 2(2.0g，0.4mmol)，在35℃干燥氩气下搅拌72h，使其发生聚合反应。然后旋转蒸发溶剂，将产物溶解于25mL CHCl 3中，置于过量乙醚中沉淀析出，得到产物MAL-PEG-b-PZLL。
为了脱除苄基保护基，去除对氨基的保护，以便于后续氨基上的 氢原子可以被酰胺基取代，发生酰胺化反应：将2.0g MAL-PEG-b-PZLL溶于20mL CF 3COOH中，加入HBr(33wt.％溶于HOAc，2mL)。混合物在0℃搅拌2h，然后置于冷乙醚中沉淀析出。将此沉淀物溶于DMF中，然后在过量乙醚中再沉淀，以去除残留的CF 3COOH，得到产物MAL-PEG-b-PLL，产物在室温真空干燥。
将MAL-PEG-b-PLL(100mg，0.18mmol)溶解于10mL含D-甘露醇(D-mannitol，100mg，0.55mmol)的碳酸氢钠溶液(50mM,pH 8.5)中，随后加入1.7mL溶于甲醇的3-氟-4-羧基菲硼酸(3-fluoro-4-carboxy-phyenylboronic acid，FPBA)溶液，再加入耦合剂4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate(DMT-MM)(254mg，0.92mmol)，将反应混合物在25℃搅拌12h，使其发生酰胺化反应以将FPBA与MAL-PEG-b-PLL连接。使用透析袋(MWCO 3500)以0.01N NaOH溶液、0.01N HCl溶液和去离子水对反应溶液透析，对透析产物进行冷冻干燥，获得产物MAL-PEG-b-PLL/PBA。测得MAL-PEG-b-PLL/PBA的数均分子量Mn为4458g/mol，重均分子量Mw为5642g/mol。
上述方法得到的MAL-PEG-b-PLL/PBA进行核磁共振，所得谱图如图1所示。由图1可知，约7.3ppm处的峰为FPBA亚苯基的特征峰，证实了FPBA成功通过与胺基共轭连接在聚合物上。MAL-PEG-b-PLL/PBA的透射电镜结果如图2a、2d、2g所示，图中可以看到较大粒径聚合物周围有大量散在的小粒径颗粒，据图中比例尺 得到较大聚合物粒径仅100nm左右，且尚未形成胶束的结构。
(2)氟化钠的负载：将5mg MAL-PEG-b-PLL/PBA溶于4mL去离子水中，得到载体水溶液；同时将548μL氟化钠(Sodium fluoride,NaF)水溶液(5mg/mL)滴加至载体水溶液中，搅拌使其充分溶解，获得氟化钠-载体水溶液。
(3)胶束粒子的合成：配制浓度为5mg/mL的单宁酸(Tannic acid,TA)水溶液，取274μL单宁酸水溶液缓慢匀速滴加至氟化钠-载体水溶液中，室温下搅拌4h，所得溶液经20分钟超滤离心收集，获得负载氟化钠与单宁酸的载药胶束粒子(CLM@NaF)，CLM@NaF的透射电镜结果如图2b、2e、2h所示。
(4)多肽的连接：将1mg唾液蛋白多肽DpSpSEEKC(Pep)和载药胶束粒子(CLM@NaF)共分散于纯三乙醇胺(TEA)(pH＝8.0)中，室温下大力搅拌反应2h后，超滤离心20分钟收集，得到负载氟化钠、单宁酸并连接有唾液蛋白多肽的多肽载药胶束粒子(CLM@NaF-Pep)，CLM@NaF-Pep的透射电镜结果如图2c、2f、2i所示。
通过图2b、2e、2h与图2c、2f、2i可知，所合成的胶束粒子粒径为300nm左右，呈胶束经典的球形核-壳结构，形貌规则、分散性好。单宁酸和氟化钠的负载、多肽的连接并不会对胶束粒子的形貌、分散性产生不利影响。
对照品CLM(MAL-PEG-b-PLL/PBA+TA)的制备：直接将单宁酸水溶液滴加至聚合物MAL-PEG-b-PLL/PBA水溶液，搅拌混合自组装， 超滤纯化后得到CLM(MAL-PEG-b-PLL/PBA+TA)胶束粒子，其与CLM@NaF的区别仅在于未添加氟化钠水溶液，即合成了仅载TA的胶束粒子结构，未负载NaF。
上述方法得到的水溶性聚合物MAL-PEG-b-PLL/PBA、CLM(MAL-PEG-b-PLL/PBA+TA)、CLM@NaF、Pep和CLM@NaF-Pep的红外光谱检测结果如图3所示，结果显示，MAL-PEG-b-PLL/PBA、CLM(MAL-PEG-b-PLL/PBA+TA)和CLM@NaF在1653cm -1、1540cm -1、3426cm -1处出现峰值，对应于整个重复、分支的聚合物结构中的酰胺延伸段；多肽(Pep)在1640cm -1和1540cm -1处也有酰胺峰出现，在1720cm -1处有一个小峰，与半胱氨酸的巯基相对应；CLM@NaF-Pep巯基带消失，并在1034cm -1和916cm -1处产生两个新的峰，这些新的延伸出现在Pep与CLM@NaF表面接合的硫醚基团的产生过程中，此外，酰胺III的吸收也被掩盖，表明氨基酸的相互作用发生了实质性的改变。证实了CLM@NaF和Pep通过硫醚基团成功连接。
实施例2：药物pH控释测试
使用高效液相色谱仪和离子交换色谱仪检测CLM@NaF-Pep中的药物在不同pH条件下的释放情况。将2mL新制备的CLM@NaF-Pep分散液转移到一个透析袋(MWCO 3500)中，然后浸泡在不同pH的15mL的10mM PBS中，在37℃摇晃(转速100r/min)。在固定的时间间隔(每0.5h，1h，2h，4h，8h，12h，24h)收集1mL透析液直至24h，每收集一次透析液就再加入等体积的新鲜缓冲液，使透析液总体积保持不变。分别用高效液相色谱仪和离子交换色谱仪检测透析液中 单宁酸与氟化钠的含量。所有实验都设置了3个平行对照组，计算其平均值并进行比较，实验重复三次。
CLM@NaF-Pep对单宁酸和氟化钠的pH控释效果如图4所示：pH分别为7.4、5.0时，单宁酸的控释效果如图4A所示；pH分别为7.4、6.5、5.0时，氟化钠的控释效果如图4B所示。由图4可知，在口腔正常生理条件下(pH 7.4)，单宁酸和氟化钠在24h内的累计释放率分别约为40％和50％，具有长期缓释的效果，且体现了CLM@NaF-Pep较高的稳定性，而当龋损发生口腔微环境酸化(pH 5.0)时，单宁酸因硼酸酯键断裂而释放，呈现一初期突释与长期缓释的效果，24h内累计释放率可达近70％，而包裹其中的氟化钠也随着pH酸化、胶束裂解而快速释放，24h内累计释放率约80％。
实施例3：抑制变异链球菌生长效果测试
将培养至指数期的变异链球菌离心3min(5000rpm)收集，使用BHI培养基稀释至10 6CFU/mL，获得细菌悬浮液。
在pH分别为7.4、6.5和5.0的BHI培养基中添加CLM@NaF-Pep，配制成实验组分散液；以PBS为空白对照组溶液，以氯己定(CHX)为阳性对照组溶液，并分装到96孔板中(每孔160μL)。随后将40μL上述细菌悬浮液添加至实验组分散液和对照组溶液中混合均匀。将96孔板置于37℃培养箱中，分别培养0.5、1、2、4、8、12、24h，随后从每组中取100μL培养液，接种于一新96孔板中，使用酶标仪在600nm处记录各孔吸光度评价抗菌活性。所有实验都设置了3个复孔，计算其平均值进行比较，实验重复三次。
抑制变异链球菌(Streptococcus mutans)的生长曲线如图5所示，由图5可知，CLM@NaF-Pep对变异链球菌表现出高效的抗菌活性，且随着pH的降低，其抑制变异链球菌生长活性更强，变异链球菌在pH为5.0时杀菌效果甚至高于氯己定。
实施例4：抗致龋生物膜活性检测
制备一定量羟基磷灰石(HA)片(直径5mm，厚度2mm)，使用高压蒸汽灭菌法对HA片进行灭菌。分别用等量去离子水(ddH 2O)、CLM@NaF水分散液、Pep水溶液和CLM@NaF-Pep水分散液处理HA片，用PBS冲洗三次以去除未黏附在HA片上的物质，得到四组预处理HA片。将增长至指数期的变异链球菌用BHI培养基稀释至OD 600为0.5，然后将该细菌悬浮液接种至预处理HA片上，并置于48孔板中，使预处理HA片完全浸入变链悬浮液中，37℃下厌氧培养1.5h。随后吸出菌液，PBS小心漂洗去除未黏附菌液，加入适量含糖培养基，在37℃下厌氧培养24-48h。最后PBS冲洗样本，超声收集HA片上生物膜，将生物膜悬浮液连续稀释，铺于BHI琼脂平板上，培养48h后，计数菌落数。实验重复三次。抗变异链球菌生物膜能力的CFU(colony forming units)计数图如图6所示。
图6结果显示，与仅用去离子水处理的HA片(图6中用control代表)相比，单独使用多肽水溶液处理的HA片(图6中用Pep代表)无明显杀菌及抗生物膜活性，单独使用CLM@NaF水分散液处理的HA片(图6中用CLM@NaF代表)有一定的杀菌活性，但由于唾液的缓冲作用无法在口腔中留存，抗生物膜能力较微弱。使用CLM@NaF-Pep 水分散液处理的HA片(图6中用CLM@NaF-Pep代表)不仅具有抗菌黏附性，并且CLM@NaF-Pep能渗入生物膜并杀灭生物膜内细菌，证实所构建的CLM@NaF-Pep具有较高的牙表面致龋生物膜的清除效果。
实施例5：抑制釉质脱矿并促进其再矿化检测
收集无龋人第三磨牙，用慢速锯去除牙根，从近远中方向垂直于牙体长轴，将牙冠切割成唇舌两部分，然后将样本切成5*4*1.5mm 3牙釉质片。用600目和1200目砂纸抛光，然后在去离子水(ddH 2O)中超声20min。将牙片的釉质面用37％磷酸酸蚀1min，然后用ddH 2O冲洗1min。将牙片随机分成4组(空白对照组、CLM@NaF组、Pep组、CLM@NaF-Pep组)，分别浸入去离子水、CLM@NaF水分散液、Pep水溶液、CLM@NaF-Pep水分散液，然后用PBS冲洗去除未黏附物质。将各组牙片釉质面朝上，其他面用耐酸指甲油覆盖。随后分别将牙片在7mL脱矿液(2.2mM CaCl 2，2.2mM NaH 2PO 4,0.05M acetic acid,pH 4.5)中37℃培养5h；在7mL再矿化液(2.58mM CaCl 2·2H 2O,1.55mM KH 2PO 4,1mg/L NaF,180mM NaCl，50mM Tris-HCl,pH 7.6)中37℃培养24h。用等离子光谱仪检测浸润前后Ca和P浓度。计算釉质暴露面积，Ca/P损失量用μg/mm 2表示。同时釉质面和横截面的形态用扫描电镜定性分析，结果如图7所示。
由图7可知，在脱矿液中，牙釉质经多肽连接的胶束粒子处理后，钙磷离子丢失量显著低于空白对照组(图7中用control代表)、Pep组(图7中用Pep代表)和CLM@NaF组(图7中用CLM@NaF代表) (图7A)；同样在再矿化环境中，CLM@NaF-Pep组(图7中用CLM@NaF-Pep代表)也表现出更显著的钙磷离子增加效果(图7B)。扫描电镜图(图7C)也能发现，在脱矿液中空白对照组釉柱、釉柱间的结构和釉柱鞘均发生溶解，Pep组和CLM@NaF组釉柱结构也呈现不同程度的破坏，而CLM@NaF-Pep组釉质面仍相对平整光滑；在再矿化环境中，CLM@NaF-Pep组在脱矿釉质表面出现了大量新的定向有序生长的羟基磷灰石晶体。证实所构建的CLM@NaF-Pep具有显著的抑制牙釉质脱矿并促进其再矿化的效果。
以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (11)

  1. 一种口腔护理组合物，其特征在于，包括载药胶束，所述载药胶束包括聚合物胶束和物理包覆于所述聚合物胶束中的釉质修复药物，所述聚合物胶束式I所示结构的水溶性聚合物在单宁酸存在的水溶液中形成的胶束粒子，其外壳通过化学键与唾液蛋白多肽连接：
    Figure PCTCN2022131838-appb-100001
    其中，x、y、z为自然数，代表聚合度，所述水溶性聚合物的Mw重均分子量为5000～6000g/mol。
  2. 根据权利要求1所述的口腔护理组合物，其特征在于：x＝80～100、y＝2～10、z＝2～8。
  3. 根据权利要求1所述的口腔护理组合物，其特征在于：所述胶束粒子的粒径为200～400nm。
  4. 根据权利要求1所述的口腔护理组合物，其特征在于：所述釉质修复药物为氟化钠。
  5. 如权利要求1所述的口腔护理组合物，其特征在于：所述口腔护理组合物呈固体、糊剂、凝胶组合物或液体组合物形式。
  6. 如权利要求1所述的口腔护理组合物，其特征在于：所述组合物进一步包含抗龋齿剂、脱敏剂、粘度调节剂、稀释剂、表面活性剂、乳化剂、泡沫调节剂、pH调节剂、研磨剂、口感剂、甜味剂、调味剂、 着色剂、防腐剂、氨基酸、抗氧化剂、抗牙结石剂、氟离子源、增稠剂、用于预防或治疗口腔的硬或软组织的病状或病症的活性剂、粘合剂、增白剂及其组合。
  7. 权利要求1～6任一项所述的口腔护理组合物在制备用于减少或抑制患者口腔中的细菌的药物或制备用于促进釉质再矿化修复的药物中的用途。
  8. 一种在患者可拆卸的口腔装置中减少或抑制细菌或促进釉质再矿化修复的离体方法，其特征在于：包括向所述可拆卸的口腔装置的表面施用如权利要求1～6任一项所述的口腔护理组合物。
  9. 一种制备权利要求1～6任一项所述的口腔护理组合物的方法，其特征在于，包括：
    制备如式I所示结构的水溶性聚合物；
    使式I所示结构的水溶性聚合物与釉质修复药物在水溶液中混合，滴加单宁酸，得到分散在水溶液中的载药胶束；
    使载药胶束与唾液蛋白多肽在三乙醇胺中生成化学键，得到权利要求1所述的口腔护理组合物。
  10. 根据权利要求9所述的制备口腔护理组合物的方法，其特征在于：所述制备如式I所示结构的水溶性聚合物包括：
    使3-马来酰亚胺基丙酸通过酰胺键连接在聚乙二醇二胺一端，得到MAL-PEG-NH 2；
    使MAL-PEG-NH 2与ε-苄氧羰基-L-赖氨酸-N-羧基-环内酸酐发生聚合反应，得到MAL-PEG-b-PZLL；
    使MAL-PEG-b-PZLL脱苄基保护后与3-氟-4-羧基菲硼酸进行酰胺化反应，得到如式I所示结构的水溶性聚合物。
  11. 根据权利要求10所述的制备口腔护理组合物的方法，其特征在于：所述制备如式I所示结构的水溶性聚合物包括：
    将聚乙二醇二胺与3-马来酰亚胺基丙酸共反应，将MAL修饰于聚合物上，得到产物MAL-PEG-NH 2；
    将ε-苄氧羰基-L-赖氨酸-N-羧基-环内酸酐溶于N,N-二甲基甲酰胺中，通过加入MAL-PEG-NH 2，使其发生聚合反应，然后将产物溶解于CHCl 3中，置于过量乙醚中沉淀析出，得到产物MAL-PEG-b-PZLL；
    将MAL-PEG-b-PZLL溶于CF 3COOH中，加入HBr后搅拌，然后置于冷乙醚中沉淀析出，得到产物MAL-PEG-b-PLL后溶解于含D-甘露醇的碳酸氢钠溶液中，随后加入溶于甲醇的3-氟-4-羧基菲硼酸溶液，再加入耦合剂4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐，使其发生酰胺化反应以将FPBA与MAL-PEG-b-PLL连接，获得产物MAL-PEG-b-PLL/PBA。
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