Self-assembling polypeptide TKH and application thereof

Abstract

The application discloses a kind of self-assembled polypeptide TKH and its application, belong to the field of biological medicine technology.The amino acid sequence of polypeptide TKH is as follows:TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLWH LLHHLLH.The composition of the application includes self-assembled polypeptide TKH, polypeptide derivative or polypeptide pharmaceutically acceptable salt, ester, and pharmaceutically acceptable adjuvant.The application further discloses the application of self-assembled polypeptide TKH, polypeptide derivative or its pharmaceutically acceptable salt or ester in preparing oral care composition for dental hard tissue remineralization, and in preparing medical material for treating or preventing oral diseases.The self-assembled polypeptide TKH of the application shows significantly enhanced self-assembly ability, liquid phase separation behavior and hydroxyapatite adsorption characteristics, not only has more excellent remineralization performance, but also can simultaneously inhibit streptococcus mutans biofilm formation and acid production activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a self-assembled polypeptide TKH and application thereof. BACKGROUND

[0002] Dental caries is a global high-incidence disease characterized by pathological demineralization of hard tissue. Compared with enamel, dentin has a lower mineral content and a higher proportion of organic matrix, which makes it more susceptible to caries progression and is accompanied by a higher risk of dentin hypersensitivity (DH). Under pathological conditions, caries-causing microbial metabolites or exogenous acidic substances can destroy the dynamic balance between demineralization and remineralization on the surface of dental hard tissue, leading to irreversible mineral dissolution, dentin tubule expansion, and collagen matrix exposure. The above changes not only promote the further development of caries, but also significantly exacerbate the symptoms of DH through hydrodynamic mechanisms.

[0003] Currently, the commonly used desensitizing agents and remineralization preparations in clinical practice, such as fluoride, bioactive glass, and resin adhesives, still have a series of limitations, including suboptimal biocompatibility, shallow occlusion depth (usually limited to a range of about 10 μm below the tubule opening), poor mechanical wear resistance, and insufficient stability in acidic environments, which seriously affect their long-term efficacy. Therefore, developing a biomimetic remineralization strategy that can simultaneously achieve intrafibrillar mineralization and deep occlusion of dentin tubules has become an important research direction for promoting dental treatment from traditional repair to biological regeneration.

[0004] Biomimetic remineralization technology provides a new approach to achieving deep occlusion of dentin tubules (DTs) by introducing mineral ions or biomimetic mineralization templates into dentin to induce in situ formation of mineral phases. Under physiological conditions, the remineralization process of dentin is finely regulated by non-collagenous proteins (NCPs). In particular, intrafibrillar mineralization (i.e., deposition of hydroxyapatite (HAP) within collagen fibers) is crucial for restoring the mechanical properties of dentin. This process relies on the penetration and transformation of amorphous calcium phosphate (ACP) into collagen fibers. Therefore, ideal biomimetic remineralization should simultaneously achieve: (1) intrafibrillar mineralization to repair the mechanical integrity of the collagen network; and (2) extracellular mineralization to effectively occlude dentin tubules and reduce sensitivity.

[0005] The key mechanism of intrafibrillar mineralization is that ACP precursors need to penetrate into the nanometer gap region of about 2 nm inside the collagen fibers. This process is achieved through liquid-liquid phase separation (LLPS), and the metastable ACP droplets formed can penetrate into the collagen fibers by capillary force. This non-classical mineralization pathway enables HAP to be deposited in the intermolecular gap of collagen. The efficiency of intrafibrillar mineralization is highly dependent on the stability of NCPs to calcium and phosphate ions. In the remineralization system based on polyelectrolytes, the electrical neutrality and osmotic pressure balance of the system need to be maintained at the same time: specifically, the osmotic pressure difference established inside and outside the collagen fibers through Gibbs-Donnan equilibrium can further drive the ACP to penetrate inward and promote intrafibrillar mineralization.

[0006] Self-assembling peptides are a class of biomaterials that can form charged aggregates through environmentally responsive LLPS, and their mechanism of action exactly fits the two key links of intrafibrillar mineralization: (1) LLPS-mediated penetration of ACP precursors into collagen; (2) mineral deposition regulated by Gibbs-Donnan equilibrium. Therefore, the technical route based on peptide self-assembly shows broad prospects in achieving biomimetic regeneration of dentin.

[0007] Therefore, providing a self-assembling peptide capable of simulating the function of natural non-collagen proteins, synchronously achieving deep dentin tubule occlusion and efficient intrafibrillar mineralization, has become a technical problem to be solved by those skilled in the art. SUMMARY

[0008] The purpose of the present application is to provide a novel alpha-hairpin peptide TKH with enhanced self-assembly ability, which is obtained by introducing a designed GKG connecting fragment into the alpha-helix peptide TVH-19 with antibacterial and mineral deposition promoting functions, and has significantly enhanced self-assembly ability, liquid phase separation behavior (LLPS) and hydroxyapatite adsorption characteristics. The peptide shows better remineralization performance than TVH-19, can effectively induce mineral deposition, occlude dentin tubules in collagen fibers and demineralized dentin models, and synchronously inhibit Streptococcus mutans biofilm formation and acid production activity, and is a novel polypeptide-based biomaterial with efficient remineralization and broad-spectrum antibacterial functions.

[0009] Another purpose of the present application is to provide the application of the polypeptide TKH.

[0010] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0011] The first aspect of the present application discloses a self-assembling polypeptide TKH, a polypeptide derivative or a pharmaceutically acceptable salt or ester of a polypeptide, the amino acid sequence of the polypeptide TKH is shown in SEQ ID No. 1, and is specifically as follows:

[0012] TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLWHLLHHLLH.

[0013] The amino acid sequence of the polypeptide TVH-19 is shown as SEQ ID No. 2.

[0014] In some embodiments of the present application, the polypeptide derivative is a C-terminal amide-modified polypeptide TKH.

[0015] In some embodiments of the present application, the pharmaceutically acceptable salt is a hydrochloride, a sulfate, an acetate, a methanesulfonate, a succinate, a fumarate, a citrate, a malate or an organic amine salt.

[0016] The second aspect of the present application discloses a composition containing the self-assembling polypeptide TKH, the polypeptide derivative or the pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable excipient.

[0017] The third aspect of the present application discloses the use of the self-assembling polypeptide TKH, the polypeptide derivative or the pharmaceutically acceptable salt or ester thereof in the preparation of an oral care composition for the remineralization of hard dental tissues.

[0018] In some embodiments of the present application, the oral care composition has at least one of the following functions:

[0019] (1) promoting the mineralization of collagen fibers;

[0020] (2) promoting mineral deposition;

[0021] (3) occluding dentin tubules;

[0022] (4) inhibiting the formation of Streptococcus mutans biofilm and acid production.

[0023] The oral care composition is a toothpaste, a mouthwash, a mouth spray, a tooth powder, an oral spray, an oral gel or a tooth whitening patch.

[0024] The fourth aspect of the present application discloses the use of the self-assembling polypeptide TKH, the polypeptide derivative or the pharmaceutically acceptable salt or ester thereof in the preparation of a medical material for treating or preventing oral diseases.

[0025] In some embodiments of the present application, the medical material has the dual functions of remineralization and antibiosis.

[0026] In some embodiments of the present application, the medical material is used for inducing dentin remineralization, treating dentin hypersensitivity, inhibiting the progression of caries or preventing enamel demineralization.

[0027] In some embodiments of the present application, the medical material comprises a dental adhesive, a desensitizer, a pit and fissure sealant, a dental hydrogel, a subgingival irrigant, or a bioactive material for dental restoration.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The polypeptide TKH retains the antibacterial and mineralization-promoting functions of its precursor polypeptide TVH-19, and significantly improves a plurality of biological properties by introducing a GKG connecting fragment. The polypeptide TKH has significantly enhanced self-assembly ability, liquid-liquid phase separation behavior (LLPS), and hydroxyapatite adsorption characteristics, and exhibits better remineralization performance and simultaneously inhibits the formation of Streptococcus mutans biofilm and acid production activity.

[0030] The self-assembly ability of the polypeptide TKH is significantly better than that of the polypeptide TVH-19. The polypeptide TKH can maintain a higher beta-sheet content in various buffer systems and can efficiently drive the self-assembly process through liquid-liquid phase separation (LLPS) behavior at a lower concentration. This advantage has been verified by ThT fluorescence experiments and the Tyndall effect experiment.

[0031] In terms of mineralization performance, the polypeptide TKH also has obvious advantages. It can effectively stabilize amorphous calcium phosphate (ACP) at a lower concentration, exhibit more excellent hydroxyapatite adsorption capacity on demineralized dentin surfaces, and more efficiently induce collagen fiber mineralization. The remineralization experiment further shows that the demineralized dentin treated with TKH not only has more uniform mineral deposition and more ideal dentin tubule sealing effect, but also forms hydroxyapatite with higher crystallinity.

[0032] In addition, TKH also exhibits enhanced biofilm inhibition ability. It can effectively inhibit the formation of Streptococcus mutans biofilm and its metabolic activity at a low concentration, and more significantly delay the pH drop to inhibit the generation of acid corrosion environment related to tooth demineralization. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The figure is a conformational diagram of the THK polypeptide and the TVH-19 polypeptide.

[0034] Figure 2 The figure is a mass spectrum of the THK polypeptide.

[0035] Figure 3 The figure is a high-performance liquid chromatogram of the THK polypeptide.

[0036] Figure 4 The figure is a microscopic image of a TKH polypeptide solution with a concentration of 0.4 mM and a pH of 7. The right image is a magnified view of the left image.

[0037] Figure 5 The figure is a phase diagram curve of Example 3.

[0038] Figure 6 Circular dichroism spectra of TKH polypeptide and TVH-19 polypeptide; the ordinate Molar ellipticity is molar ellipticity, unit deg·cm 2 ·d / mol, the abscissa Wavelength is wavelength, unit nm.

[0039] Figure 7 Statistical column chart of protein secondary structure content; wherein α-helix represents α-helix structure, and β-sheet represents β-sheet structure.

[0040] Figure 8 ThT fluorescence intensity-concentration relationship diagram of TKH polypeptide and TVH-19 polypeptide, the ordinate is ThT fluorescence value, unit FU; the abscissa Concentration is polypeptide concentration, unit μM.

[0041] Figure 9 ThT fluorescence emission spectrum of TKH polypeptide and TVH-19 polypeptide, the ordinate is ThT fluorescence value, unit FU; the abscissa Wavelength is wavelength, unit nm.

[0042] Figure 10 The results of the Tyndall effect investigation diagram.

[0043] Figure 11 The results of calcium and phosphorus stability experiment diagram, wherein the lower diagram is the micrograph of the upper diagram.

[0044] Figure 12 Infrared spectra of TKH polypeptide and TVH-19 polypeptide, the ordinate Transmitance% represents transmittance%, and the abscissa avenumber is wave number, unit cm -1 , AMIDE I and AMIDE II represent amide I and amide II respectively; AMIDEN-H Stretch represents amide N-H stretching vibration.

[0045] Figure 13 TEM diagram of collagen fiber mineralization.

[0046] Figure 14 Transmission electron microscope and element scanning diagram of demineralized dentin remineralization; each column from top to bottom is low-power diagram after demineralized dentin remineralization, high-power diagram after demineralized dentin remineralization, Ca element distribution diagram, P element distribution diagram.

[0047] Figure 15 X-ray diffraction diagram.

[0048] Figure 16 Crystal violet staining diagram of biofilm formation inhibition experiment.

[0049] Figure 17 Concentration-absorbance graph for biofilm quantitative analysis.

[0050] Figure 18 Comparison chart of the influence of different concentrations of TKH polypeptide and TVH-19 polypeptide on pH change in the biofilm formation process. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0052] The TVH-19 polypeptide described in the embodiments of the present application is prepared according to the method of patent embodiment 1 of publication number CN 107353351 A. Unless otherwise specified, the polypeptide solution in the embodiments of the present application is prepared by dissolving polypeptide powder in 10 mM PBS buffer and freshly prepared at room temperature.

[0053] The English abbreviations involved in the embodiments of the present application correspond to the following Chinese names:

[0054] DCM: dichloromethane;

[0055] DMF: N,N-dimethylformamide;

[0056] HBTU: O-benzotriazol-tetramethylurea hexafluorophosphate;

[0057] HATU: 2-(7-azobenzotriazol)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0058] DIPEA: N,N-diisopropyl ethylamine;

[0059] TFA: trifluoroacetic acid;

[0060] TIPS: tert-butyl isopropyl dimethoxy silane;

[0061] Tris: tris(hydroxymethyl)aminomethane;

[0062] SDS: sodium dodecyl sulfonate;

[0063] Example 1

[0064] In this embodiment, the polypeptide TKH is synthesized by solid-phase Fmoc-based chemical method, and its amino acid sequence is as follows: TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLHHLLH, which is synthesized by Hangzhou Bingtuo Biotechnology Co., Ltd. The specific steps are as follows:

[0065] 1. Add the target mass of Rink Amide resin into the synthesis column, add DCM to swell, and drain; then wash and replace with DMF.

[0066] 2. Resin pretreatment and Fmoc removal: swell in DCM for 30 min, replace with DMF; deprotection with 20v% piperidine / DMF for 10 min + 10 min, and wash with DMF.

[0067] 3. Coupling cycle: use Fmoc-Xaa 4 eq + HBTU (or HATU) 3.9 eq + DIPEA 6~8 eq, DMF, room temperature, 45~60 min for each residue; determine by Kaiser test, and double coupling if necessary. Sequentially couple according to the sequence: TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLHHLLH.

[0068] 4. Cleavage and precipitation: TFA / TIPS / H2O = 95:2.5:2.5 (v / v / v), room temperature, 2~3 h; precipitate with cold anhydrous ether, centrifuge, and wash with ether 1~2 times to obtain the crude peptide.

[0069] 5. Purification by preparative RP-HPLC: mobile phase water / acetonitrile + 0.1% TFA; linear gradient 5~60% B (acetonitrile), 30~60 min; collect the main peak, remove the organic solvent by rotary evaporation, and freeze-dry to obtain the pure product.

[0070] 6. Quality confirmation: analytical RP-HPLC (C18, purity should be ≥90%); ESI-MS to confirm that the molecular weight is consistent with the theoretical value.

[0071] 7. Dispensing and storage: 5 mg / tube, dry storage at -20℃. The conformational diagram of the THK polypeptide and the TVH-19 polypeptide is shown in Figure 1 , the mass spectrum of TKH is shown in Figure 2 , and the high-performance liquid chromatogram is shown in Figure 3 .

[0072] Example 2

[0073] This example discloses a method for measuring liquid-liquid phase separation (LLPS) of the polypeptide of the present application and plotting a phase diagram thereof.

[0074] TKH peptide solutions with concentrations of 0.1, 0.2, 0.3, and 0.4 mM were prepared using buffer systems (20 mM Tris, 0.2 M NaCl, and 10 mM PBS) at different pH values ​​(5.5, 6.0, 6.5, 7.0, and 7.5). Turbidity of the samples was measured at λ=350 nm using 96-well plates at each pH and concentration. Each experiment was repeated three times (n=3). Phase diagrams were plotted on a concentration-pH coordinate system based on the corresponding phase transition pH values ​​at different concentrations, and phase transition curves were generated through fitting. Furthermore, the 0.4 mM TKH peptide solution at pH 7 was imaged under a microscope.

[0075] A micrograph of a 0.4 mM, pH 7 TKH peptide solution is attached. Figure 4 As shown in the attached figure, the phase transition curve of the TKH peptide is... Figure 5 As shown in the figure. The results indicate that the TKH peptide exhibits a significant tendency for liquid-liquid phase separation (LLPS), which may further promote its self-assembly.

[0076] Example 3

[0077] This embodiment discloses the characterization of the peptide self-assembly performance of the present invention.

[0078] 1. Circular dichroism (CD) detection

[0079] Spectral data were acquired at room temperature using a circular dichroism spectrometer in the wavelength range of 190–60 nm. Three 200 μM sample solutions of TKH peptide and TVH-19 peptide were prepared using the following buffer systems: (1) 4.2 mM PBS (pH 7.0); (2) 25 mM SDS; (3) 4.2 mM PBS (pH 7.0) containing 25 mM SDS. The samples were incubated at 37°C for 24 hours before measurements were taken. All spectral acquisitions used a 2 nm bandwidth and a 1 nm ss. -1 The scan rate was determined, and the results were normalized to an average residue ellipticity value. Finally, the circular dichroism spectral data were analyzed using CDpro software.

[0080] The results are as follows Figure 6 and Figure 7 As shown, the TKH peptide exhibits typical β-sheet conformation characteristics in circular dichroism spectroscopy, indicating that it undergoes self-assembly in solution. CDpro analysis showed that the β-sheet content of the TKH peptide was 44.5% in SDS buffer and 37.3% in PBS solution, both significantly higher than the corresponding values ​​of the TVH-19 peptide under the same conditions.

[0081] 2. ThT fluorescence detection

[0082] TKH polypeptide solution and TVH-19 polypeptide solution (both at a concentration of 0-100 mM) were mixed with 10 pg / mL of thioflavin T (ThT) solution, and incubated at room temperature in the dark for 10 minutes. Fluorescence measurement was performed using an Infinite M200 PRO multifunctional microplate reader (Tecan). One group of experiments was detected at a fixed wavelength (λex=438 nm, λem=495 nm), and the other group was scanned in the emission spectrum ranging from 400 to 580 nm under λex=380 nm excitation. All fluorescence intensity data were normalized based on the ThT group containing only PBS buffer. Three replicate wells were set for each condition.

[0083] The results are shown in the accompanying Figure 8 As the concentration of TKH polypeptide increased, the ThT fluorescence intensity increased significantly, and was higher than that of TVH-19 polypeptide at the same concentration (p<0.05), indicating that the self-assembly behavior of TKH polypeptide was concentration-dependent, and the assembly degree was higher than that of TVH-19 polypeptide.

[0084] The emission spectrum is shown in the accompanying Figure 9 The TKH polypeptide showed a clear fluorescence peak near 480 nm, further confirming its stronger self-assembly forming ability.

[0085] 3. Detection of the Tyndall effect

[0086] A laser lamp was used to irradiate the sample tube, and the Tyndall effect of TKH polypeptide (0-200 mM) and TVH-19 polypeptide (0-400 mM) solutions prepared with 10 mM PBS buffer at different concentrations were detected.

[0087] The results are shown in the accompanying Figure 10 Under laser irradiation, TKH polypeptide showed a clear Tyndall effect even at a low concentration (3.125 mM), while TVH-19 polypeptide did not show a visible Tyndall phenomenon until 50 mM. This result further confirms that TKH polypeptide has a stronger self-assembly ability. Polypeptides mimic the formation process of natural dentin by self-assembly, and precisely guide and regulate the nucleation, growth and arrangement of hydroxyapatite crystals by constructing a highly ordered nanofiber scaffold network, thereby achieving the biomimetic repair and regeneration of dental hard tissue.

[0088] Example 4

[0089] This example evaluates the adsorption capacity of TKH polypeptide of the present application on the surface of demineralized dentin and its performance in inducing mineralization within collagen fibers

[0090] 1. Calcium-phosphorus stability experiment:

[0091] Na2HPO4 and CaCl2 powders were dissolved in deionized water (DDW) to prepare phosphate and calcium ion stock solutions, respectively. TKH and TVH-19 polypeptide stock solutions were also prepared using DDW. To form the mineralization stable solution, phosphate stock solution was mixed with TKH and TVH-19 polypeptide solutions first, and then calcium stock solution was added. The final mineralization medium contained 4.5 mM CaCl2, 2.1 mM Na2HPO4, 50-400 mM TKH polypeptide, and 100-800 mM TVH-19 polypeptide. The mixed solution was incubated at 37 °C for 24 hours, and the turbidity of the solution was monitored using colorimetric method. Then the mixed solution was centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded. 10 μL of the precipitate was taken for microscope observation to detect the presence of calcium phosphate deposition.

[0092] Two control groups were set up: pure deionized water as blank control (DDW group), and solution containing only phosphate stock solution as negative control (CaP group). The results are shown in FIG. 1: Figure 11 As shown in FIG. 1, TKH polypeptide can effectively stabilize amorphous calcium phosphate (ACP) precursor at a concentration of 100 mM, while TVH-19 polypeptide needs to reach 400 mM to show similar ACP stabilizing effect.

[0093] 2. ATR-FTIR analysis

[0094] Preparation of demineralized dentin samples: Clinically discarded caries-free wisdom teeth (third molars) were stored in PBS containing thymol and stored at 4 °C. The total storage time was limited to 4 weeks before use. The teeth were cut near the pulp-enamel junction using a low-speed diamond saw to prepare dentin discs with a thickness of 3.50 ± 0.50 mm, and the discs were embedded in acrylic resin. Then, 800, 1500, 2000, and 3000 grit diamond polishing papers were used in sequence to remove the residual enamel on the dentin discs and to polish them finely. The polished dentin discs were ultrasonically cleaned for 15 minutes to remove the smear layer.

[0095] The microhardness of the dentin surface (SMH) was tested using a Vickers hardness tester. On each dentin disc surface, 5 indentation tests were performed using a diamond indenter with a 25-gf load. Dentin discs with SMH0 values of 0.60-0.80 GPa were selected for subsequent experiments, and demineralized dentin was excluded. The coronal surface of the dentin disc was sealed with acid-resistant nail polish, and a 4x4 mm 2The window was then used to demineralize dentin specimens. Subsequently, the specimens were placed in 0.50 mol / L EDTA solution (pH 8.0) for calcium ion chelation demineralization for 30 min. The demineralized dentin blocks were ultrasonically cleaned for 15 min and then soaked in PBS solution at 4°C for later use. The demineralized dentin samples were coated with 100 μL of TKH peptide (100 μM) and TVH-19 peptide (200 μM) solutions, respectively, and air-dried at room temperature for 1 hour. The FTIR spectra of the following samples were obtained using ATR-FTIR technology: untreated demineralized dentin (EDTA-eached), TKH-coated dentin, TVH-19-coated dentin, and dentin samples rinsed with double-distilled water (DDW) after coating (TKH-after washing, TVH-19-after washing). Results are attached. Figure 12 As shown, after DDW rinsing, the FTIR characteristic peaks of the TKH peptide-treated group were still significantly present, indicating that the TKH peptide has excellent hydroxyapatite (HA) adsorption capacity on the demineralized dentin surface.

[0096] 3. Collagen mineralization experiment

[0097] Collagen stock solution (3 mg / mL) was diluted to a final concentration of 50 μg / mL with assembly solution (200 mM KCl, 50 mM glycine, pH 9.2), and allowed to stand at room temperature for 15 min until no flocculent precipitate formed, yielding the diluted collagen working solution. 14 μL of this working solution was added dropwise to a 300-mesh nickel TEM grid (Beijing Zhongjing Scientific Instruments Technology Co., Ltd.) coated with carbon and ethyl formate, and incubated overnight in a sealed incubator at 37°C. Subsequently, crosslinking was performed using 0.05% (w / v) glutaraldehyde solution for 2 hours, followed by gentle rinsing with deionized water and air drying at room temperature. Polyallylamine hydrochloride (PAH) solution (200 μg / mL), TKH peptide solution (100 μM), and TVH-19 peptide solution (200 μM) were prepared, with double-distilled water (DDW) as a control. The collagen-coated TEM mesh was placed upside down onto droplets of the four mineralization solutions mentioned above and incubated in a sealed incubator at 37°C for 5 days. After incubation, the nickel mesh was gently rinsed with deionized water and allowed to air dry. The mineralization of collagen fibers was observed using transmission electron microscopy (TEM), and selected area electron diffraction (SAED) analysis was performed.

[0098] The results are as follows Figure 13 As shown, the TKH peptide treatment group exhibits a distinct periodic striation structure in collagen, and its effect on inducing mineralization within collagen fibers is superior to that of the TVH-19 peptide. Meanwhile, mineral deposit particles are also visible on the outside of collagen fibers.

[0099] Example 5

[0100] This example discloses an in vitro remineralization experiment of demineralized dentin by TKH polypeptides of the present application. Specifically as follows:

[0101] Preparation of dentin samples: Demineralized dentin samples were prepared according to the method of Example 4.

[0102] The 60 dentin samples were randomly divided into 4 groups, 15 samples in each group. According to the different treatments, they were divided into NaF solution group (positive control group), PBS group (negative control group), TKH polypeptide group, and TVH19 polypeptide group. The window surface of each group of samples was treated with 100 μL of the following solutions for 1 hour: 1000 ppm NaF solution, PBS, 100 μM TKH polypeptide solution, and 200 μM TVH19 polypeptide solution. After the treatment was completed, the samples were washed with DDW and naturally air-dried. Then, the samples were soaked in artificial saliva (AS) (7 mL AS per sample) and placed in a 37°C constant temperature shaker for mineralization for 4 weeks, and the AS solution was replaced once a day (composition: 0.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 20 mM HEPES, pH 7.0). After mineralization, healthy intact dentin (Sound dentin, SD) and demineralized dentin (EDTA-eached) were used as controls, and scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-ray diffraction (XRD) analysis were performed.

[0103] The SEM and EDS results of the remineralization of demineralized dentin are shown in Figure 14 After 4 weeks of treatment with TKH polypeptide, mineral deposition was observed in the dentin tubules, forming a tubule sealing effect. Surface EDS scanning showed that calcium and phosphorus elements were uniformly distributed on the surface of the dentin, and the remineralization effect was better than that of TVH-19 polypeptide.

[0104] The XRD mineral phase analysis results are shown in Figure 15 Compared with healthy dentin, the diffraction peaks of the EDTA etching group and the PBS group at 2θ = 25.8° (002), 31.9° (211), 32.1° (112), and 32.8° (300) were significantly weakened and widened. In contrast, the remineralization treatment groups showed sharp diffraction peaks at these positions, indicating that the newly formed crystals were mainly hydroxyapatite (HAP).

[0105] Example 6

[0106] This example discloses the biofilm formation inhibition experiment and pH reduction experiment of TKH polypeptides of the present application.

[0107] 1. Biofilm formation inhibition experiment

[0108] Streptococcus mutans (S. mutans) was inoculated in a 96-well plate and incubated at 37°C for 2 hours.Streptococcus mutans A single colony of UA159 was cultured anaerobically at 37°C in 10 mL of BHI liquid medium (80% N2, 10% H2, 10% CO2). 100 μL of the bacterial culture was then transferred to 10 mL of BHI liquid medium and anaerobically cultured for 6 hours. The culture was then diluted to 2 × 10⁻⁶. 6 CFU / mL.

[0109] In 48-well plates, 100 μL of TKH or TVH-19 peptide solution and 900 μL of bacterial culture were added to each well to achieve final peptide concentrations of (2.5, 5, 10, 20, 40 μM). An equal volume of sterile BHIS liquid medium (n=3) was added to the control group. The plates were incubated in an anaerobic incubator for 24 h to allow biofilm formation. After incubation, the supernatant was discarded, and the plates were washed three times with sterile PBS. The biofilms were fixed with paraformaldehyde for 30 min and then washed three more times with PBS. The plates were stained with 0.1% (w / v) crystal violet solution for 5 min, excess dye was washed away, and the plates were dried at room temperature for 1 h before observation and image acquisition under a microscope. 33% glacial acetic acid solution was added to each well, and the plates were shaken at room temperature for 20 min. The absorbance (OD595) at 595 nm was measured in the supernatant to quantify biofilm formation.

[0110] 2. pH decrease experiment

[0111] Add 1 mL of 1×10⁻⁶ solution to each well of a 12-well plate. 4 CFU / mL Streptococcus mutans (S. mutans) was cultured anaerobically for 24 h to form a biofilm. The supernatant was discarded, and the biofilm was washed three times with PBS to remove residual culture medium. Then, 2 mL of 10 mM glucose solution (prepared with PBS, initial pH adjusted to 6.8–7.2) containing different concentrations (12.5, 25, 50, 100, 200 μg / mL) of TKH peptide or TVH-19 peptide was added. The pH of the solution was measured and recorded every 30 min using a pH meter.

[0112] The results of the biofilm formation inhibition experiment are attached. Figure 16 and Figure 17 As shown: Crystal violet staining showed that when the concentration of TKH peptide and TVH-19 peptide was 20 μM, they could significantly inhibit the formation of Streptococcus mutans biofilm; at low concentrations (5 μM), both could also effectively inhibit the metabolic activity of biofilm.

[0113] The results of the pH decrease experiment are attached. Figure 18 As shown, both TKH peptide and TVH-19 peptide can dose-dependently delay the rate of pH decrease, indicating that they can inhibit acid metabolism during biofilm formation; and the inhibitory effect of TKH peptide is better than that of TVH-19 peptide.

[0114] The above merely illustrates the preferred embodiments of the present application, which are only illustrative but not restrictive; those skilled in the art understand that many changes, modifications, and even equivalent variations can be made to the present application within the spirit and scope defined by the claims of the present application, and all shall fall within the protection scope of the present application.

Claims

1. A self-assembling polypeptide TKH, polypeptide derivative or pharmaceutically acceptable salt, ester of a polypeptide, characterized in that, The amino acid sequence of the polypeptide is as follows: TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLWHLLHHLLH; The polypeptide derivative is a C-terminal amide modification of the polypeptide.

2. The self-assembling polypeptide TKH, polypeptide derivative or polypeptide pharmaceutically acceptable salt, ester according to claim 1, characterized in that, The pharmaceutically acceptable salt is a hydrochloride, a sulfate, an acetate, a methanesulfonate, a succinate, a fumarate, a citrate, a malate, or an organic amine salt.

3. A composition characterized in that, The self-assembling polypeptide TKH, polypeptide derivative, or pharmaceutically acceptable salt, ester of the polypeptide according to claim 1, and a pharmaceutically acceptable excipient.

4. Use of the self-assembling polypeptide TKH, polypeptide derivative, or pharmaceutically acceptable salt, ester of the polypeptide according to claim 1 in the preparation of an oral care composition for the remineralization of hard dental tissue.

5. Use according to claim 4, characterized in that, The oral care composition has at least one of the following functions: (1) promoting mineralization within collagen fibers; (2) promoting mineral deposition; (3) occluding dentin tubules; (4) inhibiting the formation of Streptococcus mutans biofilm and acid production.

6. The use of a self-assembling polypeptide TKH, polypeptide derivative or a pharmaceutically acceptable salt, ester of a polypeptide according to claim 1, characterized in that, Use in the preparation of a medical material for treating or preventing oral diseases; The medical material is used for inducing dentin remineralization, treating dentin hypersensitivity, inhibiting the progression of caries, or preventing enamel demineralization.

7. Use according to claim 6, characterized in that, The medical material includes a dental adhesive, a desensitizer, a pit and fissure sealant, a dental hydrogel, a subgingival irrigant, or a bioactive material for dental restoration. The amino acid sequence of the polypeptide is as follows: TKRQQVVGLLWHLLHHLLHGKGTKRQQVVGLLWHLLHHLLH; The polypeptide derivative is a C-terminal amide modification of the polypeptide. The pharmaceutically acceptable salt is a hydrochloride, a sulfate, an acetate, a methanesulfonate, a succinate, a fumarate, a citrate, a malate, or an organic amine salt. The self-assembling polypeptide TKH, polypeptide derivative, or pharmaceutically acceptable salt, ester of the polypeptide according to claim 1, and a pharmaceutically acceptable excipient.

4. Use of the self-assembling polypeptide TKH, polypeptide derivative, or pharmaceutically acceptable salt, ester of the polypeptide according to claim 1 in the preparation of an oral care composition for the remineralization of hard dental tissue. The oral care composition has at least one of the following functions: (1) promoting mineralization within collagen fibers; (2) promoting mineral deposition; (3) occluding dentin tubules; (4) inhibiting the formation of Streptococcus mutans biofilm and acid production. Use in the preparation of a medical material for treating or preventing oral diseases; The medical material is used for inducing dentin remineralization, treating dentin hypersensitivity, inhibiting the progression of caries, or preventing enamel demineralization. The medical material includes a dental adhesive, a desensitizer, a pit and fissure sealant, a dental hydrogel, a subgingival irrigant, or a bioactive material for dental restoration.

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