Polypeptide dental surface antifouling agent as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide dental surface antifouling agent as well as a preparation method and application thereof, and belongs to the technical field of dental care preparations. The polypeptide dental surface antifouling agent comprises a polypeptide synthesized by the following sequences: (a) a dental surface anchoring peptide sequence as shown in SEQ ID NO.1; (b) a zwitterionic peptide sequence; and (c) a connecting peptide sequence as shown in SEQ ID NO.2, wherein the connecting peptide sequence is used for connecting the dental face anchoring peptide sequence and the zwitterionic peptide sequence. The polypeptide dental surface antifouling agent disclosed by the invention has good cytocompatibility and can be quickly and firmly combined with the dental surface to form a hydration barrier layer, so that the adhesion of protein and bacteria can be effectively prevented, and the problem of unbalance / disturbance of oral flora is avoided while the formation of a dental biofilm is inhibited; meanwhile, continuous flushing of saliva can be resisted, the effect of preventing protein and bacterial adhesion for a long time in the oral environment can be achieved, frequent washing is not needed to maintain the antibacterial activity, use is convenient, and the antifouling performance is excellent.

Description

Technical Field

[0001] This application belongs to the technical field of dental care preparations, and particularly relates to a polypeptide dental surface anti-fouling agent, its preparation method and application. Background Art

[0002] When special populations cannot effectively brush their teeth to maintain oral hygiene due to various restrictions, dental biofilms will rapidly accumulate formed by microorganisms that can grow on the tooth surface. Dental biofilms are highly harmful. For example, they may cause damage to the remaining tooth bodies and periodontal tissues, and may also induce diseases in systems such as digestion, respiration, nerves, and cardiovascular systems. Therefore, it is necessary to develop a simple and feasible dental care strategy that can inhibit the formation of dental biofilms.

[0003] Currently, clinically, antibacterial mouthwashes that can inhibit cariogenic microorganisms have been developed for the oral care of special populations. For example, mouthwashes containing chlorhexidine, hydrogen peroxide, cetylpyridinium chloride, povidone iodine, and essential oils, etc. However, frequent use will cause problems such as oral flora imbalance, taste change, mucosal burning sensation, tooth discoloration, and drug resistance. For this reason, antibacterial peptides with low adverse reactions, low drug resistance, and clear and rapid bactericidal effects have been developed in related fields. For example, the lactoferrin-derived anti-caries antibacterial peptide designed and synthesized by Luo Junyuan, etc. However, there is still a risk of flora disturbance with frequent use.

[0004] Therefore, while the existing antibacterial mouthwashes and antibacterial peptides are prone to cause oral flora imbalance / disturbance problems with frequent use, they also need to be gargled regularly to maintain antibacterial activity, and the actual use effect is not ideal. Summary of the Invention

[0005] This application discloses a polypeptide dental surface anti-fouling agent, its preparation method and application, which are used to solve the technical problems in the prior art that antibacterial mouthwashes and antibacterial peptides are prone to cause the risk of oral flora imbalance / disturbance and need to be gargled regularly.

[0006] To achieve the above object, the technical solution adopted in this application is:

[0007] The first aspect of this application discloses a polypeptide dental surface anti-fouling agent, which contains a polypeptide synthesized by including the following sequences;

[0008] (a) The dental surface anchoring peptide sequence of SEQ ID NO.1;

[0009] (b) The zwitterionic peptide sequence; and

[0010] (c) The linker peptide sequence of SEQ ID NO.2, which is used to connect the dental surface anchoring peptide sequence and the zwitterionic peptide sequence.

[0011] According to the disclosure of the first aspect of the present application, the zwitterionic peptide sequence is a peptide sequence formed by alternately connecting negatively charged amino acid residues and positively charged amino acid residues;

[0012] Among them, the negatively charged amino acid residue is selected from one of aspartic acid and glutamic acid; the positively charged amino acid residue is selected from one of histidine, lysine, and arginine.

[0013] According to the disclosure of the first aspect of the present application, the zwitterionic peptide sequence contains SEQ ID NO.3.

[0014] According to the disclosure of the first aspect of the present application, the polypeptide contains the sequence of SEQ ID NO.4.

[0015] According to the disclosure of the first aspect of the present application, the polypeptide dental surface anti-fouling agent contains deionized water and the freeze-dried powder of the polypeptide dissolved in the deionized water.

[0016] According to the disclosure of the first aspect of the present application, the mass-volume concentration of the polypeptide freeze-dried powder is 0.5 - 3 mg / mL.

[0017] According to the disclosure of the first aspect of the present application, the mass-volume concentration of the polypeptide freeze-dried powder is 2 mg / mL.

[0018] The second aspect of the present application also discloses a preparation method of the above-mentioned polypeptide dental surface anti-fouling agent, which includes:

[0019] Synthesizing the polypeptide by the 9-fluorenylmethoxycarbonyl solid-phase synthesis method and dissolving the polypeptide in deionized water to prepare an aqueous solution, thus obtaining it.

[0020] In the third aspect, the present application also discloses the application of the above-mentioned polypeptide dental surface anti-fouling agent in the anti-fouling treatment of the dental surface for non-therapeutic purposes.

[0021] According to the disclosure of the third aspect of the present application, the use of the polypeptide dental surface anti-fouling agent includes releasing it to the tooth surface through the ways of gargling or spraying.

[0022] Compared with the prior art, the advantages or beneficial effects of the present application at least include:

[0023] In this application, a polypeptide is constructed by designing a tooth surface anchoring peptide, an amphoteric ion peptide, and a linking peptide for connecting them, endowing the polypeptide with the ability to selectively bind to tooth surface hydroxyapatite and bind to water molecules to form a hydration layer barrier. Thus, the polypeptide tooth surface anti-fouling agent prepared from this polypeptide can quickly and firmly bind to the tooth surface and form a hydration barrier layer on the tooth surface. It not only has the function of physically preventing protein and bacteria adhesion, effectively inhibiting the formation of dental biofilm while avoiding the problem of oral flora imbalance / disturbance, but also has the function of resisting continuous salivary flushing, and can achieve the effect of preventing protein and bacteria adhesion for a long time in the oral environment without frequent gargling to maintain antibacterial activity, with convenient use and excellent anti-fouling performance. In addition, this polypeptide also has good cell compatibility, will not be swallowed into the digestive tract to cause risks, and can effectively avoid the potential risk problem of antibacterial water being swallowed. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings required for description in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments recorded in this application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0025] Figure 1 Infrared spectra of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA provided by the embodiments of this application;

[0026] Figure 2 Scanning electron micrographs of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA provided by the embodiments of this application;

[0027] Figure 3 Statistical chart of cell viability of polypeptide SAP-KE provided by the embodiments of this application;

[0028] Figure 4 Test results of protein anti-adhesion effects of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA provided by the embodiments of this application;

[0029] Figure 5 Test results of bacteria anti-adhesion effects of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA provided by the embodiments of this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments described in the present application without creative efforts fall within the scope of protection of the present application.

[0031] In the following description of this specification, the term "and / or" is used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, B exists alone, and the situation where A and B exist simultaneously. Among them, A and B may be singular or plural; the symbol " / " means "or".

[0032] In the following description of this specification, the term "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of A, B, or C", or, "at least one of A, B, and C" may represent any one of A, B, and C, or A + B, or A + C, or B + C, or A + B + C, where A, B, and C may be single or multiple respectively.

[0033] In the following description of this specification, the sequence number precedence does not mean the precedence of the execution order. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and does not constitute any limitation to the execution process of this embodiment.

[0034] In the following description of this specification, the numerical range should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within the stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this embodiment, and the upper and lower limits of the smaller range can be independently included or excluded from the range.

[0035] Unless otherwise specified, the technical / scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. Although this specification only describes preferred materials and methods, similar or equivalent any methods and materials can also be used in specific embodiments or test examples. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] In a first aspect, an embodiment of the present application provides a polypeptide dental surface anti-fouling agent. The polypeptide dental surface anti-fouling agent described in the present application contains a polypeptide synthesized by including the following sequences;

[0037] (a) Tooth surface anchoring peptide sequence of SEQ ID NO.1;

[0038] (b) Zwitterionic peptide sequence; and

[0039] (c) Linker peptide sequence of SEQ ID NO.2, which is used to link the tooth surface anchoring peptide sequence and the zwitterionic peptide sequence.

[0040] It should be noted that the tooth surface anchoring peptide (SAP) of SEQ ID NO.1 is a salivary-derived peptide screened from the salivary acquisition membrane (the specific amino acid sequence is DDDEEK). It has the characteristics of quickly and firmly binding selectively to the hydroxyapatite on the tooth surface and effectively resisting the continuous flushing of saliva. Therefore, by constructing a polypeptide with this tooth surface anchoring peptide, the present application can endow the constructed polypeptide with the property of quickly and firmly binding to the tooth surface while enhancing the effect of the polypeptide resisting the continuous flushing of saliva.

[0041] It should be noted that the zwitterionic peptide is a functional peptide formed by alternating negatively charged amino acid residues and positively charged amino acid residues. It has the ability to combine with water molecules to form a hydration layer barrier, thereby effectively preventing the adhesion of proteins and bacteria. Therefore, by constructing a polypeptide with the zwitterionic peptide and the tooth surface anchoring peptide, the present application can enable the constructed polypeptide to quickly and firmly bind to the tooth surface and form a hydration layer barrier, thereby endowing the polypeptide with a quick and firm binding effect on the tooth surface and an effective blocking effect on the adhesion of proteins and bacteria on the tooth surface, effectively solving problems such as antibacterial mouthwashes and antibacterial peptides being prone to causing oral flora imbalance / disturbance.

[0042] It should be noted that the linker peptide of SEQ ID NO.2 is a functional peptide sequence synthesized by linking four prolines (P) (the specific amino acid sequence is PPPP). It can provide a rigid backbone connection for the tooth surface anchoring peptide sequence and the zwitterionic peptide sequence, improving the ductility and monolayer packing density of the peptide chain.

[0043] In summary, in the embodiments of the present application, by designing a polypeptide composed of a tooth surface anchoring peptide, a zwitterionic peptide, and a linker peptide for connecting them, the polypeptide is endowed with the ability to selectively bind to tooth surface hydroxyapatite and form a hydration layer barrier by binding to water molecules. Therefore, the polypeptide tooth surface anti-fouling agent prepared from the polypeptide can quickly and firmly bind to the tooth surface and form a hydration barrier layer on the tooth surface. It not only physically prevents the adhesion of proteins and bacteria, effectively inhibits the formation of dental biofilms, and avoids the problem of oral flora imbalance / disturbance, but also has the effect of resisting continuous salivary flushing, can achieve a long-term blocking effect on the adhesion of proteins and bacteria in the oral environment, does not require frequent gargling to maintain antibacterial activity, is convenient to use, and has excellent anti-fouling performance. In addition, the synthetic polypeptide also has good cell compatibility, will not be swallowed into the digestive tract to cause risks, and can effectively avoid the potential risk problem of antibacterial water being swallowed.

[0044] According to the disclosure of the present application, the zwitterionic peptide sequence is a peptide sequence formed by alternately connecting negatively charged amino acid residues and positively charged amino acid residues. Among them, the negatively charged amino acid residue is selected from one of aspartic acid (D) and glutamic acid (E); the positively charged amino acid residue is selected from one of histidine (H), lysine (K), and arginine (R). Specifically, the combined sequences of the zwitterionic peptides include, but are not limited to:

[0045] (1) The zwitterionic peptide KE sequence of the combination of glutamic acid (E) and lysine (K), such as KEKE, KEKE KEKEKEKE, KEKEKEKEKEKEKEKE (SEQ ID NO.3), KEKEKEKEKEKEKEKEKE, etc.;

[0046] (2) The zwitterionic peptide HE sequence of the combination of glutamic acid (E) and histidine (H), such as HEHE, HEH EHEHEHEHE, HEHEHEHEHEHEHEHE, HEHEHEHEHEHEHEHEHEHE, etc.;

[0047] (3) The zwitterionic peptide RE sequence of the combination of glutamic acid (E) and arginine (R), such as RERE, RERE RERERERE, RERERERERERERERE, RERERERERERERERERERE, etc.;

[0048] (4) The zwitterionic peptide DK sequence of the combination of aspartic acid (D) and lysine (K), such as DKDK, DKDKDKDKDKDK, DKDKDKDKDKDKDKDK, DKDKDKDKDKDKDKDKDKDK, etc.;

[0049] (5) Zwitterionic peptide DR sequences composed of aspartic acid (D) and arginine (R), such as DRDR, DRDRDRDRDRDR, DRDRDRDRDRDRDRDR, DRDRDRDRDRDRDRDRDRDR, etc.

[0050] Among them, there is no special limitation on the peptide chain length of the zwitterionic peptide in this application, as long as it has a net neutral charge and can combine with water molecules to form a hydration layer barrier. Of course, this application takes the zwitterionic peptide KE sequence of SEQ ID NO.3 (specific sequence: KEKEKEKEKEKEKEKE) as an example for illustration, which does not represent a specific limitation on the zwitterionic peptide sequence.

[0051] In specific embodiments, this application uses the tooth surface anchoring peptide SAP of SEQ ID NO.1 (specific amino acid sequence: DDDEEK) and the zwitterionic peptide KE of SEQ ID NO.3 (specific amino acid sequence: KEKEKEKEKEKEKEKE) as functional peptides, and constructs a polypeptide through the linker peptide of SEQ ID NO.2 (specific amino acid sequence: PPPP), namely, the SAP-KE polypeptide of SEQ ID NO.4 (specific amino acid sequence: DDD EEKPPPPKEKEKEKEKEKEKEKE).

[0052] According to the disclosure of this application, the polypeptide tooth surface anti-fouling agent contains deionized water and the polypeptide lyophilized powder dissolved in the deionized water.

[0053] It should be noted that this application uses the polypeptide with the ability to quickly and firmly bind to the tooth surface and form a hydration barrier layer on the tooth surface as the active ingredient, and prepares an aqueous solution, spray and other dosage forms of the polypeptide tooth surface anti-fouling agent by dissolving it in deionized water, so that it can be used by gargling, spraying, etc., and the use is more convenient.

[0054] According to the disclosure of this application, the mass-volume concentration of the polypeptide lyophilized powder in the deionized water is preferably 0.5 - 3 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3.5 mg / mL or any one within the concentration range, and more preferably 2 mg / mL.

[0055] In the second aspect, the embodiments of this application also provide a preparation method of the polypeptide tooth surface anti-fouling agent described above in this application, and its steps preferably include:

[0056] Synthesize the polypeptide by using the 9-fluorenylmethoxycarbonyl solid-phase synthesis method, and dissolve the polypeptide in deionized water to prepare an aqueous solution, thus obtaining it.

[0057] Among them, there is no special limitation on the specific process of synthesizing the polypeptide by using the 9-fluorenylmethoxycarbonyl solid-phase synthesis method in this application. It can be synthesized by entrusting relevant institutions according to the well-known operation steps in the art. The exemplary synthesis steps include:

[0058] 1. Coupling preparation of polypeptide

[0059] 1) Swelling of resin

[0060] Add Fmoc-E(OtBu)-chloride resin (1.2 g, substitution degree = 0.33 mmol / g) into the reactor, and add dichloromethane (DCM, 6 mL / g) and stir for swelling for 5 min.

[0061] 2) Deprotection of resin

[0062] Vacuum-dry the swelling reagent, and add 20% piperidine / n,n-dimethylformamide solution (pip / DMF) (12 mL / g) and stir for 20 min.

[0063] 3) Washing after deprotection

[0064] Vacuum-dry the deprotection reagent (pip / DMF), and wash with DMF (6 mL / g) 5 times, stirring for 1 min each time and vacuum-drying for 0.5 min to obtain the carrier resin.

[0065] 4) Detection of deprotection

[0066] Take 20 grains of carrier resin and put them into the detection test tube, add 1 mL of ninhydrin detection reagent, put the detection test tube into a water bath at 95 °C or above for 0.5 min, take it out and observe the color of the resin. If the color of the resin becomes darker, it is positive, indicating that the deprotection is successful.

[0067] 5) Condensation of the second amino acid

[0068] Add 6 mL of the prepared amino acid (Fmoc-Lys(Boc)-OH) / OXYMA / DMF solution, and then add 0.95 equivalents of 1,3-diisopropylcarbodiimide (DIC) to activate for 1 min and then start stirring and reacting for 1 h.

[0069] 6) Washing after reaction

[0070] Start the washing program and wash 4 times, stirring for 1 min each time and vacuum-drying for 0.5 min.

[0071] 7) Detection of reaction

[0072] Take it out and observe the color of the resin. If there is no obvious change in the color of the resin, it indicates that the reaction condensation is successful.

[0073] 8) Repeat steps 2) to 7), and condense the amino acids in the sequence from right to left in turn

[0074] The deprotection reagent was vacuum dried, and then washed with DMF (10 mL / g) 7 times, stirring for 2 min each time and draining for 5 min.

[0075] 9) The last amino acid condensation was completed after 4 h, and deprotection was performed in sequence. Methanol (6 mL / g) was used again after the deprotection washing step.

[0076] The resin was washed twice and dried under vacuum for 10 min.

[0077] 2. Peptide cleavage

[0078] The dried resin was loaded into a boat-shaped reactor, and the lysis solution (8 mL / g) was added. The reaction solution was lysed at room temperature for 3 h, and the reaction solution was filtered out and added dropwise to glacial ether (8 mL / g). The solution was centrifuged and precipitated, and washed three times with glacial ether (5 mL / g) to obtain a crude polypeptide solid to be purified.

[0079] 3. Purification of polypeptide:

[0080] 1) Crude product filtration: Dissolve the crude peptide sample, filter it with a 0.45 μm filter membrane, and set the filtrate aside.

[0081] 2) Set purification conditions: Use mobile phase A: mobile phase B = 100:0 (v / v): phase A (0.1% ACE / water v / v), phase B (ACN) and equilibrate the system to the baseline level for 15 min before loading.

[0082] Sample loading: Manual loading, crude peptide solution at a flow rate of 12 mL / min.

[0083] Gradient elution: Set the corresponding elution gradient according to the peak elution time of the crude product, perform gradient elution according to the setting, and judge the peak shape at a wavelength of 220nm. If there is an obvious inflection, change the tube to collect the liquid. If there is no obvious inflection, change the tube appropriately according to the situation.

[0084] 3) Inject the crude peptide liquid sample into a high performance liquid chromatograph (HPLC), and collect each fraction that appears in the spectrum separately. Perform molecular weight and purity tests on all fractions to confirm the target fraction. Take an average of 30 μL of sample from the estimated target fraction and mix evenly. Perform fraction analysis using a high performance liquid chromatograph. The chromatographic column is a C18 reverse phase silica gel column; column temperature: 40°C; detection wavelength: 220nm and 254nm; flow rate: 1.0mL / min; automatic injection needle injection volume: 10μL. Judge the purity of the pure product at a wavelength of 220nm. If it is unqualified, reduce the sampling range and continue the analysis until a qualified fraction range is found, and record the preparation conditions and sampling analysis results.

[0085] 4. Freeze-drying of the polypeptide: The qualified fractions after purification are filled into freeze-drying vials and freeze-dried using a freeze dryer. After the freeze-drying is completed, the freeze-drying vials are removed, the polypeptide samples are weighed and sub-packaged, and stored at -20°C.

[0086] In a third aspect, the embodiments of the present application also provide the use of the polypeptide dental surface anti-fouling agent described in the above application for non-therapeutic dental surface anti-fouling treatment of the dental surface. Among them, since the polypeptide dental surface anti-fouling agent of the present application has the characteristics of quickly and firmly binding to the dental surface and forming a hydrated barrier layer on the tooth surface, it can replace mechanical brushing, antibacterial mouthwash, antibacterial peptides, etc. that are released to the tooth surface through gargling or spraying for non-therapeutic dental surface anti-fouling treatment, and is convenient to use and has excellent anti-fouling effect.

[0087] The technical solutions of the present application will be further described below in conjunction with specific embodiments.

[0088] Example 1

[0089] This example provides the preparation of a polypeptide dental surface anti-fouling agent, specifically including:

[0090] The freeze-dried powder of the polypeptide of SEQ ID NO.4 is dissolved in deionized water to prepare a 2 mg / mL aqueous solution of SAP-KE, which is the polypeptide dental surface anti-fouling agent.

[0091] Example 2

[0092] This example provides the preparation of a polypeptide dental surface anti-fouling agent, specifically including:

[0093] The freeze-dried powder of the polypeptide of SEQ ID NO.4 is dissolved in deionized water to prepare a 0.5 mg / mL aqueous solution of SAP-KE, which is the polypeptide dental surface anti-fouling agent.

[0094] Example 3

[0095] This example provides the preparation of a polypeptide dental surface anti-fouling agent, specifically including:

[0096] The freeze-dried powder of the polypeptide of SEQ ID NO.4 is dissolved in deionized water to prepare a 3 mg / mL aqueous solution of SAP-KE, which is the polypeptide dental surface anti-fouling agent.

[0097] To clarify the actual effects of the polypeptide dental surface anti-fouling agent described in the present application, this specification also provides Comparative Example 1 and Comparative Example 2. Among them, Comparative Example 1 is an aqueous solution of the freeze-dried powder of the dental surface anchoring peptide of SEQ ID NO.1, specifically a 2 mg / mL aqueous solution of SAP; Comparative Example 2 is an aqueous solution of the freeze-dried powder of the zwitterionic peptide of SEQ ID NO.3, specifically a 2 mg / mL aqueous solution of KE.

[0098] The simulated tooth coating treatment was carried out using the SAP-KE aqueous solution, SAP aqueous solution, and KE aqueous solution described above (all with a concentration of 2 mg / mL), and the obtained coatings were characterized. Specifically:

[0099] 1. Coating characterization

[0100] Since human tooth enamel contains up to 96% hydroxyapatite, hydroxyapatite tablets were used to simulate the tooth surface to characterize and test each coating. The specific tests were as follows:

[0101] Set the untreated hydroxyapatite tablets (abbreviated as Bare HA) as the blank group;

[0102] The hydroxyapatite tablets were immersed in the SAP-KE aqueous solution, SAP aqueous solution, and KE aqueous solution for 10 min respectively to obtain hydroxyapatite tablets coated with SAP-KE, SAP, and KE (abbreviated as SAP-KE coated HA, SAP coated HA, and KE coated HA) in sequence, which were the coating groups;

[0103] 1.1 Infrared spectroscopy tests were carried out on the blank group and the coating groups respectively, and the results are Figure 1 shown. Among them, Figure 1 are the infrared spectra of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA.

[0104] According to Figure 1 it can be seen that for the SAP-KE group, the peak of the amide bond appears at a wavelength of 1637 cm -1 , indicating the presence of a polypeptide coating on the surface of the hydroxyapatite tablets and still existing after 1 week in artificial saliva. In contrast, no amide bond peak was seen in the KE group, indicating that it does not have the ability to adsorb hydroxyapatite.

[0105] 1.2 Scanning electron microscopy characterizations were carried out on the blank group and the coating groups respectively, and the results are Figure 2 shown. Among them, Figure 2 are the scanning electron micrographs of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA.

[0106] According to Figure 2 it can be seen that compared with the bare hydroxyapatite tablets, many nanoparticles are randomly distributed on the surface of the SAP-KE group, forming a coating; compared with the SAP group, the particle size is smaller and evenly dispersed. And the polypeptide KE may have poor adhesion to hydroxyapatite due to the lack of specific adhesion motifs, showing a morphology similar to that of the bare hydroxyapatite.

[0107] 2. Cell compatibility test

[0108] The cytotoxicity of polypeptide SAP-KE was evaluated in MC 3T3 cells using the Cell Counting Kit-8. The specific test was as follows:

[0109] MC 3T3 cells were seeded at a density of 5000 cells per well in 100 μL of α-MEM medium and cultured for 24 h. Then, the medium was replaced with 100 μL of fresh medium containing 2 mg / mL SAP-KE. At the same time, media without SAP-KE and without cells were set as the control group (control) and the blank group (blank), respectively.

[0110] On the 1st, 2nd, and 3rd days, respectively, the optical density (OD) of the incubation solution was measured at a wavelength of 450 nm using a microplate reader, and the cell viability of polypeptide SAP-KE was calculated according to formula (1). The results are Figure 3 as shown. Among them, Figure 3 is the statistical chart of the cell viability of polypeptide SAP-KE.

[0111]

[0112] According to Figure 3 it can be seen that there was no significant difference in the effect of polypeptides SAP-KE at different concentrations on the viability of 3T3 cells. The cytotoxicity of SAP-KE was not concentration-dependent and could be safely applied to the oral environment.

[0113] 3. Effect of polypeptide SAP-KE on the proliferation activity of human oral flora

[0114] Dental plaque was collected from the tooth surfaces of volunteers and transferred to BHI medium. The bacteria in the dental plaque were cultured in a medium containing 2 mg / mL SAP-KE, and the medium without SAP-KE was used as the control group. On the 1st, 2nd, and 3rd days, the bacteria were stained with the LIVE / DEAD BacLight Bacterial Viability Kit, and the detection wavelengths were 530 nm and 630 nm, and the excitation wavelength was 485 nm. The values of green and red fluorescence were recorded, and the ratio of viable bacteria representing the bacterial viability in the plaque was obtained according to the standard curve.

[0115] Conclusion: The SAP-KE polypeptide antifouling agent can form a uniform coating on the surface of hydroxyapatite tablets, has good cell compatibility, high safety, and does not affect the balance of the oral flora.

[0116] 4. Evaluation of coating effect

[0117] 4.1 Evaluation of the effect on preventing protein adhesion

[0118] The bare HA, the coated SAP coated HA, KE coated HA, and SAP-KE coated HA were separately immersed in a fluorescein isothiocyanate-labeled bovine serum albumin solution (0.1 mg / mL), incubated at 37 °C for 90 min, washed three times with phosphate buffered saline (PBS), and dried at room temperature. The attached BSA was observed under a confocal laser scanning microscope, and the results were Figure 4 as shown. Among them, Figure 4 are the test results of the anti-protein adhesion effects of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA.

[0119] According to Figure 4 it can be seen that compared with other control groups, only a small amount of protein adhered to the surface of the SAP-KE coating, indicating that the SAP-KE polypeptide antifouling coating can effectively prevent protein adhesion.

[0120] 4.2 Evaluation of the effect on anti-bacterial adhesion

[0121] The anti-fouling properties of Streptococcus mutans on different coatings were evaluated using a scanning electron microscope. 400 μL of a Streptococcus mutans suspension (1×10 6 CFU / mL) was separately added onto Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA, and cultured in BHI medium at 37 °C for 24 h. Then the sections were rinsed with PBS, immersed in 2.5% glutaraldehyde overnight at 4 °C, dehydrated in gradient ethanol, and finally the bacterial adhesion was observed using a scanning electron microscope. The results were Figure 5 as shown. Among them, Figure 5 are the test results of the anti-bacterial adhesion effects of Bare HA, SAP coated HA, KE coated HA, and SAP-KE coated HA.

[0122] According to Figure 5 it can be seen that only scattered bacteria were found on the surface of the SAP-KE coating, while a large number of bacteria aggregated and formed thick biofilms on the surfaces of bare HA, KE, and SAP coatings, indicating that our SAP-KE polypeptide dental antifouling agent can effectively prevent bacterial adhesion, thereby protecting the tooth surface.

[0123] Based on the above characterization tests, it can be seen that the polypeptide dental anti-fouling agent prepared from the polypeptide shown in SEQ ID NO.4 can quickly and firmly bind to the tooth surface and form a hydrated barrier layer on the tooth surface. It not only has the function of physically preventing the adhesion of proteins and bacteria, effectively inhibiting the formation of dental biofilms while avoiding the problem of oral flora imbalance / disturbance, but also has the function of resisting the continuous flushing of saliva, and can achieve a long-term effect of preventing the adhesion of proteins and bacteria in the oral environment, without the need for frequent gargling to maintain antibacterial activity, which is convenient to use and has excellent anti-fouling performance. In addition, this synthetic polypeptide also has good cell compatibility, will not be swallowed into the digestive tract to cause risks, and can effectively avoid the potential risk problem of antibacterial water being swallowed.

[0124] The various embodiments in this specification are described in a progressive manner. For the same or similar parts between the various embodiments, reference can be made to each other. Each embodiment focuses on the differences from other embodiments.

[0125] The above embodiments are only used to illustrate the technical solutions of the present application, rather than limiting the present application; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application.

Claims

1. A polypeptide tooth surface antifouling agent, characterized in that: Comprising a polypeptide synthesized by comprising the following sequence; (a) tooth surface anchoring peptide sequence of SEQ ID NO.1; (b) a zwitterionic peptide sequence; and (c) A connecting peptide sequence of SEQ ID NO. 2, which is used to connect the tooth surface anchoring peptide sequence and the zwitterionic peptide sequence.

2. The polypeptide tooth surface antifouling agent according to claim 1, characterized in that: The zwitterionic peptide sequence is a peptide sequence formed by alternating connection of negatively charged amino acid residues and positively charged amino acid residues; Wherein, the negatively charged amino acid residue is selected from one of aspartic acid and glutamic acid; the positively charged amino acid residue is selected from one of histidine, lysine and arginine.

3. The polypeptide tooth surface antifouling agent according to claim 2, characterized in that: The zwitterionic peptide sequence comprises SEQ ID NO.

3.

4. The polypeptide tooth surface antifouling agent according to claim 1, characterized in that: The polypeptide comprises the sequence of SEQ ID NO.

4.

5. The polypeptide tooth surface antifouling agent according to any one of claims 1 to 4, characterized in that: The method comprises deionized water and the polypeptide lyophilized powder dissolved in the deionized water.

6. The polypeptide tooth surface antifouling agent according to claim 5, characterized in that: The mass volume concentration of the polypeptide lyophilized powder is 0.5-3 mg / mL.

7. The polypeptide tooth surface antifouling agent according to claim 6, characterized in that: The mass volume concentration of the polypeptide lyophilized powder is 2 mg / mL.

8. A method for preparing a polypeptide tooth surface antifouling agent, characterized in that: Include: The polypeptide is synthesized by using 9-fluorenylmethoxycarbonyl solid phase synthesis method, and the polypeptide is dissolved in deionized water to prepare an aqueous solution.

9. Use of the polypeptide tooth surface antifouling agent according to any one of claims 1 to 7 or the polypeptide tooth surface antifouling agent prepared by the preparation method according to claim 8 for non-therapeutic antifouling treatment of tooth surface.

10. The use according to claim 9, characterized in that: The use of the polypeptide tooth surface anti-stain agent includes releasing it to the tooth surface through mouthwash or spraying.

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