Anti-infection wound repair promoting hydrogel as well as preparation method and application thereof

Abstract

The invention discloses anti-infection wound repair promoting hydrogel, a preparation method and application, firstly, polydopamine microspheres are prepared according to a dopamine oxidation auto-polymerization principle, then antibacterial peptide is embedded into the polydopamine microspheres, and antibacterial peptide-coated polydopamine microspheres are obtained; and then the antibacterial peptide coated polydopamine microspheres are introduced into pullulan and gelatin composite hydrogel, so that the anti-infection hydrogel capable of promoting wound repair is prepared. Experimental results show that the anti-infection hydrogel capable of promoting wound repair has no stimulation effect on wounds, is safe and free of toxic and side effects, can be attached to the wounds, has long-acting and stable antibacterial, anti-inflammatory, pain-relieving and repair-promoting effects, effectively promotes wound healing and repair, protects wounds from infection, and has excellent biocompatibility and biodegradability. In addition, the preparation method is simple to operate and convenient for large-scale popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an anti-infection wound repair-promoting hydrogel, a preparation method and an application thereof. Background Art

[0002] Chronic wounds caused by bacterial infection have become a major problem and challenge in the medical community. Bacterial infection is one of the important factors that lead to the inability of wounds to heal, and if the infection is severe, it can also cause sepsis. Antibiotic resistance has become an urgent public health challenge worldwide. About 700,000 people die each year from bacterial infections caused by the failure of antibiotics. It is estimated that by 2050, this number will reach 10 million per year. Although some antibacterial alternatives have emerged, such as quaternary ammonium salts, metal ions and antibacterial complexes. But their cytotoxicity and immunogenicity cannot be ignored. Therefore, there is still an urgent need to develop new antibacterial agents to combat bacterial infections, especially wound infections.

[0003] Antimicrobial peptides are short peptides that have a wide range of inhibitory effects on bacteria, fungi and viruses. Antimicrobial peptides interact with bacterial cell membranes to destroy the structural integrity of bacterial cell membranes, leading to the outflow of bacterial contents, thereby achieving a rapid antibacterial effect. This special mechanism of action is not easy to produce drug resistance. Antimicrobial peptides also show unique advantages in preventing wound infection and promoting healing, but their high toxicity and low stability limit their practical application. In order to expand the scope of application of antimicrobial peptides, it is necessary to carry out reasonable design to maintain or enhance the antibacterial activity of antimicrobial peptides and reduce their hemolysis and cytotoxicity. Such as compounding with suitable carriers.

[0004] Hydrogel is a new type of functional polymer material with high water content. Its three-dimensional network structure gives it elasticity similar to that of tissue, which enables it to combine well with skin tissue and relieve pain, while the high water content helps to maintain wound moisture to accelerate wound healing. Loading antimicrobial peptides into suitable hydrogels can slowly release antimicrobial peptides, thereby avoiding degradation by biological enzymes and the cytotoxicity of high concentrations of antimicrobial peptides. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide an anti-infection and wound repair-promoting hydrogel, a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a method for preparing an anti-infection wound repairing hydrogel, comprising the following steps:

[0008] S1, preparation of antimicrobial peptide@polydopamine microspheres, denoted as AMP@PDA microspheres;

[0009] S2. Preparation of pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres:

[0010] S21, dissolving pullulan in a sodium hydroxide solution to prepare a pullulan solution;

[0011] S22, adding antimicrobial peptide @ polydopamine microspheres to the pullulan solution, mixing, and reacting to obtain an antimicrobial peptide @ polydopamine microsphere / pululan mixed solution;

[0012] S23, dispersing gelatin in water to prepare a gelatin solution;

[0013] S24, adding the gelatin solution to the antimicrobial peptide@polydopamine microspheres and pullulan mixed solution, mixing, and preparing an antimicrobial peptide@polydopamine microspheres / pululan / gelatin mixed solution;

[0014] S25. Add ethylene glycol diglycidyl ether to the antimicrobial peptide@polydopamine microspheres / pullulan / gelatin mixed solution, stir the reaction, and let it stand to obtain pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres, i.e., an anti-infection and wound repairing hydrogel.

[0015] Preferably, the pullulan, gelatin, antimicrobial peptide @ polydopamine microspheres, and ethylene glycol diglycidyl ether are used in a ratio of 0.2-25 g: 0.2-25 g: 1-60 mg: 50-1000 μL, and more preferably 2.5-12.5 g: 2.5-12.5 g: 5-30 mg: 100-600 μL.

[0016] Preferably, in step S1, the preparation of antimicrobial peptide@polydopamine microspheres comprises the following steps:

[0017] S11, adding dopamine hydrochloride into ultrapure water, mixing evenly, and preparing a dopamine hydrochloride aqueous solution;

[0018] S12, adding the antimicrobial peptide to the dopamine hydrochloride aqueous solution, mixing evenly, to prepare an antimicrobial peptide / dopamine hydrochloride mixed solution;

[0019] S13. Drop the antimicrobial peptide / dopamine hydrochloride mixed solution into the ethanol base solution at room temperature, stir overnight, and centrifuge to obtain antimicrobial peptide@polydopamine microspheres.

[0020] Preferably, the mass concentration of dopamine hydrochloride in the dopamine hydrochloride aqueous solution is 0.5 to 15%, and more preferably 2 to 10%.

[0021] Preferably, the antimicrobial peptide is a cationic polypeptide, more preferably one or a combination of two or more of FFRKVLKLIRKILR (SEQ ID NO.1), FFRKVLKLIRKIFR (SEQ ID NO.2) and FFRKVLKLIRKIWR (SEQ ID NO.3);

[0022] Preferably, the mass concentration of the antimicrobial peptide in the antimicrobial peptide / dopamine hydrochloride mixed solution is 0.01-2%, more preferably 0.1-1%.

[0023] Preferably, the ethanol alkali solution is a solution obtained by mixing ultrapure water, anhydrous ethanol and alkali, and the volume fraction of ethanol in the ethanol alkali solution is 5 to 80%, more preferably 15 to 50%;

[0024] Preferably, the mass concentration of the alkali in the ethanol alkali solution is 1 to 15%, and more preferably 3 to 10%; the alkali is one or a combination of two or more of CaCO3, Ca(OH)2, Na2CO3, NaHCO3, K2CO3, KHCO3, monoethanolamine, diethanolamine and triethanolamine, and more preferably one or a combination of two or more of NaHCO3, KHCO3, diethanolamine and triethanolamine.

[0025] Preferably, the volume ratio of the dopamine hydrochloride aqueous solution to the ethanol base solution is 1:1-25, more preferably 1:5-15; the dripping speed of the antimicrobial peptide / dopamine hydrochloride mixed solution is 1-20 mL / min, more preferably 2-10 mL / min.

[0026] Preferably, the mass concentration of pullulan in the pullulan solution is 1 to 50%, more preferably 5 to 25%.

[0027] Preferably, the mass concentration of gelatin in the gelatin solution is 1 to 50%, more preferably 5 to 25%.

[0028] The second aspect of the present invention is to provide an anti-infection and wound repair-promoting hydrogel prepared by the above preparation method.

[0029] The third aspect of the present invention is to provide the use of the above-mentioned anti-infection and wound repair-promoting hydrogel in the preparation of drugs for wound infection and wound repair.

[0030] The present invention has the following beneficial effects:

[0031] (1) The present invention firstly utilizes the principle of dopamine oxidation self-polymerization to obtain polydopamine microspheres, and then embeds antimicrobial peptides into polydopamine microspheres to obtain antimicrobial peptides @ polydopamine microspheres; then, inspired by the mussel adhesion mechanism, antimicrobial peptides @ polydopamine microspheres are introduced into pullulan and gelatin composite hydrogels to prepare an anti-infection wound repair hydrogel, i.e., pullulan / gelatin hydrogel loaded with antimicrobial peptides @ polydopamine microspheres. Among them, by successfully embedding antimicrobial peptides into polydopamine microspheres, not only can the degradation of biological enzymes and the like be avoided, and its stability be improved; it can also achieve slow and continuous release of antimicrobial peptides, achieve a long-term antibacterial effect, and at the same time avoid hemolytic activity and cytotoxicity caused by excessive concentration. The experimental results show that the anti-infection wound repair hydrogel has no irritating effect on the wound, is safe and has no toxic side effects, can adhere to the wound, and has long-lasting and stable antibacterial, anti-inflammatory, pain-relieving and repair-promoting effects, effectively promoting wound healing, repairing and protecting wounds to prevent infection, and has excellent biocompatibility and biodegradability.

[0032] (2) The preparation method of the present invention is simple to operate and is convenient for large-scale promotion and application.

[0033] In summary, the present invention provides the obtained hydrogel with excellent biocompatibility, mechanical properties, adhesion properties, sustained release properties, long-acting antibacterial properties and infection wound healing properties by compounding antimicrobial peptide@polydopamine microspheres, pullulan and gelatin. It is expected to be used in the preparation of drugs for wound infection and wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 :(a) and (b) are SEM images of antimicrobial peptide @ polydopamine (AMP@PDA) prepared in Examples 1-2, respectively; (c) is a SEM image of polydopamine (PDA) microspheres prepared in Comparative Example 1;

[0036] Figure 2 The Fourier transform infrared spectra are as follows: (a) dopamine hydrochloride (DA), PDA microspheres, AMP@PDA microspheres, AMP powder; (b) gelatin (Gel) powder, Pu / Gel hydrogel, pullulan (Pu), and pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres prepared in Examples 1, 9, and 10;

[0037] Figure 3 is the release curve of AMP in AMP@PDA microspheres;

[0038] Figure 4 This is the result of histological analysis of the wound using H&E staining (red arrows represent inflammatory cells, yellow arrows represent hair follicles, and black arrows represent epithelial tissue);

[0039] Figure 5 This is the result of histological analysis of the wound using Masson staining;

[0040] Figure 6 The figure shows the expression results of TNF-α in rat burn wounds;

[0041] Figure 7 This is the expression result of TGF-β1 in rat burn wounds;

[0042] Figure 8 This is the expression result of TGF-β3 in rat burn wound. DETAILED DESCRIPTION

[0043] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0044] Example 1

[0045] A method for preparing an anti-infection wound repairing hydrogel, comprising the following steps:

[0046] S1. Preparation of antimicrobial peptide@polydopamine microspheres, referred to as AMP@PDA microspheres, including the following specific steps:

[0047] S11, preparing a dopamine hydrochloride aqueous solution: adding 5 g of dopamine hydrochloride to 100 mL of water, stirring until completely dissolved, to obtain a dopamine hydrochloride aqueous solution;

[0048] S12, preparing an antimicrobial peptide / dopamine hydrochloride mixed solution: adding 0.5 g of an antimicrobial peptide (sequence FFRKVLKLIRKIWR) to 100 ml of an aqueous solution of dopamine hydrochloride, stirring until completely dissolved, to obtain an antimicrobial peptide / dopamine hydrochloride mixed solution;

[0049] S13, prepare ethanol alkali solution: add 20ml ethanol to 80ml water, then add 5g triethanolamine, stir evenly, to obtain ethanol alkali solution;

[0050] S14, add 10 ml of the antimicrobial peptide / dopamine hydrochloride mixed solution to 100 ml of ethanol base solution at a speed of 5 ml / min, keep a constant water bath at room temperature, stir at high speed, react for 24 hours, after the reaction is completed, centrifuge at a speed of 10000 r / min for 10 minutes, wash the precipitate with ultrapure water and ethanol alternately in sequence until the solution is clear, and dry it in a 50°C forced air drying oven to obtain antimicrobial peptide@polydopamine microspheres;

[0051] S2. Preparation of pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres:

[0052] S21, preparing a pullulan solution: weighing 10 g of pullulan powder and dissolving it in 100 ml of sodium hydroxide solution (mass concentration of 4.0%), stirring at room temperature for 20 min until completely dissolved, to obtain a pullulan solution;

[0053] S22, adding 10 mg of the antimicrobial peptide @ polydopamine microspheres (AMP@PDA) prepared in step S1 to 100 ml of the prepared pullulan solution, stirring until fully dispersed, to obtain a mixed solution of antimicrobial peptide @ polydopamine microspheres and pullulan;

[0054] S23, preparing gelatin solution: adding 10 g of gelatin (Gel) to 100 ml of deionized water for pre-swelling for 40 minutes, stirring at 50° C. until completely melted, and preparing a uniform gelatin solution;

[0055] S24, adding 100 ml of the mixed solution of antimicrobial peptide @ polydopamine microspheres and pullulan to 50 ml of the gelatin solution, stirring evenly to obtain a mixed solution of antimicrobial peptide @ polydopamine microspheres / pululan / gelatin;

[0056] S25. At room temperature, 400 μL of ethylene glycol diglycidyl ether was dropped into the antimicrobial peptide@polydopamine microspheres / pullulan / gelatin mixed solution, stirred for 10 min, and allowed to stand for 3 h to obtain pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres, recorded as AMP@PDA / Pu / Gel-10.

[0057] Example 2

[0058] The steps are basically the same as those in Example 1, except that "0.5 g antimicrobial peptide (sequence FFRKVLKLIRKIWR)" in step S12 is replaced by "0.1 g antimicrobial peptide (sequence FFRKVLKLIRKIWR)".

[0059] Example 3

[0060] The steps are basically the same as those in Example 1, except that "0.5 g antimicrobial peptide (sequence FFRKVLKLIRKIWR)" in step S12 is replaced by "0.5 g antimicrobial peptide (sequence FFRKVLKLIRKIWA)".

[0061] Example 4

[0062] The steps are basically the same as those in Example 1, except that "0.5 g antimicrobial peptide (sequence FFRKVLKLIRKIWR)" in step S12 is replaced by "0.5 g antimicrobial peptide (sequence FFRKVLKLIAKIWA)".

[0063] Example 5

[0064] The steps are basically the same as those in Example 1, except that, when preparing the ethanol alkaline solution in step S13, "add 20 ml of anhydrous ethanol to 80 mL of water" is replaced by "add 40 mL of anhydrous ethanol to 60 mL of water".

[0065] Example 6

[0066] The steps are basically the same as those in Example 1, except that, when preparing the ethanol alkali solution in step S13, "5 g triethanolamine" is replaced by "8 g triethanolamine".

[0067] Example 7

[0068] The steps are basically the same as those in Example 1, except that in step S14, "10 ml of antimicrobial peptide / dopamine hydrochloride mixed solution" is replaced by "20 mL of antimicrobial peptide / dopamine hydrochloride mixed solution".

[0069] Example 8

[0070] The steps are basically the same as those in Example 1, except that when preparing the pullulan solution, "10 g of pullulan powder" is replaced by "5 g of pullulan powder".

[0071] Example 9

[0072] The steps are basically the same as those in Example 1, except that when the antimicrobial peptide @ polydopamine microspheres are dispersed in the pullulan solution, "10 mg antimicrobial peptide @ polydopamine microspheres" are replaced with "2 mg antimicrobial peptide @ polydopamine microspheres" to obtain pullulan / gelatin hydrogel loaded with antimicrobial peptide @ polydopamine microspheres, which is recorded as AMP@PDA / Pu / Gel-2.

[0073] Example 10

[0074] The steps are basically the same as those in Example 1, except that: in step S24, when preparing the antimicrobial peptide @ polydopamine microspheres / pullulan / gelatin hydrogel loaded with antimicrobial peptide @ polydopamine microspheres, "10 mg antimicrobial peptide @ polydopamine microspheres" is replaced with "30 mg antimicrobial peptide @ polydopamine microspheres" to obtain pullulan / gelatin hydrogel loaded with antimicrobial peptide @ polydopamine microspheres, recorded as AMP@PDA / Pu / Gel-30.

[0075] Embodiment 11

[0076] The steps are basically the same as those in Example 1, except that when preparing the antimicrobial peptide@polydopamine microspheres / pullulan / gelatin mixed solution, "50 mL gelatin solution" is replaced with "100 mL gelatin solution".

[0077] Example 12

[0078] The steps are basically the same as those in Example 1, except that in step S24, "400 μL ethylene glycol diglycidyl ether" is replaced with "100 μL ethylene glycol diglycidyl ether".

[0079] Comparative Example 1

[0080] The steps are basically the same as those in Example 1, except that no antimicrobial peptide is added in step S1.

[0081] Comparative Example 2

[0082] The steps are basically the same as those in Example 1, except that no antimicrobial peptide@polydopamine microspheres are added in step S2.

[0083] Comparative Example 3

[0084] The steps are basically the same as those in Example 1, except that no gelatin solution is added in step S2.

[0085] Comparative Example 4

[0086] The steps are basically the same as those in Example 1, except that: "adding the antimicrobial peptide @ polydopamine microspheres (10 mg) to 100 mL of pullulan solution" is replaced by "dispersing the antimicrobial peptide into the pullulan solution; wherein, 6.86 g of the antimicrobial peptide (sequence FFRKVLKLIRKIWR), equivalent to 10 mg of the antimicrobial peptide @ polydopamine microspheres) is added to 100 mL of the pullulan solution.

[0087] 1. Structural characterization and performance research

[0088] (a) Surface morphology analysis (SEM)

[0089] The morphology of the polydopamine microspheres prepared in Comparative Example 1 and the AMP@PDA microspheres prepared in Examples 1-2 were observed using a scanning electron microscope. Figure 1 .

[0090] Depend on Figure 1 (a) The results show that the polydopamine (PDA) microspheres prepared in Comparative Example 1 have a clear spherical morphology with an average diameter of 125±10.9 nm.

[0091] Depend on Figure 1 (b) and 1(c) show that the antimicrobial peptide @ polydopamine (AMP@PDA) microspheres prepared in Examples 1 and 2 also have a clear spherical morphology. The average diameter of the AMP@PDA microspheres prepared in Example 1 is 336±4.6nm, and the average diameter of the AMP@PDA microspheres prepared in Example 2 is 193±19.7nm. Comparing the results of Comparative Example 1 and Examples 1-2, it can be seen that the diameter of the AMP@PDA microspheres obtained after embedding the antimicrobial peptide is significantly increased, and increases with the increase of the concentration of the antimicrobial peptide; compared with PDA microspheres, the surface of AMP@PDA microspheres is rougher, which indicates that there is a good interaction between the antimicrobial peptide and the polydopamine molecules.

[0092] (b) Fourier transform infrared spectroscopy

[0093] Fourier transform infrared spectroscopy was performed on dopamine hydrochloride (DA), PDA microspheres, AMP@PDA microspheres, AMP powder, pullulan (Pu) powder, gelatin (Gel) powder, Pu / Gel hydrogel and AMP@PDA / Pu / Gel hydrogel prepared in Examples 1 and 9-10, respectively. The results are shown in Figure 2 .

[0094] Depend on Figure 2 (a) The results show that in the PDA spectrum, 3000-3500 cm -1 The broad peak at 1599 cm -1 The band centered at 3000-3500cm is the indole-related structure formed after dopamine oxidation and self-polymerization. -1 The narrowing of the (-OH absorption peak) broadband may be the result of intermolecular hydrogen bonding interaction; and no additional peaks appear in the AMP@PDA spectrum, indicating that no covalent bond is formed. However, the absorption peak amide I band of AMP (1672cm -1 ) is significantly weakened, and the wave number shifts to 1595cm -1 ; and 1595cm -1 The absorption peak at AMP is significantly weaker than that of PDA; these results indicate that there is a strong interaction between AMP and PDA.

[0095] Depend on Figure 2 (b) The results show that Pu / Gel and AMP@PDA / Pu / Gel hydrogels have a high affinity to AMP@PDA at 3500 cm -1 Up to 3250cm -1 The broad peak at 2918 cm is caused by CC and OH stretching vibrations, and the intensity of the absorption peak of AMP@PDA / Pu / Gel-30 (Example 10) hydrogel is stronger, indicating that the hydrogen bonding between AMP@PDA microspheres and Pu / Gel hydrogel molecules is strengthened. -1 The absorption peaks at 1654, 1668 and 1601 cm-1 are due to the stretching vibration of CH in the methyl group. Pu, Gel and AMP@PDA appear at 1654, 1668 and 1601 cm-1, respectively. -1 The absorption peak at 1660 cm -1 This indicates that there is an interaction between AMP@PDA microspheres, Pu and Gel.

[0096] (c) Determination of microsphere particle size and embedding efficiency

[0097] The particle sizes and embedding rates of the AMP@PDA microspheres prepared in Examples 1 to 7 and the PDA microspheres prepared in Comparative Example 1 were measured using a nanoparticle size analyzer and an ultraviolet spectrophotometer, respectively. The results are shown in Table 1.

[0098] It can be seen from the results in Table 1 that in the process of preparing AMP@PDA microspheres, the type and amount of AMP, the concentration of DA, the concentration of ethanol solution and the pH value have a significant effect on the particle size of AMP@PDA microspheres and the encapsulation rate of AMP.

[0099] Table 1

[0100]

[0101] (d) Release performance of AMP from AMP@PDA microspheres

[0102] PBS (pH 7.4) was used as the release medium for AMP, and the release behavior of AMP in the microspheres prepared in Example 1 was detected using a micro-BCA protein quantification kit. The results are as follows: Figure 3 shown.

[0103] Depend on Figure 3The results show that the release of AMP in AMP@PDA microspheres is divided into two stages: slow release and rapid release. In the 1-48h period, there is a rapid release, and AMP shows a nearly linear release behavior in this stage. This is mainly due to the AMP adsorbed on the surface of the microspheres, with a release rate of 47%. The rapid release of AMP in the early stage is conducive to the rapid inhibition / sterilization of the wound surface and effectively controls wound infection. The next 48-168h is a slow release process, which is mainly due to the slow release of AMP embedded in the microspheres, and the total release rate can reach 75%. The slow release of AMP in the later stage can achieve long-term antibacterial effects, promote wound healing, and also solve the problem of easy degradation of antimicrobial peptides.

[0104] (e) Analysis of hydrogel swelling

[0105] The swelling properties of the AMP@PDA / Pu / Gel hydrogels prepared in Examples 1-12 and the hydrogels prepared in Comparative Examples 1-4 were tested. The results are shown in Table 2.

[0106] Table 2

[0107]

[0108] As shown in Table 2, the Pu / Gel hydrogel (Comparative Example 2) itself has a certain water absorption capacity, and the swelling rate is 3069.1±156.9%. After adding microspheres, its swelling performance changes significantly. As the amount of microspheres added increases, the swelling rate increases from 3688.5±108.2% (Example 9) to 4235.±111.7% (Example 1). However, when the amount of microspheres added continues to increase (Example 10), the water absorption capacity decreases significantly, and the swelling rate is 3323.2±118.5%, which is related to the internal structure of the microsphere hydrogel composite material. This is because the microspheres themselves also have a certain water absorption capacity, but as the number of microspheres increases, the space originally accommodating water molecules decreases, preventing water molecules from entering the hydrogel. In addition, after soaking in PBS for 24 hours, it can be clearly observed that the volume of all hydrogels is significantly increased, and the Pu / Gel hydrogel (Comparative Example 1) even has cracks, while the hydrogel with the addition of microspheres does not have cracks. The above observations indicate that the addition of microspheres not only improves the swelling properties of the hydrogel, but also enhances the intermolecular interactions, making the intermolecular connections tighter.

[0109] (f) Evaluation of mechanical properties of hydrogels

[0110] As an ideal wound dressing, it is essential to have good mechanical properties. The mechanical properties of the AMP@PDA / Pu / Gel hydrogels prepared in Examples 1-12 and the hydrogels prepared in Comparative Examples 1-4 were tested by a universal material testing machine. The results are shown in Table 2.

[0111] As can be seen from the results in Table 2, with the addition of AMP@PDA microspheres, the tensile strength of the obtained hydrogel continues to increase, and the more microspheres are added, the greater the tensile strength. However, when the amount of microspheres added reaches 30 mg (Example 10), the tensile strength begins to decrease. This may be because a large amount of antimicrobial peptides are released from the microspheres, which affects the hydrogen bonding between the catechol groups on the surface of polydopamine and the Pu / Gel hydrogel, thereby reducing the tensile strength of the AMP@PDA / Pu / Gel hydrogel. Therefore, the addition of an appropriate amount of AMP@PDA microspheres is beneficial to improving the mechanical properties of AMP@PDA / Pu / Gel hydrogel, thereby improving its application performance in the field of wound dressings.

[0112] (g) Adhesion properties of hydrogels

[0113] For wound repair materials with excellent performance, a certain degree of adhesion is essential without causing secondary damage. The adhesion strength between the hydrogel and pig skin was tested by the lap shear method. The results are shown in Table 2.

[0114] From the results in Table 2, we can see that as the amount of AMP@PDA microspheres increases, the adhesion strength of AMP@PDA / Pu / Gel hydrogel gradually increases, which can make AMP@PDA / Pu / Gel hydrogel fit perfectly with the skin. In addition, AMP@PDA / Pu / Gel hydrogel can adhere to steel, pig skin, glass and plastic surfaces, and can also adhere to fingers and wrist joints, and can remain motionless after bending and straightening.

[0115] In summary, the type and amount of AMP (antimicrobial peptide), the amount of AMP@PDA microspheres, the amount of Pu, the amount of Gel and the amount of cross-linking agent ethylene glycol diglycidyl ether have a significant effect on the swelling, mechanical properties and adhesion properties of the hydrogel.

[0116] (h) Biocompatibility of hydrogels

[0117] Biomaterials must undergo safety testing before being used in the human body, and hemolysis is one of the testing methods. Generally speaking, the lower the hemolysis rate, the less impact on red blood cells, and the safer the material. After the hydrogel is cultured with red blood cells for a period of time, the absorbance of the supernatant is measured to determine whether the material is hemolytic. The results are shown in Table 3.

[0118] Table 3

[0119]

[0120] From the results in Table 3, it can be seen that the hemolysis rate of the AMP@PDA / Pu / Gel hydrogel prepared in the present invention is less than 5%, which meets the national safety regulations on the blood compatibility of biomaterials. Therefore, the AMP@PDA / Pu / Gel hydrogel has good biocompatibility.

[0121] (i) Cytotoxicity assay

[0122] In order to evaluate the cytotoxicity of the hydrogel, the MTT method was used to analyze the effect of the hydrogel on the viability of L929 mouse fibroblasts. The results are shown in Table 3.

[0123] As shown in Table 3, Pu / Gel hydrogel has certain toxicity to cells (cell survival rate <80%), but the toxicity is significantly reduced after adding microspheres, especially the hydrogel added with AMP@PDA, the cell survival rate is above 100%. This shows that the AMP@PDA / Pu / Gel hydrogel prepared by the present invention has good biological cell compatibility, and has a certain effect of promoting cell proliferation, does not produce toxic effects on normal cells, and also has a certain ability to promote cell migration.

[0124] 2. Application of AMP@PDA / Pu / Gel hydrogel in wound repair

[0125] 2.1 Construction of rat burn model

[0126] Six male SD rats weighing about 220 g (purchased from Guangzhou Ruige Biotechnology Co., Ltd.) were selected as experimental animals. The entire experimental process complies with the ethical standards of animal experiments.

[0127] Male SD rats weighing about 220g were selected, and 0.5mL 1% sodium pentobarbital solution was injected intraperitoneally and 0.02mL of Su Mianxin was injected intramuscularly to anesthetize the rats. The hair on the back of the rats was then shaved with a shaver, and the back skin with an exposed area of ​​5×7cm was removed with a depilatory cream and washed. The exposed skin on the back was disinfected with iodine tincture, and then a copper rod with a diameter of 1.0cm was used to treat it at high temperature to a temperature of about 98°C, and 4 burn wounds were evenly scalded on the back of the rats (holding the copper rod for about 30s), with each wound 2cm apart. After 3h, the necrotic skin of the burns was cut off with surgical scissors. After taking pictures of the wound surface at each time point, 20μL (108CFU / mL) of Escherichia coli and Staphylococcus aureus mixed bacterial solution was dripped in the center of the wound after debridement treatment. After 24h of infection, the corresponding grouped hydrogel material was used to cover the surface of the rat wound. The wound was covered with 3M medical film, and the edges were fixed with tape. After observation until the anesthesia was awakened, the rats were raised in cages. Dressings were changed and samples were collected at set times. Dressings were changed every three days. The degree of wound healing (area) was observed and the healing time was recorded at 0, 3, 7, 10, and 14 days. The wound area was calculated using ImageJ software and statistical analysis was performed. The formula for calculating wound healing rate (HR) is as follows:

[0128] HR (%) = (W0-W n ) / W0×100%

[0129] Where, W0: initial wound area, W n : Wound area on the 3rd, 7th, 10th and 14th days.

[0130] 2.2 Application Performance Evaluation

[0131] (a) Evaluation of wound repair effect

[0132] The wound healing ability of AMP@PDA / Pu / Gel hydrogel was evaluated by using a rat skin burn infection wound model. The results are shown in Table 4.

[0133] Table 4

[0134]

[0135] As shown in Table 4, compared with the control group, the wound area of ​​the hydrogel group (Examples 1, 2, 9 and 10) with the addition of AMP@PDA microspheres was significantly reduced on the third day, and scabs had formed. This is mainly because the hydrogel can keep the wound moist, and the sustained-release AMP has the ability to resist bacteria and promote wound healing. On the 7th day, the healing rate of the AMP@PDA / Pu / Gel hydrogel group had reached more than 85%. On the 14th day, the new epithelial tissue in the AMP@PDA / Pu / Gel group had completely covered the wound, and the healing rate reached more than 97%. However, the central part of the wound in the control group and other groups has not yet completely healed, and the healing rate is below 90%. By comparing Example 1 with Comparative Example 1, it can be seen that AMP has a significant improvement in promoting wound repair; at the same time, it can be seen from Example 1 and Comparative Example 4 that the healing effect of AMP directly added to Pu / Gel hydrogel is significantly weaker than that of AMP@PDA microspheres, because the free AMP concentration is too high, has certain cytotoxicity, and is easily degraded. Therefore, the AMP@PDA / Pu / Gel hydrogel prepared by the present invention has the function of promoting the repair of infected wounds.

[0136] (b) Evaluation of antibacterial activity in vivo

[0137] Bacterial infection can affect wound healing. Excellent wound dressings should not only consider in vitro antibacterial ability, but also in vivo antibacterial performance. First, a copper rod was used to burn the wound evenly on the back of the rat at high temperature, and then the necrotic skin was removed. After debridement, a mixed bacterial solution of Escherichia coli and Staphylococcus aureus was dripped to infect the wound surface. Finally, the hydrogel material was covered on the surface of the rat wound for treatment. The colony count method was used to evaluate the in vivo antibacterial ability of the hydrogel material after 3 and 7 days of treatment. The results are shown in Table 5.

[0138] Table 5

[0139]

[0140]

[0141] As can be seen from the results in Table 5, all the hydrogels in this experiment have a certain inhibitory effect on Staphylococcus aureus and Escherichia coli. After adding AMP@PDA microspheres (Examples 1, 2, 9 and 10), the antibacterial effect of the hydrogel is significantly improved, and increases with the increase in the amount of AMP, with a sustained antibacterial effect. The hydrogel to which AMP is directly added (Comparative Example 4) also has significant antibacterial ability at first, which is mainly due to the rapid release of AMP; but on the seventh day, its antibacterial effect has decreased, which is due to the degradation of AMP. In summary, AMP@PDA not only has good sustained-release properties for AMP, but also helps to improve the stability of AMP. Therefore, the AMP@PDA / Pu / Gel hydrogel prepared by the present invention has a sustained antibacterial ability.

[0142] (c) Histological evaluation

[0143] In order to further evaluate the wound healing, H&E and Masson staining were used to perform histological analysis of the wound to analyze whether there was epithelialization, inflammation, granulation tissue formation, and capillary formation in the wound. Figure 4 shown.

[0144] Depend on Figure 4 The results show that on the 7th day, the necrotic tissue of all wounds was significantly reduced, and the granulation tissue tended to increase. Some inflammatory cells were observed in the wound of the control group, and fracture areas appeared in the epidermis of the rats. The epithelial tissue in the hydrogel group recovered better, especially in the AMP@PDA / Pu / Gel group, where hair follicle structures had begun to appear. By the 14th day, the epidermis and new blood vessels of each group were basically formed. Compared with the control group, the hair follicle structure in the Pu / Gel group had a better growth trend, the density of new blood vessels in the AMP@PDA / Pu / Gel group was higher, and the wound healed faster.

[0145] Collagen is the main component of the dermis, and the balance between its synthesis and degradation plays an important role in wound healing. Figure 5 Masson staining can be used to observe the formation of collagen in the wound surface of each group.

[0146] Depend on Figure 5 The results showed that on the 7th day, collagen appeared in all three groups, but the collagen in the control group showed excessive synthesis and accumulation, which is a manifestation of poor healing. On the 14th day, compared with the control group, the new collagen content in the Pu / Gel group and the AMP@PDA / Pu / Gel group was significantly higher, and the collagen fibers in the AMP@PDA / Pu / Gel group were more neatly stained and had a uniform density. These results indicate that the AMP@PDA / Pu / Gel group can better promote collagen fiber deposition.

[0147] (d) Immunomic analysis

[0148] Wound healing is a complex process, which is mainly divided into four stages: homeostasis, inflammatory response, granulation tissue proliferation, and epithelial remodeling. As the first step in the wound healing process, the inflammatory response is very important for the elimination of microorganisms. If it is not handled properly, the inflammatory stage is likely to be prolonged, resulting in the inability of the wound to heal quickly. Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by mononuclear macrophages and is the main mediator of host inflammatory response. TNF-α can delay wound healing by inhibiting growth hormone and releasing a large amount of itself. Figure 6 The expression of TNF-α in each group on days 7 and 14 is shown. Figure 6The results showed that on the 7th day, all groups showed different degrees of inflammatory response, among which the Pu / Gel group had the strongest positive expression. On the 14th day, the positive expression of each group weakened, especially the AMP@PDA / Pu / Gel group had the least inflammatory factors, indicating that AMP@PDA / Pu / Gel hydrogel can reduce the inflammatory response of infected wounds, thereby shortening the wound healing time.

[0149] Transforming growth factor (TGF-β) is essential for many processes in wound healing, including inflammation, stimulation of angiogenesis, collagen synthesis and accumulation, and myofibroblast proliferation. Overexpression of TGF-β1 can easily cause hyperplastic scarring, leading to massive proliferation of fibroblasts. TGF-β3 can promote collagen formation and fibroblast regeneration, reducing scarring. The expression of TGF-β1 and TGF-β3 can be seen in Figure 7 and Figure 8 .

[0150] Depend on Figure 7 and Figure 8 It can be seen that on the 7th day, TGF-β1 positive staining can be observed in all groups. On the 14th day, the positive expression of the AMP@PDA / Pu / Gel group was the weakest. On the 7th day, the positive expression of TGF-β3 in the AMP@PDA / Pu / Gel group was the strongest, and on the 14th day, it was significantly weakened, which may be because with the release of antimicrobial peptides, the wound began to heal and the scar fell off. The difference in positive expression between the control group and the Pu / Gel group on the 7th and 14th days was small. Therefore, it shows that AMP@PDA / Pu / Gel hydrogel can promote tissue regeneration and accelerate wound healing by upregulating the expression of TGF-β3 and downregulating TGF-β1.

[0151] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A method for preparing an anti-infection wound repair hydrogel, comprising the following steps: S1. Preparation of antimicrobial peptide@polydopamine microspheres; S2. Preparation of pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres: S21, dissolving pullulan in a sodium hydroxide solution to prepare a pullulan solution; S22, adding antimicrobial peptide @ polydopamine microspheres to the pullulan solution, mixing, and reacting to obtain an antimicrobial peptide @ polydopamine microsphere / pululan mixed solution; S23, dispersing gelatin in water to prepare a gelatin solution; S24, adding the gelatin solution to the antimicrobial peptide@polydopamine microspheres and pullulan mixed solution, mixing, and preparing an antimicrobial peptide@polydopamine microspheres / pululan / gelatin mixed solution; S25. Add ethylene glycol diglycidyl ether to the antimicrobial peptide@polydopamine microspheres / pullulan / gelatin mixed solution, stir to react, and let stand to obtain pullulan / gelatin hydrogel loaded with antimicrobial peptide@polydopamine microspheres.

2. The method for preparing the anti-infection wound repairing hydrogel according to claim 1, characterized in that: The dosage ratio of the pullulan, gelatin, antimicrobial peptide@polydopamine microspheres, and ethylene glycol diglycidyl ether is 0.2-25 g: 0.2-25 g: 1-60 mg: 50-1000 μL.

3. The method for preparing the anti-infection wound repairing hydrogel according to claim 1, characterized in that: In step S1, the preparation of antimicrobial peptide@polydopamine microspheres comprises the following steps: S11, adding dopamine hydrochloride into ultrapure water, mixing evenly, and preparing a dopamine hydrochloride aqueous solution; S12, adding the antimicrobial peptide to the dopamine hydrochloride aqueous solution, mixing evenly, to prepare an antimicrobial peptide / dopamine hydrochloride mixed solution; S13. Drop the antimicrobial peptide / dopamine hydrochloride mixed solution into the ethanol base solution at room temperature and stir overnight to obtain antimicrobial peptide@polydopamine microspheres.

4. The method for preparing the anti-infection wound repairing hydrogel according to claim 3, characterized in that: The mass concentration of dopamine hydrochloride in the dopamine hydrochloride aqueous solution is 0.5-15%.

5. The method for preparing the anti-infection wound repairing hydrogel according to claim 3, characterized in that: The mass concentration of the antimicrobial peptide in the antimicrobial peptide / dopamine hydrochloride mixed solution is 0.01-2%; the antimicrobial peptide is a cationic polypeptide.

6. The method for preparing the anti-infection wound repairing hydrogel according to claim 3, characterized in that: The ethanol alkali solution is a solution obtained by mixing water, anhydrous ethanol and alkali, wherein the volume fraction of ethanol in the ethanol alkali solution is 5-80%; the mass concentration of alkali in the ethanol alkali solution is 1-15%; the alkali is one or a combination of two or more of CaCO3, Ca(OH)2, Na2CO3, NaHCO3, K2CO3, KHCO3, monoethanolamine, diethanolamine and triethanolamine.

7. The method for preparing the anti-infection wound repairing hydrogel according to claim 1, characterized in that: The volume ratio of the dopamine hydrochloride aqueous solution to the ethanol base solution is 1:1-25; the dripping speed of the antimicrobial peptide / dopamine hydrochloride mixed solution is 1-20 mL / min.

8. The method for preparing the anti-infection wound repairing hydrogel according to claim 1, characterized in that: The mass concentration of pullulan in the pullulan solution is 1-50%, and the mass concentration of gelatin in the gelatin solution is 1-50%.

9. An anti-infection and wound repairing hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the anti-infection wound repairing hydrogel according to claim 9 in the preparation of drugs for wound infection and wound repair.

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