Efficient antibacterial peptide, preparation method thereof and application of efficient antibacterial peptide in preparation of medicine for treating vaginitis

Abstract

The invention discloses an efficient antibacterial peptide, a preparation method of the efficient antibacterial peptide and an application of the efficient antibacterial peptide in preparation of a medicine for treating vaginitis. The novel antibacterial peptide is Leu-Arg-Trp-Lys-Phe-His-Val-Lys-Trp-Arg (LRWKFHVKWR. The antibacterial peptide has a remarkable inhibition effect on candida albicans, staphylococcus aureus and escherichia coli, and the antibacterial peptide not only has excellent biocompatibility and stability, but also shows excellent drug resistance to the candida albicans. The antibacterial peptide is combined with hydroxypropyl methyl cellulose and glycerol through a physical cross-linking method to prepare an injectable hydrogel. The hydrogel is used for treating candida vaginitis induced by candida albicans, and mouse model verification shows that the hydrogel can remarkably reduce the level of vagina inflammatory factors (IL-6 and TNF-alpha) and is free of organ toxicity. The problems that traditional antibiotics are prone to drug resistance and uncontrollable in release are solved, and important clinical application value is achieved.

Description

Technical field:

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to an antimicrobial peptide based on a specific amino acid sequence Leu-Arg-Trp-Lys-Phe-His-Val-Lys-Trp-Arg (LRWKFHVKWR) and a preparation method thereof, as well as an application of a hydrogel composition containing the antimicrobial peptide in the treatment of vaginitis. The hydrogel composition contains hydroxypropylmethylcellulose (HPMC), glycerol, and deionized water, and is suitable for the treatment of bacterial vaginitis, candidal vaginitis, and aerobic bacterial vaginitis. Background technology:

[0002] Vaginitis is a common gynecological infection that occurs when a woman's immune system is weakened or she uses antibiotics for a long time. Among them, Candidal vaginitis (VVC) accounts for 75% of infections in women of childbearing age worldwide, and many of them suffer from repeated infections of Candidal vaginitis throughout their lives. Currently, commonly used drugs for the treatment of Candidal vaginitis include antibiotics such as clotrimazole and metronidazole. However, long-term use of antibiotics can damage healthy tissue cells in the vagina and may cause an imbalance in vaginal flora and acid-base balance, and produce drug-resistant bacterial strains, which increases the difficulty of treatment. Therefore, the need for new treatments is critical.

[0003] Antimicrobial peptides (AMPs) are small peptides abundant in nature that play an important role in the innate immune defense system against infection in most organisms. AMPs are usually amphiphilic and positively charged, with excellent spectrum antimicrobial activity, low biotoxicity, and most importantly, low resistance. AMPs can be screened using cell membrane chromatography, a biomimetic chromatography technique that uses active cell membranes as stationary phases. It is widely used due to its high efficiency and low cost. In this study, we used homemade cell membrane chromatography to screen an antimicrobial peptide with excellent antimicrobial effect and excellent biocompatibility.

[0004] The porous structure of hydrogels enables them to absorb large amounts of water and swell without decomposing

[15] .

[0005] Its soft performance characteristics are very similar to human soft tissue. Hydrogel materials are often used in biomedicine and other fields due to their unique properties. Hydroxypropyl methylcellulose (HPMC) is a stable, relatively viscoelastic polymer. It has good film-forming ability, is biocompatible, and is soluble in water regardless of pH, making it suitable for topical vaginal administration.

[0006] In this invention, we prepared an antimicrobial peptide (LRWKFHVKWR) with high antimicrobial activity and excellent cell compatibility by solid phase synthesis, and combined it with hydroxypropyl methylcellulose (HPMC) and glycerol to prepare an antimicrobial hydrogel with sustained release function. The composition not only has excellent antimicrobial properties, but also effectively avoids drug resistance problems, and is suitable for the treatment of various vaginal infections such as candidal vaginitis, bacterial vaginitis and aerobic bacterial vaginitis. Summary of the invention:

[0007] In order to address the above problems, the purpose of the present invention is to provide a highly effective antimicrobial peptide and a preparation method thereof, as well as a hydrogel composition containing the antimicrobial peptide for treating and preventing vaginitis.

[0008] The technical solution of the present invention is as follows:

[0009] Preparation of antimicrobial peptides: The antimicrobial peptides with the amino acid sequence of LRWKFHVKWR were prepared by solid phase synthesis. The natural or synthetic amino acid matrix containing carboxyl groups and amino groups was mainly polycondensed by amidation reaction, and its molecular weight was verified to be 1456.79Da by mass spectrometry. In addition to being used directly, the synthesized antimicrobial peptides can also be designed as peptides with other functions according to different occasions. Alternatively, reactive functional groups such as carboxyl or hydroxyl groups can be introduced into the polypeptide molecule by partial bonding of ester bonds and amide bonds.

[0010] Performance of antimicrobial peptides:

[0011] (1) The antimicrobial peptide has significant antibacterial effects on Staphylococcus aureus, Escherichia coli and Candida albicans, with minimum inhibitory concentrations (MIC) of 6 μg / mL, 20 μg / mL and 10 μg / mL, respectively.

[0012] (2) Antimicrobial peptides exhibit good biocompatibility both in vitro and in vivo and are not prone to induce drug resistance.

[0013] Preparation of hydrogel loaded with antimicrobial peptides: A porous hydrogel composed of hydroxypropyl methylcellulose and glycerol is simultaneously loaded with cationic antimicrobial peptides, wherein the concentration of the antimicrobial peptides is 25 mg / kg.

[0014] The present invention also provides a method for preparing the hydrogel, the steps of which are as follows:

[0015] (1) Dissolve the antimicrobial peptide in an appropriate amount of deionized water and stir evenly;

[0016] (2) Then add 5% glycerol, heat to 65°C, add 3% hydroxypropyl methylcellulose, stir evenly and wait for mixing, then heat for 15 minutes, turn off the heat after the hydroxypropyl methylcellulose is completely dissolved, and wait for natural cooling;

[0017] (3) Stirring continuously until the mixture is cooled to room temperature and then stopped. After cooling to room temperature, stirring is stopped and the mixture is left overnight. When the hydrogel is diluted to a pH value of about 4, hydrochloric acid is used to adjust the pH value to obtain an injectable hydrogel containing antimicrobial peptides.

[0018] In general, compared with the prior art, the technical solution described in the present invention has the following beneficial results:

[0019] (1) Broad-spectrum antibacterial activity: The antimicrobial peptide has significant antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and fungi, and is suitable for the treatment of a variety of vaginal infections.

[0020] (2) Not prone to drug resistance: Compared with existing antibiotics, the antimicrobial peptides are not prone to induce drug resistance and are suitable for long-term use.

[0021] (3) Sustained release function: The hydrogel composition can slowly release antimicrobial peptides, prolong the drug action time, and improve the therapeutic effect.

[0022] (4) Good biocompatibility: The composition is non-toxic to cells and is compatible with the vaginal environment, and can effectively maintain the balance of vaginal flora.

[0023] (5) Multiple dosage forms: The composition can be prepared into injectable hydrogels, vaginal suppositories, nanoparticle suspensions, and soluble films for easy clinical use.

[0024] (6) Compared with the prior art, the method of the present invention is easy to operate, does not require an initiator, and as a hydrogel material has the advantages of treating vaginitis infection and having a simple preparation process. Description of the drawings:

[0025] Figure 1 (a) is a mass spectrum of the antimicrobial peptide of the present invention, Figure 1 (b) is the structural formula of the antimicrobial peptide;

[0026] Figure 2 The antibacterial zone effect diagram of the antimicrobial peptide of the present invention on Staphylococcus aureus, Escherichia coli, and Candida albicans;

[0027] Figure 3 Minimum inhibitory concentration of antimicrobial peptides against Staphylococcus aureus, Escherichia coli, and Candida albicans

[0028] Figure 4 (a) is HPMC hydrogel, Figure 4(b) is P2 / HPMC hydrogel, Figure 4 (c) is the SEM image of HPMC hydrogel. Figure 4 (d) is the SEM image of P2 / HPMC hydrogel;

[0029] Figure 5 Fourier transform infrared spectroscopy was used to detect the differences in chemical bonds between HPMC and P2 / HPMC hydrogels;

[0030] Figure 6 To test the stability of hydrogel in artificial vaginal fluid;

[0031] Figure 7 is the antimicrobial peptide release rate of the hydrogel;

[0032] Figure 8 Live / dead staining inverted fluorescence microscope images of L929 cells treated with peptide hydrogels of different concentrations at 1, 2, and 3 days;

[0033] Fig. 9 Testing the peptide hydrogels for cell proliferation;

[0034] Fig.10 To test the cytotoxicity of peptide hydrogels to cells;

[0035] Fig.11 is the hemolysis rate of mouse blood cells by peptide hydrogels of different concentrations;

[0036] Fig.12 This is a photo of the LB plate used to test the drug resistance of antimicrobial peptides to Candida albicans;

[0037] Fig.13 This is a photo of the LB plate of mouse vaginal lavage fluid;

[0038] Fig.14 (a) shows the changes of IL-6 in different groups of mice. Fig.14 (b) Changes of TNF-α in different groups of mice;

[0039] Fig.15 H&E and Masson staining of sections of mouse wound healing skin and various tissues and organs. Specific implementation method:

[0040] The present invention is further described in detail below in conjunction with embodiments:

[0041] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0042] Example 1

[0043] This example provides a method for preparing the antimicrobial peptide LRWKFHVKWR (P2), comprising the following steps:

[0044] First, fill the extraction tube with 0.5g of dichlorotriphenyl resin balls and then dissolve them in DMF for 90 minutes. After adding the first amino acid and DIEA for 2 hours, wash the reactants 3 times, and then add 150μL DIEA and 5mL methanol to terminate the reaction. After rinsing 3 times, add piperidine at a concentration of 20% to remove the Fomc protecting group. Then, after washing 3 times, add the second amino acid Pybop and HOBT, add DIEA and DMF to dissolve and react for 2h, and then add 20% piperidine. Repeat the above steps to synthesize the desired peptide library, and finally add 4.75mL trifluoroacetic acid, 125μL triisopropylsilane and 125μL deionized water for peptide cleavage, then react for 2 hours, and drop the cleavage reaction solution into 30mL ice ether to precipitate the product. The resulting product is centrifuged and freeze-dried for storage.

[0045] Example 2

[0046] This example provides a method for minimum inhibitory concentration testing, comprising the following steps:

[0047] The antimicrobial peptide prepared by the present invention is dried and dissolved in PBS to conduct inhibition zone experiment and minimum inhibitory concentration (MIC) experiment. In the inhibition zone experiment, 200 μL of pre-made bacterial suspension is added to 10 mL of LB broth culture medium that has been sterilized at high temperature and cooled to body temperature, mixed and poured into a culture dish, and after the culture medium solidifies, two holes are punched in each LB culture medium, one hole is added with PBS solution, and the other is added with peptide solutions of different concentrations, and the culture plate is placed in a bacterial incubator (37°C) and observed after 24 hours. Figure 2 Shown are the inhibition zone images of antimicrobial peptides against Staphylococcus aureus, Escherichia coli, and Candida albicans, respectively.

[0048] In the MIC experiment, 10 μL of bacterial suspension was added to 1 mL of solution containing different gradient concentrations of peptide LRWKFHVKWR, and incubated in a bacterial incubator for 2 hours. Then 100 μL of each solution was evenly applied to the LB medium after high-temperature sterilization and solidification, and the LB medium was placed in a bacterial incubator (37°C) for 24 hours. Figure 3 It shows that the short peptide prepared by the present invention has good antibacterial property and obvious inhibition zone. The minimum inhibitory concentration of the peptide against Staphylococcus aureus is 6 μg / mL; the minimum inhibitory concentration against Escherichia coli is 20 μg / mL; and the minimum inhibitory concentration against Candida albicans is 10 μg / mL.

[0049] Example 3

[0050] This embodiment provides a method for preparing a hydrogel containing antimicrobial peptides, comprising the following steps:

[0051] Add a certain amount of deionized water to a water bath and heat it, then add antimicrobial peptides at a concentration of 25 mg / kg, stir well, add 5% glycerol, heat to 65°C, add 3% hydroxypropyl methylcellulose (HPMC), stir well and wait for mixing, then heat for 15 minutes, turn off the heating after HPMC is completely dissolved, wait for natural cooling, keep stirring, and finally stop after cooling to room temperature. After cooling to room temperature, stop stirring and leave overnight. When the hydrogel is diluted to a pH of about 4, use hydrochloric acid to adjust the pH. Figure 4 (d) Shown is the scanning image of the hydrogel loaded with antimicrobial peptides.

[0052] Example 4

[0053] This example provides a Fourier transform infrared spectroscopy detection method, comprising the following steps:

[0054] After freeze-drying, the chemical bond composition of the hydroxypropyl methylcellulose hydrogel sample containing antimicrobial peptides was analyzed by Fourier transform infrared spectroscopy. Figure 5 As shown, the peak is 3517cm -1 OH stretching vibration at 2900-3000cm -1 CH stretching vibration at 1032 cm -1 The peak at 3517 cm corresponds to the stretching vibration of COC and CO. -1 The OH intensity at did not change, indicating that the synthesis process of P2 / HPMC hydrogel was physical cross-linking.

[0055] Example 5

[0056] This example provides a method for determining the stability of artificial vaginal fluid (SVF), comprising the following steps:

[0057] Prepare the hydrogel sample in Experimental Example 1. In order to evaluate the stability of P2 / HPMC hydrogel in simulated vaginal fluid (SVF), we weighed 20 mg of hydrogel and placed it in SVF for 12 h. Samples were taken every hour, and the peak area changes were observed after HPLC detection. Figure 6 As shown, after being placed in the artificial vaginal fluid for 10 hours, the content of antimicrobial peptide P2 is still above 50%, indicating that the antimicrobial peptide can be released continuously and stably in the vagina.

[0058] Example 6

[0059] This example provides a method for determining the slow release rate of antimicrobial peptides, comprising the following steps:

[0060] Determination of the standard curve of antimicrobial peptide concentration: Take 1 mg of LRWKFHVKWR powder and dissolve it in 1 mL of ultrapure water to obtain a 1 mg / mL antimicrobial peptide solution. Then, take 500 μL of the 1 mg / mL antimicrobial peptide solution and add it to 500 ultrapure water to dilute it into a 500 μg / mL P2 solution, and then perform gradient dilutions in sequence to prepare a series of solutions with a concentration gradient of 125 μg / mL-1000 μg / mL. Use a UV spectrophotometer to measure the absorbance of the LRWKFHVKWR solution, that is, the ABS value (absorbance value) at 280 nm.

[0061] Hydrogel antimicrobial peptide release assay: Add 1 mL of PBS buffer solution to the 24-well plate containing the hydrogel and immerse the gel. Place the 24-well plate on a shaker to achieve uniform distribution of LRWKFHVKWR in each well. Set up three parallel replicate wells for each group, take the immersion solution every hour, and measure the concentration of antimicrobial peptide LRWKFHVKWR released in the solution. Figure 7 As shown, after 24 hours, the release rate of antimicrobial peptides can reach more than 45%.

[0062] Example 7

[0063] This example provides a biocompatibility testing method, comprising the following steps:

[0064] The short peptide prepared by the present invention is tested for its cytotoxicity using the MTT method. First, L929 cells are placed in a 96-well plate and cultured for 24 hours, and then a series of antimicrobial peptides LRWKFHVKWR at different concentrations are added to each well. After incubation for 24 hours, the cell survival rate is measured using an ELISA instrument, and untreated cells are used as a control. The peptide is incubated in L929 cells for 24, 48, and 72 hours, and then stained with AM / PI, and the cell survival is observed under an inverted fluorescence microscope. The results show that the short peptide prepared by the present invention is non-toxic to L929 cells and has a high cell survival rate, indicating that it has good biocompatibility. Figure 8 Live / dead staining inverted fluorescence microscopy images of L929 cells treated with hydrogels of different concentrations on day 1, 2, and 3; Fig. 9 Cell proliferation test for hydrogels; Fig.10 Cytotoxicity test of hydrogel on L929 cells.

[0065] Example 8

[0066] This example provides a test method for a hemolysis experiment, comprising the following steps:

[0067] The antimicrobial peptide hydrogel prepared by the present invention was tested for its hemolytic activity using mouse blood cells. The antimicrobial peptide hydrogel was treated with PBS to a concentration gradient of 0, 1, 2, 5, 10, 15, and 20 mg / mL to treat blood cells, and blood cells treated with Triton X-100 were used as a positive control. After two hours of treatment, the cells were photographed, centrifuged, and the supernatant was measured with an ELISA reader. Finally, the blood cells were fixed with 2.5% glutaraldehyde and dehydrated with ethanol gradients before being scanned. The results showed that the antimicrobial peptide hydrogel prepared by the present invention had good blood compatibility. Fig.11 The hemolysis rate of mouse blood cells by hydrogels of different concentrations.

[0068] Example 9

[0069] This example provides a method for testing the anti-drug resistance performance of a peptide, comprising the following steps:

[0070] The pre-prepared Candida albicans suspension (1×10 7 CFU / mL) as the solvent in the centrifuge tube, prepare the MIC solution for Candida albicans, and prepare the MIC solution of clotrimazole as a comparison. Incubate in a bacterial incubator (37°C, 100rpm), take a sample (200μL) every 2 hours as a generation, then take 100μL of the mixture and spread it evenly on the LB culture plate, incubate (37°C, 24 hours), count the colonies, take pictures, and record the MIC n / MIC1 values ​​were plotted as dotted line graphs to observe the changes. Within 10 generations of Candida albicans, the minimum inhibitory concentration of the peptide against Candida albicans did not change, while the corresponding clotrimazole showed a 2-fold increase in the minimum inhibitory concentration against Candida albicans in the 10th generation, indicating that Candida albicans began to show strong resistance to the drug. Such results indicate that the new short peptide we discovered has excellent anti-Candida albicans resistance. Fig.12 The LB plate photo of the peptide resistance test on Candida albicans is compared with the MIC change curve of the peptide and clotrimazole.

[0071] Example 10

[0072] This example provides a method for establishing a mouse vaginitis model, comprising the following steps:

[0073] In this example, we will use KM mice infected with Candida albicans to evaluate the therapeutic effect of P2 / HPMC hydrogel in the in vivo antibacterial test in the mammalian anti-infection model. First, four equal groups were created from mice: saline group, HPMC hydrogel group, clotrimazole group, and P2 / HPMC hydrogel group. The mice were induced to enter pseudoestrus by preparing 5 mg / mL estradiol benzoate dissolved in vegetable oil and injecting 0.1 mL subcutaneously every day for one week. 20 μL of Candida albicans suspension (1×109 CFU / mL) was injected into the vagina for 5 days. On the last day, the vulva of the mice was observed for swelling, and the plates were coated with vaginal washings diluted with PBS to observe the success of modeling, which was the same as day 0. Group treatment was carried out from the first day and continued for 25 days. The plates were coated with vaginal washings from different groups of mice every two days. Fig.13 This is a photo of the LB plate of mouse vaginal lavage fluid. On the 15th day, no Candida albicans was produced in the antimicrobial peptide hydrogel group.

[0074] Embodiment 11

[0075] This example provides a method for pathological analysis of wound tissue and organ toxicology testing, including the following steps:

[0076] On the last day, after each mouse was killed, the kidneys, liver, spleen, lungs, heart, and vagina were taken and preserved in 4% paraformaldehyde. The sections were then examined using H&E and Masson staining and examined under an inverted fluorescence microscope. Mouse plasma was taken and the amount of inflammatory factors IL-6 and TNF-α was analyzed using an enzyme-linked immunosorbent assay kit. The levels of inflammatory factors in the P2 / HPMC hydrogel group were quite low, indicating that the antimicrobial peptide hydrogel group can well reduce vaginal inflammation and is more conducive to the repair of vaginal epithelial cells. In addition, to determine how the drug affects other organs of mice, we performed H&E staining and analyzed the heart, liver, spleen, lungs, and kidneys of mice, and the P2 / HPMC hydrogel group had no adverse immune effects on mice. Fig.14 (a) shows the changes of IL-6 in different groups of mice. Fig.14 (b) Changes of TNF-α in different groups of mice; Fig.15 H&E and Masson staining of sections of mouse wound healing skin and various tissues and organs.

Claims

1. A highly effective antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is Leu-Arg-Trp-Lys-Phe-His-Val-Lys-Trp-Arg (LRWKFHVKWR).

2. A method for preparing the highly effective antimicrobial peptide according to claim 1, characterized in that: The preparation method comprises the following steps: a. According to the amino acid sequence LRWKFHVKWR, the antimicrobial peptide chain was synthesized by solid phase synthesis; b. The antimicrobial peptide was mixed with deionized water at a concentration of 25 mg / kg, glycerol and hydroxypropyl methylcellulose were added, heated and stirred, and after cooling, the pH was adjusted to 4 to obtain an injectable hydrogel; The antimicrobial peptide is composed of 10 amino acid residues and has a molecular weight of 1456.79 Da (verified by mass spectrometry).

3. An antibacterial composition for treating or preventing vaginitis, characterized in that: The composition comprises the following components: the antimicrobial peptide according to claim 1, hydroxypropylmethylcellulose, glycerol and deionized water.

4. The antibacterial composition according to claim 3, characterized in that The antimicrobial peptide has an inhibitory effect on the following pathogenic microorganisms: Staphylococcus aureus, Escherichia coli, and Candida albicans.

5. The antibacterial composition according to claim 4, characterized in that The composition is suitable for preparing at least one of an in vivo therapeutic drug or an in vitro therapeutic drug.

6. The antibacterial composition according to claim 4, characterized in that The composition is used for preparing a method for locally treating diseases caused by bacterial or fungal infection.

7. The antibacterial composition according to claim 4, characterized in that The composition can be used in combination with probiotics and / or antibacterial agents to prepare medicines for treating diseases related to bacterial or fungal infections.

8. The antibacterial composition according to claim 4, characterized in that The dosage form of the composition is selected from any one or more of the following: injectable gel, vaginal suppository, nanoparticle suspension, soluble film.

9. The antibacterial composition according to claim 4, characterized in that The composition is used for preparing medicines for treating diseases related to Gram-positive bacteria, Gram-negative bacteria or fungal infections.

10. The antibacterial composition according to claim 4, characterized in that: The composition is used for preparing at least one of the drugs for treating the following diseases: bacterial vaginitis, candidal vaginitis, and aerobic bacterial vaginitis.

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