Composite polypeptide targeting hydroxyapatite and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a composite polypeptide of targeted hydroxyapatite and application thereof. The invention provides a composite polypeptide. The composite polypeptide comprises a hydroxyapatite binding peptide and an antibacterial peptide, the amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO. 1; the amino acid sequence of the hydroxyapatite binding peptide is as shown in SEQ ID NO. 2. According to the invention, the hydroxyapatite binding peptide and the antibacterial peptide are compounded, so that the formed composite polypeptide has good hydroxyapatite binding capacity, can keep specific binding with a hydroxyapatite material, is in targeted binding with tooth hard tissue, has good biocompatibility for oral cells, can prevent the formation of an oral microbial biofilm, and can be used for preparing a dental implant. The growth of oral microorganisms is inhibited, biological membranes formed by the oral microorganisms are killed, and a potential new strategy is provided for prevention and / or treatment of oral infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composite polypeptide targeting hydroxyapatite and its application. Background Technology

[0002] Oral biofilms are microbial communities embedded in a macromolecular matrix secreted by the microorganisms themselves. They are widely present on the oral mucosa, tongue, dental restorations, and tooth surfaces, especially accumulating near the gingival margin. Biofilm formation not only increases the resistance of microorganisms to traditional antibacterial agents, but their presence is also a direct cause of oral infectious diseases and may even trigger more serious systemic diseases.

[0003] Given that oral infectious diseases are caused by pathogenic bacteria, the use of antibiotics is essential. However, the widespread use of antibiotics can lead to many side effects, such as allergic reactions, dysbiosis, and drug resistance. Antimicrobial peptides (AMPs) and their synthetic derivatives constitute a class of compounds with diverse antimicrobial mechanisms. These compounds possess antimicrobial properties and, due to their unique mechanisms of action, are less likely to induce resistance in microorganisms. However, the antimicrobial effects of existing antimicrobial peptides have several shortcomings. For example, while the antimicrobial decapeptide KSL and its analogues can prevent biofilm formation, they have little effect on the formed biofilm. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composite polypeptide targeting hydroxyapatite and its application. It has a high affinity for hydroxyapatite, prevents the formation of oral microbial biofilms, inhibits the growth of oral microorganisms, kills the biofilms formed by oral microorganisms, prevents and / or treats oral infectious diseases, and has good biocompatibility with oral cells.

[0005] This invention provides a composite polypeptide comprising a hydroxyapatite-bound peptide and an antimicrobial peptide;

[0006] The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1;

[0007] The amino acid sequence of the hydroxyapatite-binding peptide is shown in SEQ ID NO.2.

[0008] Preferably, the amino acids of the antimicrobial peptide are D-type amino acids; and the amino acids of the hydroxyapatite-binding peptide are L-type amino acids.

[0009] Preferably, the amino acid sequence of the composite polypeptide is as shown in SEQ ID NO.4 or SEQ ID NO.14.

[0010] The present invention also provides the application of the composite polypeptide described in the above technical solution in the preparation of one or more of antibacterial products, anti-biofilm products and targeted hydroxyapatite products.

[0011] Preferably, the antimicrobial product includes products that inhibit pathogens and / or inhibit the formation of pathogen biofilms.

[0012] Preferably, the pathogenic bacteria include one or more of Streptococcus mutans, Enterococcus faecalis, and oral plaque.

[0013] The present invention also provides an antibacterial agent comprising the composite polypeptide described in the above technical solution.

[0014] The present invention also provides the application of the composite polypeptide or antibacterial agent described in the above technical solution in the preparation of products for the prevention and / or treatment of oral infectious diseases.

[0015] Preferably, the oral infectious disease includes diseases caused by pathogenic bacteria; the pathogenic bacteria include one or more of Streptococcus mutans, Enterococcus faecalis, and oral plaque.

[0016] Beneficial effects:

[0017] This invention provides a composite polypeptide comprising a hydroxyapatite-binding peptide and an antimicrobial peptide; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1; the amino acid sequence of the hydroxyapatite-binding peptide is shown in SEQ ID NO.2. This invention combines the hydroxyapatite-binding peptide and the antimicrobial peptide to form a composite polypeptide with excellent hydroxyapatite binding ability, maintaining specific binding with hydroxyapatite materials, targeting and binding to dental hard tissues, exhibiting good biocompatibility with oral cells, preventing the formation of oral microbial biofilms, inhibiting the growth of oral microorganisms, and killing the biofilms formed by oral microorganisms, providing a potential new strategy for the prevention and / or treatment of oral infectious diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 To investigate the inhibitory effects of different peptides on the formation of biofilms of Streptococcus mutans on HA tablets;

[0020] Figure 2 To investigate the inhibitory effects of different peptides on the formation of Enterococcus faecalis biofilm on HA tablets;

[0021] Figure 3 To investigate the inhibitory effects of different peptides on the formation of oral plaque biofilms by multiple bacterial species on HA tablets;

[0022] Figure 4 The killing effect of different peptides on the formation of biofilms by Streptococcus mutans on HA tablets;

[0023] Figure 5 To investigate the inhibitory effects of different peptides on the formation of Enterococcus faecalis biofilm on HA tablets;

[0024] Figure 6 The killing effect of different peptides on the formation of oral plaque biofilm by multiple bacterial species on HA tablets;

[0025] Figures 1-6 Red represents dead bacteria, green represents live bacteria, and different lowercase letters indicate significant differences (p value ≤ 0.05).

[0026] Figure 7 Results of hydroxyapatite binding ability tests for different peptides;

[0027] Figure 8 The results show the biocompatibility of different peptides; where A represents relative cell activity, with the red line indicating good relative cell activity; and B represents the results of optical microscopy. Detailed Implementation

[0028] This invention provides a composite polypeptide comprising a hydroxyapatite-bound peptide and an antimicrobial peptide;

[0029] The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1;

[0030] The amino acid sequence of the hydroxyapatite-binding peptide is shown in SEQ ID NO.2.

[0031] In this invention, as one embodiment, the amino acids of the antimicrobial peptide are D-type amino acids. As one embodiment, the amino acids of the hydroxyapatite-binding peptide are L-type amino acids. As one embodiment, the amino acid sequence of the complex polypeptide is shown in SEQ ID NO.4 or SEQ ID NO.14.

[0032] The composite polypeptide provided by this invention exhibits excellent biocompatibility, antibacterial and anti-biofilm capabilities, and can maintain specific binding with hydroxyapatite materials, targeting and binding to dental hard tissues. This invention does not have strict requirements regarding the source of the composite polypeptide; it can be obtained using conventional methods in the art, such as artificial synthesis.

[0033] Given the advantages of the composite polypeptide provided by this invention, its application in the preparation of one or more antibacterial products, anti-biofilm products, and targeted hydroxyapatite products, as well as its application in the preparation of products for the prevention and / or treatment of oral infectious diseases, also falls within the scope of protection of this invention.

[0034] In this invention, as one embodiment, the antibacterial product includes a product that inhibits pathogenic bacteria and / or inhibits the formation of pathogenic biofilms; as another embodiment, the antibacterial product is a product that inhibits both pathogenic bacteria and the formation of pathogenic biofilms. As one embodiment, the pathogenic bacteria include one or more of *Streptococcus mutans*, *Enterococcus faecalis*, and oral plaque; as another embodiment, the pathogenic bacteria include *Streptococcus mutans*, *Enterococcus faecalis*, and oral plaque. As another embodiment, inhibiting pathogenic bacteria means inhibiting the growth of pathogenic bacteria. As another embodiment, inhibiting the formation of pathogenic biofilms includes preventing the formation of pathogenic biofilms and killing biofilms formed by oral pathogens. The oral plaque described in this invention is a mixed multi-species bacteria, including but not limited to *Streptococcus mutans* and *Enterococcus faecalis*.

[0035] The present invention also provides an antibacterial agent comprising the composite polypeptide described in the above technical solution.

[0036] The present invention also provides the application of the composite polypeptide or antibacterial agent described in the above technical solution in the preparation of products for the prevention and / or treatment of oral infectious diseases.

[0037] In this invention, as one embodiment, the oral infectious disease includes diseases caused by pathogenic bacterial infection. As one embodiment, the pathogenic bacteria include one or more of Streptococcus mutans, Enterococcus faecalis, and oral plaque; as another embodiment, the pathogenic bacteria include Streptococcus mutans, Enterococcus faecalis, and oral plaque.

[0038] To further illustrate the present invention, a composite polypeptide targeting hydroxyapatite and its application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] The peptides shown in Table 1 were synthesized by Shanghai Sangon Biotech Co., Ltd. using the standard Fmoc protocol.

[0041] Table 1 Information on artificially synthesized peptides

[0042]

[0043] In this context, uppercase letters represent L-type amino acids, and lowercase letters represent D-type amino acids.

[0044] Test Example 1

[0045] Antibacterial activity and anti-biofilm performance testing

[0046] (1) Bacterial culture

[0047] Streptococcus mutans UA159, Enterococcus faecalis ATCC 29212, and oral plaque (taken from the lingual surface of the second molar of healthy adults) were cultured in BHI (brain and heart infusion medium) under anaerobic conditions at 37°C. During the logarithmic growth phase, the culture medium was diluted to 10⁻⁶. 6 and 10 7 CFU / mL available for use.

[0048] (2) Detection of minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC)

[0049] The peptides from Example 1 were serially diluted in BHI liquid medium in 96-well plates, with each well containing 100 μL of liquid, ranging from 500 μg / mL to 0.98 μg / mL. 3% NaClO was used as a positive control, and the medium served as a growth control (negative control). 100 μL of the 10 μg / mL peptides obtained in step (1) was added. 6 CFU / mL bacterial suspension, mix thoroughly. Final bacterial concentration: 5 × 10⁻⁶ 5 CFU / mL, peptide concentration 250 μg / mL~0.49 μg / mL. Anaerobic culture for 24 hours, OD measured using a microplate reader. 595 The inhibition rate was calculated using the formula (1 - ODtreated / ODnegative control) × 100%; where ODtreated represents the OD of the bacterial culture after 24 hours of culture of the peptide or positive control group. 595 The OD value represents the OD value of the negative control group. 595 The test results are shown in Table 2, where MIC indicates an antibacterial rate of 100%.

[0050] (3) Minimum Inhibition Biofilm Concentration (MBIC) Detection

[0051] After anaerobic culture for 24 hours in step (2), discard the upper layer of culture medium in the 96-well plate, wash twice with PBS, fix with methanol, stain with 0.1 wt.% crystal violet, wash away the excess stain, resuspend in 95% v / v ethanol, and detect OD using a microplate reader. 595 The biofilm inhibition rate was calculated as (1 - ODtreated / ODnegative control) × 100%; where ODtreated represents the OD of the bacterial culture after 24 hours of culture of the peptide or positive control group. 595 The OD value represents the OD value of the negative control group. 595 The test results are shown in Table 2, where MBIC indicates a biofilm inhibition rate of 100%.

[0052] Table 2. Minimum inhibitory concentration and minimum biofilm inhibition concentration of peptides (μg / mL)

[0053]

[0054] As shown in Table 2, DJK-5@SVA and SVA@DJK-5 exhibited the best antibacterial activity, with minimum inhibitory concentrations (MICs) and minimum biofilm inhibition concentrations (MICs) of 31.25 μg / mL against Streptococcus mutans and Enterococcus faecalis; and MICs and MICs against multi-species plaques of 250 μg / mL.

[0055] (4) Inhibition of biofilm formation on hydroxyapatite (HA) sheets (Sichuan University Biomaterials Engineering Research Center)

[0056] After ultrasonic cleaning and disinfection, the HA plates were placed in 24-well plates and randomly divided into 5 treatment groups for the following treatment:

[0057] Treatment Group 1 (water): Add the same volume of water as the DJK-5 peptide solution in Treatment Group 2;

[0058] Treatment group 2 (SVA): SVA peptide solution was added;

[0059] Treatment group 3 (DJK-5): DJK-5 peptide solution was added;

[0060] Treatment group 4 (DJK-5@SVA): DJK-5@SVA peptide solution was added;

[0061] Treatment group 5 (SVA@DJK-5): Add SVA@DJK-5 peptide solution;

[0062] In this embodiment, when verifying the inhibitory effect against Streptococcus mutans or Enterococcus faecalis, the effective concentration of the peptide in treatment groups 2 to 5 was 20 μM; when verifying the inhibitory effect on oral plaque, the effective concentration of the peptide in treatment groups 2 to 5 was 161 μM.

[0063] After treatments 1-5 were completed, each group was incubated overnight at 4°C. Take 1 mL of the 10... 7CFU / mL bacterial suspension was added dropwise to HA slides. After inoculation for 1.5 hours, the inoculation was replaced with fresh, sterile BHI. The slides were then anaerobically incubated at 37°C for a specified time (24 hours for verifying inhibitory effects against Streptococcus mutans or Enterococcus faecalis; 4 hours for verifying inhibitory effects on oral plaque). After rinsing the biofilm with PBS, the slides were stained with a LIVE / DEAD BacLight Bacterial Viability Kit in the dark for 20 minutes. The slides were then scanned and photographed using a laser confocal microscope. The live-to-dead bacterial ratio was calculated using IMRIS. The results are shown below. Figures 1-3 As shown.

[0064] according to Figure 1 It can be seen that DJK-5@SVA and SVA@DJK-5 can bind to HA to exert anti-mutagenic effects against Streptococcus mutans; according to Figure 2 It can be seen that DJK-5@SVA and SVA@DJK-5 can exert anti-Enterococcus faecalis effects by binding to HA, but the DJK-5@SVA group has a higher Enterococcus faecalis mortality rate, which is significantly higher than that of DJK-5 and SVA@DJK-5; according to Figure 3 It can be seen that DJK-5@SVA and SVA@DJK-5 can combine with HA to exert anti-clinical plaque effects against multiple oral bacteria. The DJK-5@SVA group has a higher rate of dead bacteria in oral plaque, which is significantly higher than that of DJK-5 and SVA@DJK-5.

[0065] (5) Killing effect on biofilm formation on HA tablets

[0066] After ultrasonic cleaning and sterilization, the HA tablets were placed in a 24-well plate, and 2 mL of the 10-well plate obtained in step (1) was added. 7 CFU / mL bacterial culture was anaerobically cultured at 37℃ for 3 days. After rinsing the biofilm with ultrapure water, the individuals were randomly divided into 5 treatment groups for the following treatments:

[0067] Treatment Group 1 (water): Add the same volume of water as the DJK-5 peptide solution in Treatment Group 2;

[0068] Treatment group 2 (SVA): SVA peptide solution was added;

[0069] Treatment group 3 (DJK-5): DJK-5 peptide solution was added;

[0070] Treatment group 4 (DJK-5@SVA): DJK-5@SVA peptide solution was added;

[0071] Treatment group 5 (SVA@DJK-5): Add SVA@DJK-5 peptide solution;

[0072] In this embodiment, when verifying the inhibitory effect against Streptococcus mutans or Enterococcus faecalis, the effective concentration of the peptide in treatment groups 2 to 5 was 20 μM; when verifying the inhibitory effect on oral plaque, the effective concentration of the peptide in treatment groups 2 to 5 was 161 μM.

[0073] After treatments 1-5, the cells were rinsed once with PBS, stained with the LIVE / DEAD BacLight Bacterial Viability Kit in the dark for 20 minutes, and then scanned and photographed using a laser confocal microscope. The live-to-dead bacterial ratio was calculated using IMRIS. The results are as follows: Figures 4-6 As shown.

[0074] according to Figure 4 It can be seen that DJK-5@SVA and SVA@DJK-5 can kill Streptococcus mutans resistant to mutation within the biofilm already formed on HA, and the bactericidal effect of DJK-5@SVA is significantly higher than that of DJK-5 and SVA@DJK-5; according to Figure 5 It can be seen that DJK-5@SVA and SVA@DJK-5 can kill Enterococcus faecalis in the biofilm already formed on HA, and DJK-5@SVA has a better bactericidal effect than DJK-5 and SVA@DJK-5; according to Figure 6 It can be seen that DJK-5@SVA and SVA@DJK-5 can kill oral plaque of multiple species in the biofilm that has formed on HA, and DJK-5@SVA has a slightly better bactericidal effect than DJK-5 and SVA@DJK-5.

[0075] Test Example 2

[0076] Detection of hydroxyapatite binding capacity of peptides

[0077] Hydroxyapatite (HA) powder was sequentially ultrasonically washed with ultrapure water, anhydrous ethanol, and ultrapure water. The HA powder was then treated with 10 wt.% NaOH solution at 90°C for 2 hours, followed by washing with ultrapure water until neutral. The HA powder was dispersed in 10 mM HEPES solution, and peptides (DJK-5@SVA, SVA@DJK-5) at different concentrations (0, 5, 10, 15, 20, 25, 30 μM) were added and incubated overnight at 37°C and 180 rpm. The centrifuge tubes were then removed and centrifuged at 12000 rpm for 10 min. The supernatant was added to a 96-well plate (three replicates per tube), and the prepared BCA working solution was added and incubated at 60°C for 1 hour. OD562 was measured, and the amount of unbound peptide in the supernatant was calculated based on the standard curve. The absorbance was calculated using the formula: Qe = C0 (initial sample volume) - Ce (unbound amount in supernatant). The obtained data were fitted to the Freundlich adsorption model, and the affinity constant k was calculated. The results are as follows: Figure 7 As shown.

[0078] according to Figure 7 It can be seen that DJK-5@SVA and SVA@DJK-5 fit the Freundlich adsorption model well, and the coefficient of determination R of the fitting curve is high. 2 Both are close to 1, and DJK-5@SVA's R 2 The value reached 0.9151. The affinity constant k was calculated based on the fitted curve. The affinity of DJK-5@SVA was higher than that of SVA@DJK-5, which is consistent with the results of the anti-biofilm experiment in Test Example 1.

[0079] Test Example 3

[0080] Biocompatibility testing of peptides

[0081] Biocompatibility of the peptides was assessed using a CCK-8 assay kit: Human gingival fibroblasts (HGF-1) were seeded at 5000 cells / well in 96-well plates and incubated for 24 h. Then, DMEM basal medium containing different concentration gradients of peptides (DJK-5, DJK-5@SVA, SVA@DJK-5) was added. After another 24 h of incubation, the supernatant was discarded, and CCK-8 working solution was added. The cells were then examined under an optical microscope, and absorbance (A) was measured at 450 nm using a microplate reader. Statistical analysis was performed to calculate relative cell viability. The results are shown below. Figure 8 As shown.

[0082] according to Figure 8 It can be seen that the relative activity of each group of cells was not significantly different from that of the control group without peptides (P>0.05), and all were above 80%, indicating that the compound peptides have good biocompatibility. Furthermore, compared with the control group without peptides, the cell morphology after peptide treatment did not change significantly, indicating that the peptides have no significant effect on cell morphology, further demonstrating their good biocompatibility.

[0083] As can be seen from the above, the composite polypeptide provided by the present invention has good biocompatibility, antibacterial and anti-biofilm capabilities, and can specifically bind to hydroxyapatite materials and target and bind to dental hard tissues.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite polypeptide, It is characterized in that including hydroxyapatite-binding peptides and antimicrobial peptides; The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1; The amino acid sequence of the hydroxyapatite binding peptide is shown in SEQ ID NO.

2.

2. The composite polypeptide according to claim 1, It is characterized in that The amino acids of the antimicrobial peptide are D-type amino acids; and the amino acids of the hydroxyapatite-binding peptide are L-type amino acids.

3. The composite polypeptide according to claim 1 or 2, It is characterized in that The amino acid sequence of the composite polypeptide is shown as SEQ ID NO.4 or SEQ ID NO.

14.

4. Use of the composite polypeptide according to any one of claims 1 to 3 in the preparation of one or more of an antibacterial product, an anti-biofilm product and a targeted hydroxyapatite product.

5. The use according to claim 4, It is characterized in that The antimicrobial products include products that inhibit pathogens and / or inhibit the formation of biofilms of pathogens.

6. The use according to claim 5, It is characterized in that The pathogenic bacteria include one or more of Streptococcus mutans, Enterococcus faecalis and oral plaque.

7. An antimicrobial agent, It is characterized in that It comprises the composite polypeptide according to any one of claims 1 to 3.

8. Use of the composite polypeptide according to any one of claims 1 to 3 or the antibacterial agent according to claim 7 in the preparation of a product for preventing and / or treating oral infectious diseases.

9. The use according to claim 8, It is characterized in that The oral infectious diseases include diseases caused by pathogenic bacteria; the pathogenic bacteria include one or more of Streptococcus mutans, Enterococcus faecalis and oral plaque.

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