Preparation method and application of antibacterial peptide hydrogel dressing with good drug resistance

Abstract

The invention discloses a preparation method and application of an antibacterial peptide hydrogel dressing with good drug resistance, and relates to the technical field of functional materials. Comprising antibacterial peptide, an initiator, a sodium alginate matrix, a reaction aid, a reaction activator and the like. The preparation method has the beneficial effects that the antibacterial peptide is screened, and the prepared antibacterial peptide is added into a sodium alginate matrix, so that rapid gelation under a normal-temperature condition can be realized; the gel has the characteristics of good stability, short coagulation time, long sterilization time, excellent drug resistance and the like, has good adhesive force and lasting antibacterial effect, can realize efficient sterilization, realizes the dual effects of efficient antibacterial and difficult generation of drug resistance, and solves the problem of drug-resistant bacteria in the process of treating skin bacterial infection. The polypeptide has potential application value in the aspect of treating bacterial infectious diseases.

Description

Technical field:

[0001] The present invention belongs to the field of biomedical materials, and more particularly relates to a preparation method and application of a hydrogel based on natural sodium alginate and containing antimicrobial peptides. Background technology:

[0002] Infectious diseases caused by drug-resistant bacteria cause a significant number of deaths worldwide and have become a major medical issue. A range of antimicrobial drugs has been developed. Antibiotics are an important class of antimicrobial agents. It is well known that typical antibiotics inhibit cellular protein synthesis, affect cell membrane permeability, or specifically interfere with bacterial biochemical metabolism, causing them to lose their ability to grow and thrive, thereby inhibiting or killing bacteria and ultimately leading to cell death. However, the easy availability and overuse of antibiotics have led to an increase in antibiotic-resistant bacteria. The emergence of drug-resistant pathogens has rendered most currently used antibiotics ineffective, making the treatment of bacterial infectious diseases a major challenge. Currently, pathogenic bacteria cause approximately 90 billion infections annually and kill approximately 20,000 children each year. The World Health Organization has designated drug-resistant bacteria as a serious challenge facing healthcare systems. Therefore, the development of alternative antimicrobial drugs to treat drug-resistant bacteria is urgent.

[0003] In recent years, antimicrobial peptides have emerged as potential alternatives to traditional antibiotics due to their excellent biocompatibility, rapid and efficient broad-spectrum antimicrobial activity, and low resistance potential. Therefore, the design of antimicrobial peptides with broad-spectrum antimicrobial properties has become an important area of ​​research. Furthermore, antimicrobial peptides are also needed to increase their in vivo half-life in mammalian cells and reduce their cytotoxic or hemolytic activity. To date, nearly 5,000 natural antimicrobial peptides have been discovered from animals, plants, and marine organisms. Approximately 50% of these antimicrobial peptides are short cationic peptides consisting of 10-50 amino acids. It is generally believed that antimicrobial peptides bind to bacteria through electrostatic interactions, inducing pore formation and breaking down cell membranes, thereby killing the bacteria.

[0004] Disruption of the cell membrane is considered difficult to develop drug resistance. Therefore, the use of antimicrobial peptides as antimicrobial drugs to treat infectious diseases caused by pathogenic bacteria will not develop drug resistance. We have recently developed a new class of short cationic peptides as a new class of antimicrobial peptides. Although antimicrobial peptides have the potential to be new antimicrobial agents, their poor stability is mainly due to the high degree of proteolysis, which limits the clinical application of antimicrobial peptides. Other limitations, such as poor interaction with host cells, may lead to systemic toxicity. Therefore, AMPs are actually not feasible in some environments because infected tissues often show high proteolytic activity. One of the common approaches to overcome these limitations is to encapsulate the AMP in a carrier

[0005] In view of the problems existing in the existing technology and materials, the present invention has studied a new short cationic peptide with good biocompatibility and hemolytic properties, as well as excellent antibacterial properties. More importantly, compared with the current antimicrobial peptides, it exhibits excellent antibacterial effects and has long-term stable effects. At the same time, we immobilized the cationic peptide on an ordered mesoporous hydrogel composed of sodium alginate and cationic monomers to prepare an antibacterial hydrogel. These materials form an antibacterial hydrogel with high liquid absorption properties through covalent fixation of AMP. At the same time, experiments show that AMP physically loaded into the hydrogel can exert higher bactericidal activity and improve the stability of enzymatic degradation without releasing the peptide. In the local open wound model of bacterial infection in mice, it promoted wound healing to a certain extent. Therefore, we believe that this strategy has great prospects in the development of in vivo infectious wound treatment platforms and can be applied to other treatment methods. Summary of the invention:

[0006] To address the above-mentioned problems, the present invention provides a method for preparing a hydrogel that can be used to treat skin wounds and inhibit bacteria, effectively solving the problem of drug-resistant bacteria. Specifically, the method involves preparing a hydrogel containing the following ingredients.

[0007] The technical solutions of the present invention are as follows:

[0008] Preparation of antimicrobial peptides: Utilizing solid-phase synthesis, natural or synthetic amino acid matrices containing carboxyl and amino groups are polycondensed primarily through amidation reactions. These synthesized antimicrobial peptides can be used directly or designed to possess various other functionalities depending on the application. Alternatively, reactive functional groups such as carboxyl or hydroxyl groups can be introduced into the peptide molecules through the formation of ester and amide bonds.

[0009] Preparation of hydrogel loaded with antimicrobial peptides: An ordered mesoporous hydrogel composed of sodium alginate and CaCl2 is loaded with cationic antimicrobial peptides, wherein the above-mentioned antimicrobial peptide molecules account for 15-20% of the weight of the entire hydrogel.

[0010] According to one embodiment of the present invention, the antibacterial polypeptide described is an antibacterial polypeptide containing tryptophan at the N-terminus and a polypeptide sequence containing continuous alternating positive and negative charges at the C-terminus.

[0011] The present invention also provides a method for preparing the hydrogel, which comprises the following steps:

[0012] (1) Dissolve the antimicrobial peptide in an appropriate amount of deionized water and stir evenly to obtain solution ①;

[0013] (2) Dissolve the reaction aid, calcium chloride, completely with an appropriate amount of deionized water and stir evenly to obtain solution ②;

[0014] (3) Stirring the sodium alginate matrix with an appropriate amount of deionized water at 30-50° C. to dissolve it completely, and removing bubbles during the stirring process using an ultrasonic cleaning machine to obtain solution ③; freeze-drying the solution;

[0015] (4) Dissolve the freeze-dried sodium alginate matrix in a buffer solution and adjust the pH to about 7-7.5. Then, add a small amount of solution ② by feeding and stir to obtain a sol.

[0016] (5) Add solution ① to the sol obtained in step (4) and continue stirring to obtain antimicrobial peptide-modified sodium alginate hydrogel through electrostatic adsorption.

[0017] In general, the technical solution described in the present invention has the following beneficial results compared with the existing technology:

[0018] (1) The hydrogel has a three-dimensional network structure, which is characterized by the antibacterial effect of the hydrogel after loading the antimicrobial peptide can reach 98%, and the antimicrobial peptide can be slowly released in the hydrogel. The hydrogel can be used to treat bacterial infections of wounds and promote wound healing to a certain extent.

[0019] (2) Compared with the prior art, the method of the present invention is easy to operate and, as a hydrogel material, has the advantages of treating bacterial infections, having a large saturated adsorption capacity, and a simple preparation process.

[0020] (3) The present invention combines the one-strain-one-substance chemical synthesis method with the solid phase synthesis method to screen cationic short peptide chains. Utilizing the cationic properties of the polypeptide chain, an antibacterial hydrogel based on antimicrobial peptides and sodium alginate was studied. In particular, the sodium alginate used is a monomer with important application value and good biological activity. The negative ions of sodium alginate react with multivalent cations (such as Ca 2+ 、Al 3+ The antimicrobial peptides (e.g., cationic peptides) interact electrostatically and form a cross-linked network, thus forming a gel. The loaded antimicrobial peptide is also a cationic peptide chain. Based on this property, the prepared antimicrobial hydrogel can inhibit bacterial infection in damaged skin by slowly releasing the antimicrobial peptides, while also accelerating wound healing to a certain extent. Description of the drawings:

[0021] Figure 1 Schematic diagram of the hydrogel gelation process prepared in Example 1

[0022] Figure 2 Scanning electron microscopy image of the hydrogel prepared in Example 1

[0023] Figure 3 Detecting chemical bond differences between freeze-dried hydrogels and raw hydrogels using Fourier transform infrared spectroscopy

[0024] Figure 4 Rheometer testing of frozen hydrogels

[0025] Figure 5 To detect the release rate of antimicrobial peptides from the gel surface in PBS buffer solution with a pH of 7.5

[0026] Figure 6 (a) is a diagram showing the inhibition zone effect of the antimicrobial peptide of the present invention on Staphylococcus aureus and Escherichia coli. Figure 6 (b) is the minimum inhibitory concentration of the peptide against Staphylococcus aureus, Figure 6 (c) is the minimum inhibitory concentration of the peptide against Escherichia coli;

[0027] Figure 7 Live / dead staining inverted fluorescence microscope images of L-929 cells treated with peptide hydrogels at different concentrations on days 1, 2, and 3;

[0028] Figure 8 To test the cytotoxicity of peptide hydrogels to cells;

[0029] Figure 9 Scanning electron microscopy (SEM) images of mouse blood cells treated with peptide hydrogels at different concentrations;

[0030] Figure 10 is the hemolysis rate of mouse blood cells by peptide hydrogels at different concentrations;

[0031] Figure 11 To investigate the healing process of mouse wounds treated with different methods;

[0032] Figure 12 To create a graph of mouse weight changes after wounding;

[0033] Figure 13 Photo of LB plate for drug resistance test (Staphylococcus aureus and Escherichia coli);

[0034] Figure 14 H&E staining of sections of mouse wound healing skin and various tissues and organs; Specific implementation method:

[0035] The present invention is described in further detail below with reference to the embodiments:

[0036] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0037] Example 1 Preparation of RKIIIRW-loaded hydrogel

[0038] Preparation of frozen alginate hydrogels:

[0039] Weigh sodium alginate and dissolve it in ultrapure water under stirring at a certain temperature to prepare a sodium alginate solution with a mass concentration of 1-2%. Use an ultrasonic cleaner to ultrasonically treat it to remove bubbles. Subsequently, prepare a calcium chloride solution with a concentration of 1%-2%, and mix the two solutions. Add the above mixed solution to a 1mm thick glass membrane, place it in a -80℃ refrigerator and freeze it for 12 hours, and freeze-dry it. Place the freeze-dried hydrogel at room temperature, wait for it to return to room temperature, and then cut the gel. The final product is a frozen sodium alginate hydrogel.

[0040] Preparation of hydrogels containing antimicrobial peptides:

[0041] Weigh RKIIIRW and dissolve it in PBS to obtain a concentration gradient of 150ug / mL to 1000ug / mL. Then soak the sodium alginate gel obtained above in the RKIIIRW solution, incubate it at room temperature for 2 hours with stirring, and then use it. Wash off the excess RKIIIRW with PBS buffer solution to obtain a hydrogel containing antimicrobial peptides. Figure 2 As shown, the prepared hydrogel was subjected to ethanol gradient dehydration, and the morphology of the gel was observed using a scanning electron microscope after complete dehydration.

[0042] Example 2 Fourier transform infrared spectroscopy detection

[0043] After freeze-drying, 25 mg of the sodium alginate macroporous frozen hydrogel sample was taken and the chemical bond composition of the freeze-dried powder was analyzed by Fourier transform infrared spectroscopy. The selected spectra were recorded in the range of 4000-500 cm -1 The chemical bond differences between the hydrogel freeze-dried powder and the raw materials were detected by Fourier transform infrared spectroscopy. The hydrogel freeze-dried powder was detected at 2850 cm -1 The characteristic peaks appeared at , indicating that the hydrogel was successfully loaded with antimicrobial peptides, such as Figure 3 The results show that the hydrogel prepared by freeze drying has a relative strength to meet the application of wound dressing

[0044] Example 3 Rheometer Test Performance

[0045] Prepare the hydrogel sample in Experimental Example 1. Place the gel sample on the sample stage of the rheometer and evaluate the time-varying properties of the hydrogel at 37°C and 1 Hz. Figure 4As shown, it can be seen that during the test, the storage modulus (G') of the hydrogel was greater than the loss modulus (G"), indicating that the gels all exhibited solid gel properties, which was mainly attributed to the cross-linking effect induced by CaCl2 and the cationic peptide RKⅢRW. It is worth noting that the storage modulus of the hydrogel is related to the cross-linking density, and the G' of sodium alginate is higher than that of the antimicrobial peptide hydrogel, indicating that the presence of antimicrobial peptides reduces the cross-linking density of the hydrogel. In addition, within the experimental range, the storage modulus and loss modulus of the hydrogel did not change much, indicating that the two types of gels have relatively stable structures. This proves the successful preparation of the hydrogel.

[0046] Example 4 Determination of slow release rate of antimicrobial peptides

[0047] To determine the antimicrobial peptide concentration standard curve: Dissolve 1 mg of RKIIIRW powder in 1 mL of ultrapure water to obtain a 1 mg / mL antimicrobial peptide solution. Then, pipette 500 μL of this 1 mg / mL antimicrobial peptide solution into 500 mL of ultrapure water to dilute it to a 500 μg / mL DP7 solution. This solution is then serially diluted to prepare a series of solutions with a concentration gradient ranging from 125 μg / mL to 1000 μg / mL. The absorbance of the RKIIIRW solution is measured using a UV spectrophotometer (ABS) at 280 nm.

[0048] Gel surface peptide release assay: Add 1 ml of PBS buffer solution with a pH of 7.5 to a 24-well plate containing frozen gel and immerse the gel. Place the 24-well plate on a shaker to achieve uniform distribution of RKIIIRW in each part of the well. Set up three parallel replicate wells for each group, and take the immersion solution at 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h and 96h to measure the concentration of antimicrobial peptide RKIIIRW released in the solution. Figure 5 As shown in the release curve, this slow release characteristic can ensure the continuous therapeutic effect at the wound and achieve the effect of controlled release, which is beneficial to reduce toxic side effects.

[0049] Example 5 Minimum Inhibitory Concentration Test

[0050] The short 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 solution of different concentrations. The culture plate is placed in a bacterial incubator (37 ° C) and observed after 24 hours. In the MIC experiment, 10 μL of bacterial suspension is added to 1 mL of a solution containing different gradient concentrations of peptide RKIIIRW, placed in a bacterial incubator and incubated for 2 hours, and then 100 μL of each solution is evenly applied to the LB culture medium after high temperature sterilization and solidification, and the LB culture medium is placed in a bacterial incubator (37 ° C) and cultured for 24 hours. The results showed that the short peptide prepared by the present invention had good antibacterial properties and obvious inhibition zones. The minimum inhibitory concentration of the peptide against Staphylococcus aureus was 80 μg / mL; the minimum inhibitory concentration against Escherichia coli was 100 μg / mL. Figure 6 (a) is a diagram showing the inhibition zone effect of the short peptide of the present invention on Staphylococcus aureus and Escherichia coli. Figure 6 (b) is the minimum inhibitory concentration of the short peptide against Staphylococcus aureus, Figure 6 (c) is the minimum inhibitory concentration of the short peptide against Escherichia coli.

[0051] Example 6 Biocompatibility

[0052] The short peptides prepared by the present invention were tested for cytotoxicity using the MTT assay. 929 cells were first cultured in a 96-well plate for 24 hours, and then a series of different concentrations of the antimicrobial peptide RKIIIRW were added to each well. After 24 hours of incubation, cell viability was measured using a microplate reader, with untreated cells serving as a control. The peptides were incubated in 929 cells for 24, 48, and 72 hours, followed by AM / PI staining, and cell viability was observed under an inverted fluorescence microscope. The results showed that the short peptides prepared by the present invention were non-toxic to 929 cells and had a high cell survival rate, indicating good biocompatibility. Figure 7 Live / dead staining inverted fluorescence microscopy images of L-929 cells treated with different concentrations of peptides on day 1, 2, and 3; Figure 8 The cytotoxicity of the peptides to 929 and LO2 cells was tested.

[0053] Example 7 Hemolysis Experiment

[0054] The hemolytic activity of the short peptides prepared in this invention was measured using mouse blood cells. The antimicrobial peptides were treated with a gradient of concentrations of 0, 100, 200, 400, 600, 800, and 1000 μg / mL in PBS. Triton X-100-treated blood cells served as a positive control. After two hours of treatment, the cells were photographed, centrifuged, and the supernatant analyzed using a microplate reader. Finally, the cells were fixed with 2.5% glutaraldehyde, dehydrated with a gradient of ethanol, and then photographed and scanned. The results demonstrated good hemocompatibility of the short peptides prepared in this invention. Figure 9 SEM images of mouse blood cells treated with different concentrations of peptides; Figure 10 is the hemolysis rate of mouse blood cells at different concentrations of peptide.

[0055] Example 8 Mouse Wound Healing Model

[0056] In the in vivo antibacterial test of the peptide prepared in this example in the mammalian anti-infection model, mice were divided into four groups: the mice were treated with equal amounts of sterilized PBS solution, peptide solution, Staphylococcus aureus suspension (1×10 7 CFU / mL), peptide and Staphylococcus aureus mixed suspension (1×10 7 CFU / mL) treatment, wherein the concentration of the peptide was 200μg / mL (3*MIC concentration), and all solutions used PBS as solvent. Throughout the experiment, mice in all groups were free to drink water and eat. In each group, sterilized scissors were used to make excision wounds on the dorsal surface of the mouse. Then 20μL of each solution was inoculated into each wound of the mouse. The wound area and weight of the mice were measured every day. The results showed that in the group with added peptide, the wound healed significantly faster than the group with only bacteria added. The group using peptide alone had no effect on the mice, and the mice's eating and weight were not affected, so the peptide has good biosafety. Figure 11 To investigate the healing process of mouse wounds treated with different methods; Figure 12 This is a graph showing the weight changes of mice after wounding.

[0057] Example 9 Anti-drug resistance properties of peptides

[0058] The pre-prepared bacterial suspension (1×10 7 CFU / mL) was used as the solvent in the centrifuge tube to prepare the MIC solution for Escherichia coli and the MIC solution for Staphylococcus aureus. At the same time, the MIC solution of penicillin-streptomycin dual antibody solution was prepared 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 evenly spread it on the LB culture plate, incubate (37°C, 24 hours), count the colonies, take pictures, and record the MIC nThe MIC / MIC1 values ​​were plotted as a dotted line graph to observe changes. The peptide's minimum inhibitory concentration (MIC) for bacteria remained unchanged within 10 bacterial passages. However, the corresponding penicillin-streptomycin dual-antibiotic solution's MIC against Staphylococcus aureus doubled by the 10th passage, signaling the beginning of strong bacterial resistance to the drug. The MIC for Escherichia coli increased to 1.5-fold by the 7th passage, and resistance against E. coli further increased by the 10th passage. These results demonstrate that our novel short peptide possesses excellent antibacterial resistance. Figure 13 Photos of LB plates for peptide resistance testing (Staphylococcus aureus and Escherichia coli) and comparison of MIC change curves of peptide and penicillin-streptomycin dual antibody solution.

[0059] Example 10 Pathological Analysis of Wound Tissue and Organ Toxicology Testing

[0060] To more clearly visualize the structural changes in wound tissue after wound healing, skin sections from the healed wounds of mice were sectioned, stained, and observed under an inverted fluorescence microscope. H&E staining of the sections revealed the formation of new skin tissue in both the experimental and control groups, but the growth of cells and blood vessels within the tissue varied. In the bacterial infection and PBS groups, only epidermal growth occurred, without the growth of internal cells and blood vessels, indicating incomplete healing. In contrast, the density of hair follicles, myofibroblasts, and capillaries in the tissues of the peptide + bacteria and pure peptide groups gradually increased. This suggests that the peptide promotes fibroblast proliferation and differentiation, thereby accelerating wound healing. To verify the peptide's side effects on mouse organs, organ sections from the heart, liver, spleen, lung, and kidney of the four treated groups were stained to observe the effects of the peptide on these organs after entering the circulation. As shown in the figure, no pathological signs such as fibrosis, inflammation, or localized bleeding were observed in any of the five organ types in the four groups of mice. These results demonstrate that the RKGAK peptide has no adverse effects on major organs and is safe for use as an open wound dressing. Figure 14 H&E staining of sections of mouse wound healing skin and various tissues and organs.

Claims

1. A hydrogel of antimicrobial peptide with good drug resistance, characterized in that: The system includes antimicrobial peptide, sodium alginate matrix, initiator, reaction aid and deionized water, etc. The amino acid sequence of the antimicrobial peptide is: RKIIIRW; the reaction aid is calcium chloride.

2. The antimicrobial peptide-containing hydrogel according to claim 1, characterized in that The antimicrobial peptide is prepared by using a solid-phase synthesis method to synthesize an antimicrobial peptide chain according to the amino acid sequence RKIIIRW, and purifying it by HPLC column chromatography. The peptide contains 7 amino acid residues and has a molecular weight of 984.32 Da.

3. The antimicrobial peptide hydrogel according to claim 1, characterized in that The minimum inhibitory concentration of the antimicrobial peptide hydrogel against Escherichia coli is 100 μg / mL.

4. The antimicrobial peptide hydrogel according to claim 1, characterized in that The minimum inhibitory concentration of the antimicrobial peptide hydrogel against Staphylococcus aureus is 80 μg / mL.

5. The antimicrobial peptide-containing hydrogel according to claim 1, characterized in that The concentration of the antimicrobial peptide substance in the antimicrobial peptide hydrogel is 1-500 μg / mL.

6. The antimicrobial peptide-containing hydrogel according to claims 1-5, characterized in that The preparation method of the hydrogel containing antimicrobial peptides comprises the following steps: (1) Dissolve the antimicrobial peptide in an appropriate amount of deionized water and stir evenly to obtain solution ①; (2) Dissolve the reaction aid, calcium chloride, completely with an appropriate amount of deionized water and stir evenly to obtain solution ②; (3) Stirring the sodium alginate matrix with an appropriate amount of deionized water at 30-50° C. to dissolve it completely, and removing bubbles during the stirring process using an ultrasonic cleaning machine to obtain solution ③; freeze-drying the solution; (4) Dissolve the freeze-dried sodium alginate matrix in a buffer solution and adjust the pH to about 7-7.

5. Then, add a small amount of solution ② by feeding and stir to obtain a sol. (5) adding solution ① to the sol obtained in step (4), continuing stirring, and obtaining antimicrobial peptide-modified sodium alginate hydrogel by electrostatic adsorption.

7. The antimicrobial peptide-containing hydrogel according to claim 6, characterized in that The hydrogel containing the antimicrobial peptide can be used to quickly treat small wounds, abrasions, cuts and infected wounds by applying the hydrogel on the wound surface, and the infection is caused by Staphylococcus aureus.

8. The use according to claim 6, characterized in that: The screened antimicrobial peptides and antimicrobial peptide hydrogels have good drug resistance.

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