Self-assembled polypeptide material for bionic remineralization of demineralized enamel and application of self-assembled polypeptide material

Abstract

The invention provides a self-assembled polypeptide material for bionic remineralization of demineralized enamel and application of the self-assembled polypeptide material, and belongs to the technical field of remineralization repair of demineralized enamel. The invention provides a self-assembled polypeptide material for bionic remineralization of demineralized enamel. The self-assembled polypeptide material comprises a self-assembled template unit, an enzyme digestion response unit and a bonding unit which are connected in sequence, the amino acid sequence of the self-assembly template unit is GYINFpSYE; the amino acid sequence of the enzyme digestion response unit is GPQGIWGQ; and the amino acid sequence of the bonding unit is DKTKREEVD. The self-assembled polypeptide material provided by the invention simultaneously realizes a template effect of an organic matter pre-organization, guides directional deposition of calcium and phosphorus ions, slowly degrades the organic matter, reduces organic occupation, and promotes dual effects of directional arrangement and phase change crystallization of amorphous calcium phosphate; the purposes of regularly and orderly arranged hydroxyapatite crystal formation, demineralized enamel structure repair and mechanical property recovery are achieved.

Description

Technical Field

[0001] This invention relates to the field of demineralized enamel remineralization repair technology, and in particular to a self-assembled polypeptide material for biomimetic remineralization of demineralized enamel and its application. Background Technology

[0002] The World Health Organization (WHO) reported in its 2022 Global Oral Health Report that nearly 3.5 billion people worldwide suffer from oral diseases, resulting in an economic burden of nearly US$387 billion. Dental caries is the most widespread non-communicable disease, affecting over 2.5 billion people in both primary and permanent teeth. The International Caries Detection and Assessment System (ICDAS) defines early-stage caries as: no obvious cavities, but a change in enamel color, appearing chalky white or brown, whether moistened or dry with saliva. Enamel demineralization, as an early manifestation of caries, requires timely and effective treatment, which is a crucial aspect of global caries prevention and control. Orthodontic treatment involves placing appliances on the smooth enamel surface. These space-occupying devices alter the oral environment, increase the difficulty of plaque removal, and promote plaque retention, thereby increasing the likelihood of enamel demineralization. Studies have shown that the incidence of enamel demineralization is as high as 79.3% in patients with an average age of 10-24 years who have received orthodontic treatment for 1-4 months.

[0003] Demineralization and remineralization occur constantly in the oral environment, and under physiological conditions, they are in balance. However, under the influence of plaque, food, or other iatrogenic factors, destructive demineralization is significantly aggravated, leading to an imbalance between demineralization and remineralization, and eventually resulting in defects in the hard tissues of the tooth. Remineralization treatment uses drugs or other techniques to guide the in-situ deposition of calcium and phosphate ions in the demineralized area, reducing demineralization and promoting restorative remineralization. Currently, the two commonly used clinical materials (sodium fluoride-calcium triphosphate and casein phosphopeptide-amorphous calcium phosphate) have somewhat limited therapeutic effects, inducing the formation of thin and loose aggregates of new enamel that cannot reproduce the unique hierarchical structure and excellent mechanical properties of natural enamel.

[0004] In the biological process of enamel development, ameloblasts secrete amelogenin and its degrading enzymes, which play a core role in enamel formation. However, the extraction, separation, and purification of human amelogenin are difficult and costly. Therefore, in recent years, research on the biomineralization mechanism of amelogenin-induced enamel formation and the design of amelogenin analogs to induce enamel regeneration has received widespread attention. Based on the study of the biomineralization mechanism of enamel, how to (1) simulate the structure and function of amelogenin and induce the directional crystallization of inorganic minerals through organic pre-organization, while achieving (2) the gradual and slow degradation of organic matter to reduce organic occupancy, has become the focus of research on biomimetic mineralization materials for enamel. However, most current studies only focus on one of these aspects and cannot take both properties into account. For example, the volatility of triethylamine and the slow reaction of RNA with RNases in the oral environment are used to simulate the degradation of organic matrix. Such research designs only simulate the role of amelogenin in capturing calcium and phosphorus ions, which is insufficient in realizing its template role in inducing directional crystallization. As a result, the original enamel prism growth extension shown in the study has limited efficacy when the enamel structure is severely damaged. For example, disulfide reducing agents induce lysozyme to undergo β-sheet transformation to form amyloid protein assemblies, and the hydrophilic ends of amphiphilic peptides are used to capture calcium and phosphorus ions. The hydrophobic interaction of alkyl chains regulates the directional arrangement of crystals. Such studies have achieved the induction of inorganic minerals with hierarchical structures by organic matter pre-organization, but the organic matter was not removed in time, and the mineralization degree and mechanical strength of the newly formed enamel need to be improved.

[0005] Therefore, research on remineralized biomaterials based on the natural process of enamel development and biomimetic amelogenin-induced biomineralization has not yet developed material designs that combine the template effect of organic pre-organization with its slow degradation function. Summary of the Invention

[0006] The present invention aims to develop remineralized materials that combine the template effect of pre-organic materials with slow degradation function, so as to achieve enamel-like structure regeneration, demineralized enamel structure repair and mechanical property restoration.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a self-assembled polypeptide material for biomimetic remineralization of demineralized enamel, the polypeptide comprising a self-assembled template unit, an enzyme cleavage response unit, and an adhesive unit connected in sequence; The amino acid sequence of the self-assembled template unit is GYINFpSYE; The amino acid sequence of the enzyme digestion response unit is GPQGIWGQ; The amino acid sequence of the adhesive unit is DKTKREEVD.

[0008] The present invention also provides a method for preparing a self-assembled polypeptide material for biomimetic remineralization of demineralized enamel, wherein the preparation method is a standard polypeptide solid-phase synthesis method.

[0009] The present invention also provides the application of the self-assembled polypeptide material described above in the preparation of demineralized enamel remineralization restoration products.

[0010] Preferably, the repair product is used to promote the regeneration of enamel-like hydroxyapatite crystals.

[0011] Preferably, the repair product is an oral administration formulation.

[0012] Preferably, the oral administration formulation uses the self-assembled polypeptide material as the main active ingredient and includes pharmaceutically acceptable excipients.

[0013] Preferably, the oral medication is a mouthwash, ointment, toothpaste, spray, patch, or chewing gum.

[0014] This invention provides a self-assembled polypeptide material for the biomimetic remineralization of demineralized enamel, used as a biomimetic remineralization material for repairing demineralized enamel. The self-assembled polypeptide material can adsorb onto the surface of demineralized enamel through electrostatic attraction and enrich calcium and phosphate ions from the salivary environment. It self-assembles into nanofibers that guide the directional deposition of calcium and phosphate ions. Utilizing the stimuli-responsive interaction between enzymatic units and intrinsic salivary matrix metalloproteinases, it promotes the directional arrangement of amorphous calcium phosphate, phase transition crystallization to form regular and ordered hydroxyapatite crystals, reduces organic matter occupancy, and increases crystallinity. This self-assembled polypeptide material can achieve the regeneration of a regular and ordered enamel structure, restoring the structural integrity and mechanical properties of the demineralized enamel surface.

[0015] Compared with existing technologies and products, the self-assembled polypeptide material provided by this invention simultaneously achieves the template effect of organic matter pre-organization, guides the directional deposition of calcium and phosphate ions, and has the dual effects of slow degradation of organic matter, reduction of organic occupancy, promotion of directional arrangement of amorphous calcium phosphate and phase transformation crystallization, thereby achieving the purpose of forming regularly ordered hydroxyapatite crystals, repairing demineralized enamel structure and restoring mechanical properties. Attached Figure Description

[0016] Figure 1 The structural formula for a self-assembled polypeptide material; Figure 2 To observe the morphological changes of self-assembled peptide materials from 0 to 24 h using TEM; Figure 3 To analyze the enzymatic digestion status of self-assembled peptide materials from 0 to 4 h using HPLC; Figure 4 To analyze the enzymatic digestion status of self-assembled peptide materials over 4-24 h using HPLC; Figure 5 MALDI-TOF MS was used to analyze the enzymatic digestion status of self-assembled peptide materials over 4–24 h. Figure 6 To observe the adhesion effect of self-assembled peptide materials on demineralized enamel surfaces using CLSM; Figure 7 To observe the effect of self-assembled peptide materials on in vitro induced demineralization and remineralization of enamel; Figure 8 To test the effect of remineralization on the mechanical properties of self-assembled peptide materials through nanoindentation testing; Figure 9 The effect of SEM self-assembled peptide materials on inducing demineralized enamel remineralization in the rat oral cavity. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1

[0019] Preparation of self-assembled polypeptide materials

[0020] The self-assembled peptide material comprises a self-assembling template unit (GYINFpSYE, SEQ ID NO: 1, where S in the sequence shown in SEQ ID NO: 1 is the pre-phosphorylated structure due to limitations of the sequence software), an enzyme digestion response unit (GPQGIWGQ, SEQ ID NO: 2), and an adhesive unit (DKTKREEVD, SEQ ID NO: 3). It was prepared using the standard solid-phase peptide synthesis method (SPPS), and the specific steps are as follows: 2-Chlorotriphenylmethyl resin was selected as the synthesis carrier, and Fmoc-amino acid (GYINFpSYEGPQGIWGQDKTKREEVD) and palmitic acid were used as raw materials.

[0021] 1) Weigh 0.5 mmol of N-fluorenylmethoxycarbonyl-aspartic acid resin into a polypeptide synthesis tube and swell it in dichloromethane (DCM) in a shaker at room temperature for 3 h. Then remove the DCM by vacuum filtration.

[0022] 2) Add 10 ml of deprotectant (DMF:DBU=98:2, anhydrous piperazine 5%) according to the ratio, and react in a shaker at room temperature for 20 min.

[0023] 3) Rinse the resin alternately with DCM and DMF, 3 times each; take a small amount of resin and the detection reagent (0.5 g ninhydrin, 10 ml anhydrous ethanol) in a centrifuge tube, heat in boiling water for 45 s, and the color turns purple, indicating that the Fmoc group has been completely removed. 4) Mix 5.0 mmol valine, 5.0 mmol HBTU and 10 mL coupling agent (DMF:N-methylmorpholine = 19:1), react in a shaker at room temperature for 10 min, then add to the reaction system of step 1), and react in a shaker at room temperature for 1 h to couple the mixture. 5) Remove the reaction solution by vacuum filtration, and wash the resin with DCM and DMF alternately, 3 times each; Take a small amount of resin and detection reagent into a centrifuge tube, heat with boiling water for 45 seconds, and if there is no color change, it indicates that the valine coupling is successful; 6) Repeat steps 3) to 5) until the last amino acid glycine of the target polypeptide sequence is successfully coupled; 7) Add 10 ml of methanol to shrink the resin, react at room temperature for 15 min, remove methanol by vacuum filtration, and transfer the resin to a serum bottle. Add 10 mL of lysis buffer (trifluoroacetic acid: triisopropylsilane: ddH2O = 9.5 : 0.25 : 0.25, volume ratio), and react in an ice-water bath with magnetic stirring for 2.5 h; 9) The mixture was separated into solid and liquid phases using a syringe and cotton. The collected reaction solution was dried under nitrogen until it reached a viscous state. This viscous solution was then added dropwise to ice-cold ether, and the resulting white precipitate was the crude polypeptide product. This was then purified by preparative liquid chromatography and freeze-dried to obtain the self-assembled polypeptide material. The specific structure is shown below. Figure 1 As shown.

[0024] After the self-assembled peptide material was prepared, it was dissolved in DMSO solvent to prepare a 10 mg / ml solution. 10 μL of the above solution was placed in a centrifuge tube, and 990 μL of ultrapure water was slowly added to the centrifuge tube. The concentration of the resulting mixed solution was 0.1 mg / ml.

[0025] Example 2

[0026] Characterization of self-assembled peptide materials

[0027] The morphological changes of self-assembled peptides (0.1 mg / ml) incubated at 37°C for 0–24 h were recorded using transmission electron microscopy (TEM) and circular dichroism spectroscopy (CD). Figure 2 The results showed that the self-assembled peptides could achieve a morphological transformation from nanoparticles to nanofibers in a solution of H2O / DMSO=99:1 (0.1 mg / ml), with β-sheet content ranging from 25.9% to 32.2% and showing a certain increasing trend over time.

[0028] Table 1. Changes in the secondary structure content of self-assembled peptide materials.

[0029] Example 3

[0030] Enzymatic degradation characterization of self-assembled peptide materials

[0031] High-performance liquid chromatography (HPLC) was used to record the enzymatic cleavage of a self-assembled peptide (0.1 mg / ml) by salivary matrix metalloproteinase (final concentration 0.1 μg / ml, purchased from Taoshu Biotechnology, human recombinant protein MMP-8, TMPY-01884). Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) was used to detect the molecular structure of the cleavage products. Results showed that the cleavage reaction gradually completed within 0-4 hours. Figure 3 ), 4—24h Figure 4 The product remained relatively stable, and MALDI-TOF MS results showed that the product consisted of the two fragments before and after the restriction site. Figure 5 This demonstrates that salivary matrix metalloproteinases have the function of precise enzymatic degradation of self-assembled peptides, and can complete the enzymatic degradation within 4 hours.

[0032] Example 4

[0033] Adhesion characterization of self-assembled peptide materials

[0034] The adhesion of self-assembled peptide materials to demineralized enamel surfaces was recorded using laser scanning confocal microscopy (CLSM). Specifically, the self-assembled peptides were fluorescently labeled with fluorescein isothiocyanate (FITC) at the same concentration as previously described (0.1 mg / ml).

[0035] The specific procedure for establishing an in vitro demineralization model is as follows: Freshly extracted human molars or premolars are cut under cold water using a low-speed diamond saw. Enamel sections are then polished sequentially with 600, 800, 1200, 2000, and 2500 grit silicon carbide sandpaper, followed by ultrasonic cleaning in deionized water for 30 minutes to obtain enamel sections. After etching with 37% phosphoric acid for 30 seconds, the sections are ultrasonically cleaned in deionized water for 30 minutes to obtain demineralized enamel sections, thus obtaining the in vitro demineralization model.

[0036] Human demineralized tooth enamel sections were soaked in fluorescent peptide solution for 10 minutes, then placed in distilled water and subjected to continuous ultrasonic stimulation for 0-120 minutes as interference conditions. After the sections were removed, CLSM was performed to observe the fluorescence distribution and characterize the adhesion of the self-assembled peptide material on the demineralized enamel surface. Figure 6 The results showed that the self-assembled polypeptide material had good adhesion to demineralized enamel surfaces and could resist the scavenging effect of continuous ultrasonic stimulation for up to 120 min in vitro.

[0037] Example 5

[0038] Characterization of mineralization properties of self-assembled peptide materials in an in vitro demineralization model

[0039] Scanning electron microscopy (SEM) was used to observe the enamel structure repair results of human demineralized tooth enamel sections treated with self-assembled peptides after 7 days of induced mineralization in artificial saliva. The improvement effect on mechanical properties was tested using a nanoindenter.

[0040] The composition of artificial saliva is: 1.5 mM K2HPO4, 2.5 mM CaCl2, 10.0 mM HEPES, 150 mM NaCl, 0.77 mM NaF, pH 7.0 ± 0.1.

[0041] SEM results ( Figure 7 The results showed that self-assembled peptide materials, when co-incubated with artificial saliva for 24 hours, could induce the formation of regular, ordered, enamel-like hydroxyapatite crystals, restoring surface integrity. Cross-sectional images showed that the newly formed and primary enamel crystals were aligned and had no clear boundaries. Nanoindentation test results ( Figure 8 The results showed that the mechanical properties of the glaze decreased significantly after demineralization, but the mechanical properties could be restored to the level of the natural glaze after 7 days of self-assembled peptide-induced mineralization, with no significant difference.

[0042] Example 6

[0043] Characterization of mineralization properties of self-assembled peptide materials in a rat demineralization model

[0044] Scanning electron microscopy (SEM) was used to observe the enamel structure repair results of human demineralized enamel sections treated with self-assembled peptides after 7 days of induced mineralization in the rat oral cavity. The improvement in mechanical properties was also observed after 7 and 14 days of induced mineralization in the rat oral cavity using a nanoindenter.

[0045] SEM results ( Figure 9 The results showed that after 7 days of induction in the rat oral environment, the self-assembled peptide material could induce the formation of regular, ordered, enamel-like hydroxyapatite crystals, restoring its surface integrity. Nanoindentation tests showed that the mechanical properties were significantly improved after 7 days of induced mineralization by the self-assembled peptide, with hardness and elastic modulus of 2.52 ± 0.40 GPa and 86.16 ± 5.00 GPa, respectively; after 14 days, the mechanical properties recovered to the level of natural enamel, with hardness and elastic modulus of 4.09 ± 0.26 GPa and 105.00 ± 0.62 GPa, respectively. These results indicate that the self-assembled peptide material can induce the formation of regular, ordered hydroxyapatite crystals in the rat oral environment, achieving demineralized enamel structure repair and improved mechanical properties.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A self-assembled polypeptide material for biomimetic remineralization of demineralized tooth enamel, characterized in that, The polypeptide comprises a self-assembled template unit, an enzyme digestion response unit, and an adhesion unit connected in sequence. The amino acid sequence of the self-assembled template unit is GYINFpSYE; The amino acid sequence of the enzyme digestion response unit is GPQGIWGQ; The amino acid sequence of the adhesive unit is DKTKREEVD.

2. The method for preparing a self-assembled polypeptide material for biomimetic remineralization of demineralized tooth enamel as described in claim 1, characterized in that, The preparation method is the standard polypeptide solid-phase synthesis method.

3. The application of the self-assembled polypeptide material of claim 1 or the self-assembled polypeptide material prepared by the method of claim 2 in the preparation of demineralized enamel remineralization restoration products.

4. The application as described in claim 3, characterized in that, The repair product is used to promote the regeneration of hydroxyapatite crystals with an enamel-like structure.

5. The application as described in claim 3, characterized in that, The repair product is an oral medication.

6. The application as described in claim 5, characterized in that, The oral administration formulation uses the self-assembled polypeptide material as the main active ingredient and includes pharmaceutically acceptable excipients.

7. The application as described in claim 5, characterized in that, The oral medication preparations are mouthwash, ointment, toothpaste, spray, patch, or chewing gum.

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