Polypeptide having anticarious property, polypeptide self-assembled hydrogel and use thereof

Abstract

Provided is a polypeptide having an anticarious property, the amino acid sequence thereof being shown as SEQ ID NO. 1. Also provided are a polypeptide self-assembled hydrogel having an anticarious property and the use thereof, the polypeptide self-assembled hydrogel comprising the polypeptide shown as SEQ ID NO. 1.

Description

A peptide with anti-caries properties, a peptide self-assembled hydrogel and its applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024108177997, filed on June 24, 2024, entitled "A polypeptide with anti-caries properties, a polypeptide self-assembled hydrogel and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biochemical technology, and specifically relates to a polypeptide with anti-caries properties, a polypeptide self-assembled hydrogel, and its applications. Background Technology

[0004] Antimicrobial peptides are a large class of bioactive polypeptides that possess resistance to harmful organisms (or pathogens) such as bacteria, fungi, parasites, and viruses. Because they generally carry a sufficient amount of positive charge and are often hydrophobic, they can bind to negatively charged biomembranes under electrostatic interactions, penetrating and disrupting the membrane structure, leading to cell death. Unlike the single-target bactericidal mechanism of traditional antibiotics, antimicrobial peptides can disrupt pathogens at multiple sites, significantly reducing the emergence of drug-resistant bacteria and exhibiting broad-spectrum antimicrobial activity, making them one of the best alternatives to antibiotics in the future. In recent years, research on the application of antimicrobial peptides in oral cavity has been widely conducted worldwide, and significant leaps have been made in the past decade in research on controlling oral pathogens through antimicrobial peptides. To date, many natural or synthetic antimicrobial peptides have shown inhibitory effects on the growth of Streptococcus mutans, planktonic bacteria, and biofilms in vitro. They prevent and treat dental caries through mechanisms such as inhibiting the growth of microorganisms like Streptococcus mutans, reducing bacterial acid resistance, inhibiting microbial acid production, inhibiting biofilm formation, and promoting remineralization. These studies suggest that antimicrobial peptides have the potential to become a safe and promising solution for dental caries. Summary of the Invention

[0005] In order to obtain a novel antimicrobial peptide against Streptococcus mutans, this application provides a polypeptide with anti-caries properties, which exhibits excellent antimicrobial activity against Streptococcus mutans, providing a new option for the prevention and treatment of early dental caries in clinical practice.

[0006] This application also provides a peptide self-assembled hydrogel with anti-caries properties and its application.

[0007] This application is achieved through the following technical solution:

[0008] This application provides a polypeptide with anti-caries properties, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Based on the same inventive concept, this application provides a polypeptide self-assembly hydrogel with anti-caries properties, wherein the polypeptide self-assembly hydrogel contains the aforementioned polypeptide with anti-caries properties.

[0010] Furthermore, the polypeptide self-assembly hydrogel is formed by the self-assembly of the polypeptide in a solvent.

[0011] Optionally, the solvent is PBS buffer.

[0012] Based on the same inventive concept, this application provides the application of a polypeptide with anti-caries properties in the preparation of anti-caries drugs.

[0013] Based on the same inventive concept, this application provides the application of a polypeptide self-assembled hydrogel with anti-caries properties in the preparation of anti-caries drugs.

[0014] Based on the same inventive concept, this application provides an anti-caries drug, wherein the effective component of the anti-caries drug includes the aforementioned polypeptide with anti-caries properties and / or a polypeptide self-assembled hydrogel with anti-caries properties.

[0015] Based on the same inventive concept, this application provides the application of a polypeptide with anti-caries properties in inhibiting or killing Streptococcus mutans.

[0016] Based on the same inventive concept, this application provides the application of a polypeptide self-assembled hydrogel with anti-caries properties in inhibiting or killing Streptococcus mutans.

[0017] Based on the same inventive concept, this application provides a Streptococcus mutans inhibitor, wherein the active ingredient of the inhibitor includes the aforementioned polypeptide with anti-caries properties and / or a polypeptide self-assembled hydrogel with anti-caries properties.

[0018] Based on the same inventive concept, this application also provides a method for preparing a polypeptide self-assembled hydrogel with anti-caries properties, the preparation method comprising:

[0019] A peptide with anti-caries properties was mixed with PBS buffer to obtain a reaction solution.

[0020] The reaction solution was reacted at 23–37°C for a period of time to obtain a polypeptide self-assembled hydrogel with anti-caries properties.

[0021] Preferably, in the reaction solution, the concentration of the polypeptide is 4–10 mmol / L and the concentration of the phosphate is 6–10 mmol / L;

[0022] The reaction time of the reaction solution is 5 to 30 minutes.

[0023] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0024] 1. This application discloses a polypeptide with anti-caries properties, named A3. Polypeptide A3 exhibits excellent antibacterial activity against a variety of pathogens, especially Streptococcus mutans. The minimum inhibitory concentration (MIC) of polypeptide A3 against Streptococcus mutans is significantly lower than that against Escherichia coli and Staphylococcus aureus. Polypeptide A3 can also be self-assembled to prepare polypeptide hydrogels, achieving adhesion and sustained release at the site of tooth decay. It can serve as an effective component of anti-caries drugs, providing a new option for the prevention and treatment of early caries in clinical practice.

[0025] 2. This application discloses a self-assembled polypeptide hydrogel with anti-caries properties. Using polypeptide A3 as a raw material, polypeptide A3 self-assembles into a polypeptide hydrogel in PBS solution. The resulting material retains the original antibacterial properties of antimicrobial peptide A3 and exhibits excellent biocompatibility. It also achieves adhesion and sustained release at the site of tooth decay. Studies on the stability of this self-assembled polypeptide hydrogel in different pH environments revealed that its in vitro degradation rate slows down as pH decreases. This facilitates the slow release of the drug in an acidic caries environment, which is of great significance for the clinical inhibition of early caries. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 shows the results of determining the minimum bactericidal concentration of compound A3 against standard Streptococcus mutans bacterial strains.

[0028] Figure 2 shows the effect of compound A3 on the proliferation of cell line L929.

[0029] Figure 3 shows the preparation process of the antimicrobial peptide self-assembled hydrogel (inverted vial experiment).

[0030] Figure 4 shows the antibacterial activity of the self-assembled hydrogel of the antimicrobial peptide.

[0031] Figure 5 shows the colony growth after the antimicrobial peptide self-assembled hydrogel.

[0032] Figure 6 shows the experimental results of the self-assembled hydrogel CCK-8 of antimicrobial peptides.

[0033] Figure 7 shows the results of the LDH release experiment from the self-assembled hydrogel of the antimicrobial peptide.

[0034] Figure 8 shows the staining results of live / dead cells in the self-assembled hydrogel of antimicrobial peptides. Detailed Implementation

[0035] The present application will be described in detail below with reference to specific embodiments and examples, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the application.

[0036] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In the event of any conflict, this specification shall prevail.

[0037] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0038] The overall approach of this application is as follows:

[0039] Traditional antimicrobial peptides, while controlling the growth of Streptococcus mutans, also inhibit other symbiotic bacteria, significantly impacting the oral flora balance and negatively affecting oral health. Furthermore, dental caries primarily occur in the crown of the tooth. Due to the vascular distribution, systemic medication is difficult to reach this area. Topical application is a relatively effective drug delivery method, but most drugs require a specific drug-carrying medium to achieve adhesion and sustained release at the tooth site.

[0040] Hydrogels are highly hydrophilic three-dimensional network gels that can rapidly expand in water but cannot dissolve. They can absorb large amounts of water while maintaining their original structure after expansion. As a soft material, hydrogels are widely used in the biomedical field. Compared with traditional synthetic hydrogels, peptide self-assembled hydrogels have many advantages. Similar to other molecular self-assembly systems, peptide self-assembly relies on non-covalent interactions. First, the structure and function of the hydrogel can be adjusted by regulating the structure of the peptide. Second, peptide hydrogels possess stable mechanical properties, a porous structure, and good biocompatibility, which are beneficial for the migration and growth of the loaded cells. Finally, the large specific surface area of ​​peptides can promote the adsorption of drugs and biomolecules.

[0041] Based on this, this application provides a polypeptide with anti-caries properties. This application uses computer-generated antimicrobial peptides to capture their antimicrobial characteristics, and artificial intelligence learns these characteristics to establish a predictive model. From a series of generated non-natural antimicrobial peptides, a polypeptide with high antimicrobial activity is selected, its amino acid sequence being RRRWRWRIGIRIY, and labeled as compound A3.

[0042] Experiments revealed that compound A3 exhibits excellent antibacterial activity against a variety of pathogens, particularly *Streptococcus mutans*. The minimum inhibitory concentration (MIC) of peptide A3 against *Streptococcus mutans* was significantly lower than that against *Escherichia coli* and *Staphylococcus aureus*. Furthermore, compound A3 can self-assemble into a peptide hydrogel in PBS solution. This material retains the original antibacterial properties of the antimicrobial peptide while possessing excellent biocompatibility, and also achieves adhesion and sustained release at dental caries sites. Studies on the stability of the peptide hydrogel in different pH environments showed that its in vitro degradation rate slows down as the pH decreases. This is beneficial for the slow release of the drug in acidic caries environments, providing a new approach for the clinical inhibition of early caries.

[0043] The following will provide a detailed description of a polypeptide and a polypeptide self-assembled hydrogel with anti-caries properties, in conjunction with embodiments and experimental data.

[0044] Example 1

[0045] This embodiment describes the synthesis and purification of a polypeptide with anti-caries properties.

[0046] The polypeptide (i.e., compound A3) was synthesized using the classic solid-phase polypeptide synthesis method, and the specific steps are as follows:

[0047] 1) Swelling resin: Weigh a certain amount of resin and add it to the peptide synthesizer. Then add an appropriate amount of redistilled DCM and stir for 30 minutes to make the resin completely swell. After drying, wash the resin 4 times with redistilled DMF for 3 minutes each time, and then dry it.

[0048] 2) Indene test: Place a small amount of resin in a test tube, add the indene test reagent (ninhydrin: pyridine: phenol = 1:2:1), heat in boiling water for 3 minutes. If the resin color does not change, proceed to the next step.

[0049] 3) Deprotection: Add the deprotection reagent (piperidine:DMF = 1:4) to the synthesizer, stir, and repeat 3 times, 5 minutes each time, to completely remove the Fmoc protecting group. Wash the resin 4 times with redistilled DMF, 3 minutes each time. Test again with indole; if the resin turns blue-purple, the deprotection is successful.

[0050] 4) Condensation reaction: Weigh out 3 times the excess of Fmoc-protected amino acids and condensing agents HOBT and HBTU, dissolve them in redistilled DMF, add 6 times the excess of initiator DIEA, stir and mix well, then add to the synthesizer. Stir for 1 hour under inert gas protection, dry under vacuum, wash the resin 4 times with redistilled DMF for 3 minutes each time, dry under vacuum, and test for indole. If there is no color change in the resin, the condensation reaction is successful. Repeat the steps of removing the Fmoc protecting group and the condensation reaction until the entire peptide chain is synthesized.

[0051] 5) Peptide cleavage: After the last amino acid condensation is complete, remove the Fmoc protecting group, wash the resin four times with redistilled DMF for 3 minutes each time, then wash the resin three times each with alternating DCM and anhydrous methanol for 3 minutes each time. Place the resin in a vacuum drying oven. After the resin is completely dry, add the cleavage reagent (TFA:Tis:H2O = 95:2.5:2.5), stir slowly for 3 hours, and collect the cleavage solution.

[0052] 6) Extraction of peptides: The cutting fluid was evaporated under reduced pressure and stored in a -20℃ refrigerator for 15 min. After removal, an appropriate amount of ice-cold ether was added for precipitation. The extract was then extracted with water, and the aqueous phase was collected in a beaker and freeze-dried to obtain crude peptide powder.

[0053] 7) Peptide purification: The peptide was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). The crude peptide powder was dissolved, filtered, and loaded onto the elution system, which was an aqueous solution of 15%-95% acetonitrile at a flow rate of 10 mL / min for 80 min. The peak shape changes at 220 nm and 254 nm were observed. The eluent from the main peak was collected and labeled. After freeze-drying, the pure peptide was obtained. Purity analysis was performed using RP-HPLC. A small amount of the pure peptide was dissolved, loaded onto the elution system, which was an aqueous solution of 5%-95% acetonitrile at a flow rate of 1 mL / min for 30 min. The chromatogram was integrated, and the purity was calculated. The actual molecular weight of the peptide was determined by mass spectrometry.

[0054] The amino acid sequence of the obtained compound A3 is shown in Table 1.

[0055] Table 1. Amino acid sequences and theoretical molecular weights of the synthesized compounds.

[0056] Example 2

[0057] This embodiment determines the minimum inhibitory concentration of compound A3.

[0058] The standard strains used for the assay of the antibacterial activity of the compounds—Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), and Streptococcus pneumoniae (ATCC 49619)—were all obtained from the U.S. Culture Collection.

[0059] The minimum inhibitory concentration (MIC) of the compound against bacteria was slightly modified according to the Clinical and Laboratory Standards Institute (CLSI) microdilution method. In short, a single bacterial colony was picked and incubated in 4 ml of the appropriate culture medium at 37°C and 180 rpm for 18 h on a shaker, and then diluted to 1 × 10⁻⁶ with the appropriate culture medium. 5CFU / ml was kept on hand. The minimum inhibitory concentration (MIC) of the compound against the selected strains was determined using the microbroth dilution method: serially diluted solutions of the compound were added to sterile 96-well plates, with a final bacterial concentration of 1 × 10⁻⁶ in each well. 4 CFU / ml. First, add 100 μl of BHI medium to each well to dilute the 2 mg / ml drug solution to 512 μM. Add 100 μl of drug solution to each well (wells 1-5 in row A) to bring the concentration to 256 μM. Perform serial dilutions to BHI well concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, and 2 μM, respectively, and add 100 μl of BHI medium to each well to bring the total volume to 200 μl. Add 20 μl of bacterial suspension at a concentration of 1 × 10⁵ CFU / ml to each well to ensure a final bacterial concentration of 1 × 10⁵ CFU / ml in each well. 4 CFU / ml. Each strain was cultured for 24 hours under its corresponding culture conditions. The minimum concentration at which no bacterial growth was observed in the well at OD 600 nm was defined as the MIC. The experiment was repeated three times. The results are shown in Table 2.

[0060] Table 2 shows the minimum inhibitory concentration of compound A3 against standard bacterial strains.

[0061] As shown in Table 2, compound A3 exhibited good antibacterial activity against all the tested bacteria.

[0062] Example 3

[0063] This embodiment conducts an experiment to determine the minimum bactericidal concentration of compound A3:

[0064] In the above MIC determination experiment, 50 μL of *Streptococcus mutans* bacterial suspension was taken from the small well and evenly spread onto a suitable agar solid medium. The plates were then incubated upside down for 24 hours under suitable conditions. The lowest concentration at which no colony growth was observed on the plate was defined as the minimum bactericidal concentration (MBC). The experiment was effectively repeated three times. The experimental results are shown in Figure 1.

[0065] As shown in Figure 1, no colony growth can be observed on the plate at a concentration of 8 MIC, which is the MBC concentration.

[0066] Example 4

[0067] This embodiment demonstrates the cytotoxicity experiment of compound A3.

[0068] The cytotoxicity of compound A3 was determined by measuring its effect on the proliferation of mouse epithelial-like fibroblasts L929 using the CCK-8 assay. The cell lines used in this example were provided by the Experimental Center of the School of Basic Medical Sciences, Lanzhou University. L929 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator.

[0069] Detection of the in vitro toxicity of compound A3 to L929 cells:

[0070] Seed cell suspension (8 × 10⁶ cells per well) into 96-well plates. 3 100 μl of high-glucose DMEM medium containing 10% fetal bovine serum was added to each well of the culture plate, and the plates were incubated in an incubator. After cell attachment, the old medium was discarded, and the compound was serially diluted with complete medium. 100 μl of the diluted compound solution was added to each well, with 3 replicates per group. A control group without compound was used. The culture plates were incubated for 24 h, 48 h, and 72 h, respectively. The original medium was then discarded, and 10 μl of CCK-8 solution and 100 μl of complete medium were added. The culture plates were gently shaken and incubated for another 1-2 h. The absorbance at 450 nm was measured. Finally, the cytotoxicity* (%) was calculated as: [A(drug-added) - A(blank)] / [A(0-drug-added) - A(blank)] × 100%. The experimental results are shown in Figure 2.

[0071] As shown in Figure 2, after antimicrobial peptide A3 was applied to L929 cells, the cell survival rate was close to 80% at the minimum inhibitory concentration (8 μM), indicating good cell compatibility.

[0072] Example 5

[0073] This embodiment describes the preparation of a peptide self-assembled hydrogel with anti-caries properties.

[0074] To investigate the effects of antimicrobial peptide A3 concentration and PBS concentration on hydrogel formation, a controlled variable method was used, and the influence of these factors on the formation of antimicrobial peptide self-assembled hydrogels was explored by inverting vials.

[0075] A certain amount of antimicrobial peptide A3 was dissolved in sterile water to prepare a 25 mM stock solution. Then, the A3 stock solution was added to PBS of different concentrations (0 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM) at pH 7.4 to prepare reaction solutions with a final concentration of 5 mM or 10 mM. The reaction was carried out at 23°C for 5 min, and the fluidity of the solution was observed. Next, the antimicrobial peptide A3 stock solution was added to 8 mM PBS at pH 7.4 at different final concentrations (0 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM) and reacted at 23°C for 5 min. The fluidity of the solution was observed. The results are shown in Figure 3.

[0076] As shown in Figure 3, when the PBS concentration is 6 mM, both 5 mM and 10 mM A3 concentrations can form peptide self-assembled hydrogels. However, at an A3 concentration of 10 mM, the hydrogel morphology is unstable due to insufficient PBS ion concentration. When the PBS concentration is 8 mM, both 5 mM and 10 mM A3 concentrations can form stable peptide self-assembled hydrogels. When the PBS concentration is controlled at 8 mM, the fluidity and transparency of the entire system gradually decrease with increasing A3 concentration. A stable peptide self-assembled hydrogel can be formed at an A3 concentration of 5 mM. Ultimately, we selected the peptide self-assembled hydrogel formed with an A3 concentration of 5 mM and a PBS concentration of 8 mM for subsequent experiments.

[0077] Example 6

[0078] This embodiment tests the antibacterial activity of the self-assembled hydrogel of the antimicrobial peptide.

[0079] The antibacterial activity of A3 self-assembled hydrogels (A3 concentration 5 mM, PBS concentration 8 mM) was tested using *Streptococcus mutans*.

[0080] Single colonies were picked from BHI agar plates and inoculated into 4 ml of BHI liquid medium. The culture was incubated at 37°C and 180 rpm for 8–12 h until the logarithmic growth phase. 700 μl of the antimicrobial peptide self-assembled hydrogel was added to a 1.5 ml sterile EP tube. An equal volume of PBS was used as a negative control. The logarithmic growth phase bacterial culture was diluted to 1 × 10⁻⁶. 5 CFU / ml, slowly add 700 μl of bacterial suspension to hydrogel or PBS, incubate at 37℃ for 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h. After each incubation, gently shake the suspension to mix. Take 100 μl of the suspension and add it sequentially to a sterile 96-well plate, setting up three parallel control wells. Measure the absorbance (OD600) at 600 nm using a multi-mode microplate reader. Simultaneously, dilute 100 μl of the suspension in BHI medium. 6 The solution was then added to BHI agar plates, spread evenly with a spreader, and incubated at 37°C. After 18 hours, the growth of colonies on the plates was recorded. The results are shown in Figures 4 and 5.

[0081] As shown in Figures 4 and 5, the antibacterial activity of the A3 self-assembled hydrogel was judged by the change in the OD600 value of the bacterial solution. The OD600 value of the control group gradually increased, indicating that the bacteria were active. However, the OD600 value of the experimental group did not change with the extension of the incubation time, indicating that the A3 self-assembled hydrogel could effectively inhibit the proliferation of Streptococcus mutans.

[0082] Based on the growth of colonies on the plates, the number of colonies on the control group plates gradually increased, indicating that the bacteria were active. After 3 hours, no bacteria grew on the hydrogel group plates, and no colonies appeared on the plates as time went on. This indicates that the A3 self-assembled hydrogel can quickly kill the Streptococcus mutans that was co-incubated with it.

[0083] Example 7

[0084] This embodiment tests the cytotoxicity of antimicrobial peptide self-assembled hydrogels.

[0085] 1. The cytotoxicity of antimicrobial peptide self-assembled hydrogels on L929 cells was determined using the CCK-8 assay: L929 cells were cultured at a concentration of 4 × 10⁶ cells / cells. 4 Seeds were cultured at a density of 1 / ml in 96-well plates and incubated in a cell culture incubator for 24 h. The supernatant was then discarded, and 100 μl of DMEM medium (equilibrated with PBS for 24 h beforehand) and the corresponding proportion of fetal bovine serum were added to each well for further incubation for 24 h, 48 h, and 72 h. After incubation, the supernatant was discarded, and 100 μl of CKK-8 application solution was prepared at a ratio of CCK-8 solution to complete medium of 1:10, and incubated for 1.5 h. OD450 nm was then measured to evaluate the cytotoxicity of the self-assembled hydrogel of the antimicrobial peptide. The results are shown in Figure 6.

[0086] As shown in Figure 6, after incubation of L929 cells with A3 self-assembled hydrogel for 24h, 48h, and 72h, no significant differences were observed compared to the control group, indicating that the A3 self-assembled antimicrobial peptide has good cell compatibility.

[0087] 2. Assessing the cytotoxicity of hydrogels using LDH (lactate dehydrogenase) release assay: L929 cells were cultured at a rate of 4 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 / ml in 96-well plates and incubated for 24 h. The supernatant was then discarded, and 100 μl of DMEM medium (pre-equilibrated with PBS for 24 h) and the corresponding proportion of fetal bovine serum were added to each well for further incubation for 24 h, 48 h, and 72 h. The LDH release reagent (C0016-1, Beyotime) from the LDH release kit was used as a maximum enzyme activity control (positive control). After incubation, the LDH release amount of all cells was measured using the Beyotime LDH release kit. LDH release from the A3 hydrogel was calculated using the following formula: LDH release = (OD490(hydrogel) - OD490(blank)) / (OD490(MAX) - OD490(blank)) to determine the effect of antimicrobial peptide gelation on its cytotoxicity. The results are shown in Figure 7.

[0088] As shown in Figure 7, the amount of cell death and cytotoxicity were determined by the amount of LDH released. There were no significant differences between the experimental group and the PBS control group at 24h, 48h, and 72h, indicating that the A3 self-assembled antimicrobial peptide has good cell compatibility.

[0089] 3. Live / dead cells were stained green / red using a live / dead cell staining method, and then the cell density and state were observed using a fluorescence microscope to determine the cytotoxicity of the antimicrobial peptide self-assembled hydrogel.

[0090] First, L929 cells were divided into groups of 4 × 10⁻⁶. 4 Cells were seeded at a density of 1 / ml in 96-well plates and incubated for 24 h in a cell culture incubator. The supernatant was then discarded, and DMEM pre-equilibrated with hydrogel and control PBS for 24 h was added for further incubation for 24 h, 48 h, and 72 h. After incubation, the supernatant was discarded, and the cells were stained with the corresponding fluorescent dyes. Finally, cell morphology was observed under a fluorescence microscope and photographed. Live cells were stained with calcein AM, and dead cells were stained with BOBO-3 iodide for 15 min. The results are shown in Figure 8.

[0091] As shown in Figure 8, the morphology and proliferation of L929 cells were not affected after co-incubation with the A3 self-assembled hydrogel. All cells in the experimental group exhibited intact and healthy adherence, and the cell density in the experimental group gradually increased with the extension of incubation time, showing a growth trend comparable to that of the control group, further demonstrating that the A3 self-assembled antimicrobial peptide has good cell compatibility.

[0092] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0093] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0094] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A polypeptide with anti-caries properties, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A polypeptide self-assembled hydrogel with anti-caries properties, characterized in that, The polypeptide self-assembled hydrogel contains a polypeptide with anti-caries properties as described in claim 1.

3. The self-assembled polypeptide hydrogel with anti-caries properties according to claim 2, characterized in that, The polypeptide self-assembly hydrogel is formed by the self-assembly of the polypeptide in a solvent.

4. The self-assembled polypeptide hydrogel with anti-caries properties according to claim 3, characterized in that, The solvent is PBS buffer.

5. The application of the polypeptide with anti-caries properties as described in claim 1 in the preparation of anti-caries drugs.

6. The application of the polypeptide self-assembled hydrogel with anti-caries properties as described in claims 2 to 4 in the preparation of anti-caries drugs.

7. An anti-caries drug, characterized in that, The active ingredient of the anticaries includes a polypeptide with anticaries properties as described in claim 1 and / or a polypeptide self-assembled hydrogel with anticaries properties as described in claims 2 to 4.

8. The application of the polypeptide with anti-caries properties as described in claim 1 in inhibiting or killing Streptococcus mutans.

9. The application of a self-assembled polypeptide hydrogel with anti-caries properties as described in claims 2 to 4 in inhibiting or killing Streptococcus mutans.

10. A Streptococcus mutans inhibitor, characterized in that, The active ingredient of the inhibitor includes a polypeptide with anti-caries properties as described in claim 1 and / or a polypeptide self-assembled hydrogel with anti-caries properties as described in claims 2 to 4.

11. A method for preparing a self-assembled polypeptide hydrogel with anti-caries properties as described in claims 2-4, characterized in that, The preparation method includes: A peptide with anti-caries properties was mixed with PBS buffer to obtain a reaction solution. The reaction solution was reacted at 23–37°C for a period of time to obtain a polypeptide self-assembled hydrogel with anti-caries properties.

12. The method for preparing a self-assembled polypeptide hydrogel with anti-caries properties according to claim 11, characterized in that, In the reaction solution, the concentration of the polypeptide is 4–10 mmol / L, and the concentration of the phosphate is 6–10 mmol / L; The reaction time of the reaction solution is 5 to 30 minutes.

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