CN114225097B - Self-healing hydrogel wound dressing loaded with antibacterial peptide and preparation method thereof - Google Patents

Abstract

The invention relates to a self-healing hydrogel wound dressing loaded with antibacterial peptides and a preparation method thereof, wherein the self-healing hydrogel wound dressing loaded with the antibacterial peptides comprises a composite hydrogel matrix and the antibacterial peptides loaded on the composite hydrogel matrix; the composite hydrogel matrix is formed by crosslinking phenylboronic acid, linear hydrophilic polysaccharide and acellular porcine small intestine submucosa matrix material, wherein the linear hydrophilic polysaccharide is naturally derived and contains carboxyl functional groups. According to the invention, based on the fact that the phenylboronic acid, the linear hydrophilic polysaccharide and the acellular porcine small intestine submucosa matrix material are esterified to generate the dynamic covalent bonds, the reversibility and balance of the dynamic covalent bonds are fully utilized, so that the obtained hydrogel matrix has good swelling performance, and can present self-repairing capability under certain conditions, thereby avoiding the problem that the existing hydrogel wound dressing is easy to crack or fracture due to exposure under the action of external tension or during tissue movement after being used.

Description

A self-healing hydrogel wound dressing loaded with antimicrobial peptides and its preparation method

technical field

The invention relates to the technical field of medical biomaterials, in particular to a self-healing hydrogel wound dressing loaded with antimicrobial peptides and a preparation method thereof.

Background technique

The skin is an important organ that covers the surface of the human body and directly contacts the external environment. It has the functions of sensing external stimuli, regulating body temperature and protecting the human body from external damage. Due to the characteristics of direct contact with the outside world, the skin is also one of the most vulnerable tissues. Although most common skin injuries can basically return to their original appearance over a period of time, it is difficult for adult skin injuries to restore 100% of the skin function, but usually accompanied by the formation of scar tissue and skin appendages, such as hair, sweat glands There is a serious lack of waiting. The repair of skin wounds is divided into four continuous and coordinated processes: hemostasis, inflammation, proliferation, and remodeling. However, the wound healing process generally does not proceed in a perfect and orderly manner, and the influence of various factors at any stage may lead to abnormal wound repair, such as excessive inflammation, burns, extensive loss of skin tissue, infection, and diabetes.

Wound dressings can cover wounds, provide a temporary barrier against external infections, and serve as inductive templates to guide skin cell reorganization and subsequent infiltration and integration of host tissues, showing significant effects on wound healing. An ideal skin wound dressing should meet the following requirements: (1) good histocompatibility, no toxicity or inflammation; (2) good moisture retention, which can maintain a moist environment of the wound, promote cell hydration, and have a certain effect on wound exudate. (3) It has sufficient physical and mechanical strength to ensure its integrity and avoid foreign bacterial invasion caused by material damage. Many wound dressings have been developed to facilitate wound repair, such as semipermeable membranes, semipermeable foams, hydrogels, and hydrogels. Among these dressings, hydrogels are the most competitive dressing candidates due to their good hydrophilicity, biocompatibility, and three-dimensional porous structure like extracellular matrix (ECM), which has also attracted many researchers interest of.

In the wound healing process, bacterial infection will prolong the inflammatory phase, and excessive inflammation is not conducive to wound healing. Therefore, it is necessary to add antibacterial agents to the dressing. Different antibacterial dressings can be obtained by adding different antibacterial agents. Conventional Although antibiotic antibacterial dressings can play a better antibacterial effect, excessive use of antibiotics can easily lead to drug resistance of bacteria; conventional inorganic nanoparticle antibacterial dressings, such as nano-silver dressings, have good antibacterial properties, but due to their Larger cytotoxicity will inevitably bring a certain degree of damage to the wound surface. Meanwhile, common hydrogel dressings are prone to cracking or breaking when exposed to external tension or tissue activity. The rupture of the hydrogel will not only lead to the deterioration or even loss of its own performance, but also further cause the invasion of foreign bacteria and lead to wound infection.

Contents of the invention

Based on this, the object of the present invention is to provide a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which has good antibacterial effect and has the advantage of certain self-healing properties.

A self-healing hydrogel wound dressing loaded with antibacterial peptides, which consists of a composite hydrogel matrix and antimicrobial peptides loaded on the composite hydrogel matrix; the composite hydrogel matrix is composed of phenylboronic acid, linear hydrophilic The linear hydrophilic polysaccharide is a polysaccharide of natural origin and contains a carboxyl functional group.

The antimicrobial peptide-loaded self-healing hydrogel wound dressing described in the embodiments of the present invention uses linear hydrophilic polysaccharides and acellular porcine small intestinal submucosa matrix material cross-linked with phenylboronic acid to form a composite hydrogel matrix, based on phenylboronic acid (PBA) undergoes an esterification reaction with the linear hydrophilic polysaccharide and the amino functional group and carboxyl functional group contained in the matrix material of the decellularized porcine small intestine submucosa, through which the reaction is cross-linked to form a gel, and then a hydrogel that can be used as a wound dressing is obtained. Through the formation of dynamic covalent bonds, the crosslinking reaction makes full use of the reversibility and balance of the dynamic covalent bonds themselves, so that the obtained hydrogel matrix has good swelling properties and can exhibit self-healing ability under certain conditions. , thus avoiding that the existing hydrogel wound dressings are easily broken or broken due to exposure to external tension or tissue activities after use, which not only leads to deterioration or even loss of their own performance, but also further causes foreign bacteria to invade and cause wound infection At the same time, the dynamic covalent bond can also endow the hydrogel matrix with suitable softness, which will not cause foreign body sensation to the wound during use, and can be easily self-created and exfoliated after the wound is healed, without causing any damage to the wound surface. Secondary damage.

In addition, in the present invention, by selecting linear hydrophilic polysaccharides from natural sources and acellular porcine small intestinal submucosa matrix material, the prepared hydrogel material has good biocompatibility, and the decellularized porcine small intestinal submucosa matrix material (SIS), as an extracellular matrix (ECM), has less immunogenicity, and most of its components are type I and type III collagen, which can provide a certain mechanical strength, and at the same time, a variety of cytokines are retained in SIS, Such as basic fibroblast growth factor (bFGF), transforming growth factor (TGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc., these components play an important role in tissue remodeling and wound healing, and are used in Wound dressing can effectively promote wound repair.

Further, antimicrobial peptide materials are loaded in the composite hydrogel matrix. Compared with inorganic nano-antibacterial particles and antibiotics, antimicrobial peptides (AMPs) are not easy to produce drug resistance because of their antibacterial mechanism and antibiotics. AMPs have attracted much attention due to their broad-spectrum antibacterial, antifungal, antiviral and immune enhancing effects. Antimicrobial peptides have excellent antibacterial performance and are not easy to produce drug resistance, and when loaded on the composite hydrogel matrix, they can also participate in the formation of dynamic covalent bonds, thereby showing the characteristics of slow release, which can play a role of long-acting antibacterial effect.

Further, the linear hydrophilic polysaccharide is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, and heparin, and has good biocompatibility.

Further, the antimicrobial peptide is cecropin antimicrobial peptide, which is an animal antimicrobial peptide, which has strong lethality to Gram-positive bacteria and some Gram-negative bacteria, and can be self-healing in the loaded antimicrobial peptide The concentration in the hydrogel wound dressing is 0.05-1.0 mg/L.

In addition, the embodiment of the present invention also provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which includes the following specific steps:

S1. Preparation of phenylborated linear hydrophilic polysaccharide

Dissolve linear hydrophilic polysaccharide powder in pure water, then add 3-aminomethylphenylboronic acid (PBA) and 4-(4,6-dimethoxy-1,3,5 -Triazin-2-yl)-4-methylmorpholine (DMTMM), adjust the pH value of the solution to 6.0-7.0 after it is completely dissolved, transfer it to a dialysis bag after stirring at room temperature, and use it under predetermined temperature conditions Ion water dialysis for a predetermined time, and the obtained dialysate is freeze-dried to obtain the phenylborated linear hydrophilic polysaccharide;

S2. Preparation of acellular porcine small intestinal submucosa matrix material (SIS) freeze-dried powder

S21. Cut the cleaned and treated pig small intestinal mucosa into small pieces, then immerse in a PBS buffer solution containing trypsin and ethylenediaminetetraacetic acid (EDTA), stir for a predetermined time under predetermined temperature conditions, centrifuge and collect the precipitate;

S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing polyethylene glycol octylphenyl ether (Triton-X-100) and ethylenediaminetetraacetic acid (EDTA) for a predetermined time, centrifuge and collect the precipitate, and use Wash the precipitate several times with PBS buffer solution;

S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing deoxyribonuclease I (DNase I) and magnesium chloride (MgCl ₂ ), stir at a predetermined temperature for a predetermined time to completely remove cells in the tissue, centrifuge and Collect the precipitate and wash the precipitate several times with PBS buffer solution;

S24. Soak and sterilize the precipitate obtained in step S23 with an ethanol solution of peracetic acid, centrifuge and collect the precipitate, wash several times with PBS buffer solution, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal submucosa tissue;

S25. Put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into the acetic acid solution, stir for a predetermined time, filter with a filter cloth to remove larger particles, and then transfer the filtrate to a dialysis bag. dialyzing with deionized water for a predetermined time under certain conditions, and freeze-drying the filtrate to obtain the freeze-dried powder of acellular porcine small intestinal submucosa matrix material;

S3. Preparation of self-healing hydrogel wound dressings loaded with antimicrobial peptides

Weighing a predetermined amount of phenylborated linear hydrophilic polysaccharide powder obtained in step S1 and dissolving it in PBS buffer solution to obtain solution A; weighing a predetermined amount of lyophilized powder of acellular porcine small intestinal submucosa matrix material obtained in step S2 and dissolving it in PBS In the buffer solution, solution B is obtained; the antimicrobial peptide powder is dispersed into the step solution B, and then solution A and solution B are uniformly mixed to obtain the antimicrobial peptide-loaded self-healing hydrogel wound dressing.

The preparation method of the antimicrobial peptide-loaded self-healing hydrogel wound dressing described in the embodiment of the present invention first uses phenylboronic acid to modify the linear hydrophilic polysaccharide, endows it with a phenylboronic acid structure, and further combines with self-made decellularized pig The amino groups rich in small intestinal submucosa matrix materials react with carboxyl functional groups to achieve the purpose of cross-linking to form dynamic covalent bonds. The preparation method is simple to operate, and has low requirements on equipment conditions and temperature conditions, low energy consumption, and low preparation costs. , which is convenient for large-scale clinical application.

Further, in step S1, 3-aminomethylphenylboronic acid (PBA) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl chloride The molar ratio of morphine (DMTMM) is 1:1～1:1.5.

Further, in step S21, the pig small intestinal mucosa is treated with a PBS buffer solution containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid;

In step S22, the precipitate obtained in step S21 is treated with a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether and 25 mmol/L ethylenediaminetetraacetic acid;

In step S23, the precipitate obtained in step S22 is treated with a PBS buffer solution containing 30U/mL deoxyribonuclease I and 10mmol/L magnesium chloride;

In step S24, 4% ethanol solution with a peracetic acid concentration of 0.1% is used to sterilize the precipitate obtained in step S23 by soaking for 1.5 to 2 hours;

In step S25, 15 mg of pepsin is used for every 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, and then added to the acetic acid solution and stirred for 22-26 hours.

Further, the solvents of solution A and solution B in step S3 are both PBS buffer solution, the pH range of which is 7.0-8.0, the mass concentration of phenylborated linear hydrophilic polysaccharide in solution A is 5%, and the decellularized polysaccharide in solution B is The mass concentration of the porcine small intestine submucosa matrix material is 5%, and the mixed mass ratio of solution A and solution B is 1:0.6˜1:1.4.

Further, the mass concentration of the antimicrobial peptide in the composite hydrogel matrix solution in step S4 is 0.01-0.1%.

Further, in step S21, stir at 35-40°C for 5-7h; in step S22, the precipitation treatment time is 23-25h; in step S23, stir at 35-40°C for 23-25h; in step S24, the stirring time is 23～25h.

For better understanding and implementation, the present invention will be described in detail below in conjunction with the accompanying drawings.

Description of drawings

Fig. 1 is a schematic diagram of the self-healing performance of the self-healing hydrogel wound dressing described in Comparative Example 1;

Fig. 2 is the nuclear magnetic hydrogen spectrogram of SA and SA-PBA prepared in embodiment 4;

Fig. 3 is the relationship diagram of elastic modulus (G') and viscous modulus (G") of three groups of self-healing hydrogel wound dressings prepared in Comparative Examples 1, 2, and 3 as a function of angular frequency;

Fig. 4 is the in vitro release curve of AMP from the antimicrobial peptide-loaded self-healing hydrogel wound dressing prepared in Example 4.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, specific implementations of the present invention will be described in detail below.

In the following description, a lot of specific details have been set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways that are different from the present invention described here, and those skilled in the art can do so without departing from the connotation of the present invention. Similar promotions are made in the following cases, so the present invention is not limited by the following disclosed embodiments.

Example 1

Embodiment 1 of the present invention provides a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which consists of a composite hydrogel matrix and antimicrobial peptides loaded on the composite hydrogel matrix; the composite hydrogel matrix consists of It is formed by cross-linking phenylboronic acid, linear hydrophilic polysaccharide and submucosa matrix material of decellularized porcine small intestine, and the linear hydrophilic polysaccharide is a polysaccharide of natural origin and containing carboxyl functional group. Specifically, in this embodiment, it is sodium alginate. Sodium alginate (SA) is a linear hydrophilic polysaccharide, which has the stability, solubility, viscosity and safety required for pharmaceutical preparation excipients. Due to its good biocompatibility and good bioabsorbability, it has been It has been widely used in the medical field. The antimicrobial peptide is cecropin antibacterial peptide, which has strong lethality to Gram-positive bacteria and some Gram-negative bacteria, and is not easy to produce drug resistance. The concentration in adhesive wound dressings is 0.05mg/L.

Example 2

Embodiment 2 of the present invention provides a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which consists of a composite hydrogel matrix and antimicrobial peptides loaded on the composite hydrogel matrix; the composite hydrogel matrix consists of It is formed by cross-linking phenylboronic acid, linear hydrophilic polysaccharide and submucosa matrix material of decellularized porcine small intestine, and the linear hydrophilic polysaccharide is a polysaccharide of natural origin and containing carboxyl functional group. Specifically, in this embodiment, hyaluronic acid. Hyaluronic acid is an acidic mucopolysaccharide, which is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine. With its unique molecular structure and physical and chemical properties, it shows a variety of important Physiological functions, such as lubricating joints, regulating the permeability of blood vessel walls, regulating protein, water and electrolyte diffusion and operation, and promoting wound healing. The antimicrobial peptide is cecropin antibacterial peptide, which has strong lethality to Gram-positive bacteria and some Gram-negative bacteria, and is not easy to produce drug resistance. The concentration in adhesive wound dressings is 0.5mg/L.

Example 3

Embodiment 3 of the present invention provides a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which consists of a composite hydrogel matrix and antimicrobial peptides loaded on the composite hydrogel matrix; the composite hydrogel matrix consists of It is formed by cross-linking phenylboronic acid, linear hydrophilic polysaccharide and submucosa matrix material of decellularized porcine small intestine, and the linear hydrophilic polysaccharide is a polysaccharide of natural origin and containing carboxyl functional group. Specifically, in this example, carboxymethyl chitosan and heparin. Carboxymethyl chitosan is an important water-soluble chitosan derivative, which has good biocompatibility and biodegradability, and is widely used in hydrogels and wound healing biomaterials. Heparin is a natural anticoagulant substance in animals. It can relieve pain, anticoagulate, inhibit inflammatory response, promote angiogenesis, restore local blood supply, and affect the synthesis and degradation of collagen. After wound healing, the skin is smooth and scars are relieved. and scar contractures. The antimicrobial peptide is cecropin antibacterial peptide, which has strong lethality to Gram-positive bacteria and some Gram-negative bacteria, and is not easy to produce drug resistance. The concentration in the glue wound dressing is 1.0mg/L.

Example 4

Embodiment 4 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which includes the following specific steps:

Preparation of S1, phenylborated sodium alginate (SA-PBA)

Weigh 1g of sodium alginate (SA), dissolve it in 20mL of pure water, then add 0.38g (0.25mmol) of 3-aminomethylphenylboronic acid (PBA) and 0.83g (0.3mmol) of chlorine to the solution 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM), after being completely dissolved, adjust the solution with 1mol/L hydrochloric acid solution pH value to 6.5, stirred at room temperature for 72 hours, then transferred to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyzed with deionized water for 3 days at room temperature, and obtained the dialysate obtained after freeze-drying with a desktop lyophilizer to obtain the Phenylborated sodium alginate (SA-PBA polymer conjugate), the product is stored in a desiccator for subsequent use;

S2. Preparation of acellular porcine small intestinal submucosa matrix material (SIS) freeze-dried powder

S21. Remove the dirt in the fresh pig small intestine, clean it, scrape off the small intestine tissue with a knife until it becomes translucent to obtain the pig small intestine mucosa, and cut the processed pig small intestine mucosa into 2cm×2cm size, then immerse it in a phosphate buffered solution (PBS buffered solution) containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid (EDTA), and stir with a magnetic stirrer at 37°C for 6h at a speed of 300rpm, Centrifuge at a speed of 8000rpm and collect the precipitate;

S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether (Triton-X-100) and 25mmol/L ethylenediaminetetraacetic acid (EDTA) for 24h, centrifuge and Collect the precipitate and wash the precipitate several times with PBS buffer solution;

S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing 30 U/mL deoxyribonuclease I (DNase I) and 10 mmol/L magnesium chloride (MgCl ₂ ), and stir at 37° C. for 24 hours to completely remove the Cells, centrifuge and collect the pellet, wash the pellet several times with PBS buffer solution;

S24. Soak and sterilize the precipitate obtained in step S23 with a 4% ethanol solution with a peracetic acid concentration of 0.1% for 2 hours, centrifuge and collect the precipitate, wash with PBS buffer solution several times, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal submucosa tissue ;

S25. Put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into a 0.5 mol/L acetic acid solution according to the ratio of using 15 mg of pepsin per 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, and stirred for 24 hours , use filter cloth to filter to remove larger particles, then transfer the filtrate to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyze with deionized water for 3 days at room temperature, freeze-dry the filtrate, and obtain the decellularized porcine small intestinal submucosa matrix Material (SIS) lyophilized powder;

S3. Preparation of self-healing hydrogel wound dressing (SA-PBA/AMP/SIS hydrogel) loaded with antimicrobial peptides

Weigh 0.05 g of SA-PBA obtained in step S1 and dissolve it in 1 mL of PBS buffer solution with pH=7.4 to obtain solution A with a mass concentration of SA-PBA of 5%; weigh a predetermined amount of decellularized porcine small intestinal mucosa obtained in step S2 The lyophilized powder of the lower matrix material was dissolved in PBS buffer solution, and a solution B with a mass concentration of SIS of 5% was configured to obtain a solution B; cecropin (AMP) powder was dispersed into solution B, and the dispersed mass concentration of the antimicrobial peptide was 0.1 %, then solution A and solution B are mixed evenly according to the mass ratio of 1:1, and the self-healing hydrogel wound dressing ( SA-PBA/AMP/SIS hydrogel), wherein the final concentration of AMP in the SA-PBA/AMP/SIS hydrogel is 0.5 mg/mL.

Example 5

Embodiment 5 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which includes the following specific steps:

Preparation of S1, phenylborated sodium alginate (SA-PBA)

Weigh 1g of sodium alginate (SA), dissolve it in 20mL of pure water, then add 0.38g (0.25mmol) of 3-aminomethylphenylboronic acid (PBA) and 0.83g (0.3mmol) of chlorine to the solution 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM), after being completely dissolved, adjust the solution with 1mol/L hydrochloric acid solution pH value to 6.5, stirred at room temperature for 72h, then transferred to a dialysis bag with a molecular weight cut-off of 6-8kDa, and dialyzed with deionized water for 2d at room temperature, and the obtained dialysate was freeze-dried with a desktop lyophilizer to obtain the described Phenylborated sodium alginate (SA-PBA polymer conjugate), the product is stored in a desiccator for subsequent use;

S2. Preparation of acellular porcine small intestinal submucosa matrix material (SIS) freeze-dried powder

S21. Remove the dirt in the fresh pig small intestine, clean it, scrape off the small intestine tissue with a knife until it becomes translucent to obtain the pig small intestine mucosa, and cut the processed pig small intestine mucosa into 2cm×2cm size, then immerse it in a phosphate buffer solution (PBS buffer solution) containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid (EDTA), and stir at a speed of 300rpm in a magnetic stirrer for 5h at 35°C, Centrifuge at a speed of 8000rpm and collect the precipitate;

S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether (Triton-X-100) and 25mmol/L ethylenediaminetetraacetic acid (EDTA) for 24h, centrifuge and Collect the precipitate and wash the precipitate several times with PBS buffer solution;

S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing 30 U/mL deoxyribonuclease I (DNase I) and 10 mmol/L magnesium chloride (MgCl ₂ ), and stir at 35° C. for 23 hours to completely remove cells in the tissue , centrifuge and collect the precipitate, wash the precipitate several times with PBS buffer solution;

S24. Soak and sterilize the precipitate obtained in step S23 in 4% ethanol solution with a peracetic acid concentration of 0.1% for 1.5 h, centrifuge and collect the precipitate, wash with PBS buffer solution for several times, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal submucosa organize;

S25. Put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into a 0.5 mol/L acetic acid solution according to the ratio of using 15 mg of pepsin per 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, and stirred for 23 hours , use filter cloth to filter to remove larger particles, then transfer the filtrate to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyze with deionized water for 2 days at room temperature, and freeze-dry the filtrate to obtain the decellularized porcine small intestinal submucosa matrix Material (SIS) lyophilized powder;

S3. Preparation of self-healing hydrogel wound dressing (SA-PBA/AMP/SIS hydrogel) loaded with antimicrobial peptides

Weigh 0.05 g of SA-PBA obtained in step S1 and dissolve it in 1 mL of PBS buffer solution with pH=7.0 to obtain solution A with a mass concentration of SA-PBA of 5%; weigh a predetermined amount of decellularized porcine small intestinal mucosa obtained in step S2 The lyophilized powder of the lower matrix material was dissolved in PBS buffer solution, and a solution B with a mass concentration of SIS of 5% was configured to obtain a solution B; cecropin (AMP) powder was dispersed into solution B, and the dispersed mass concentration of the antimicrobial peptide was 0.1 %, then solution A and solution B were uniformly mixed at a ratio of 1:0.6 to obtain the antimicrobial peptide-loaded self-healing hydrogel wound dressing (SA-PBA/SIS hydrogel).

Example 6

Embodiment 6 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which includes the following specific steps:

Preparation of S1, phenylborated sodium alginate (SA-PBA)

Weigh 1g of sodium alginate (SA), dissolve it in 20mL of pure water, then add 0.38g (0.25mmol) of 3-aminomethylphenylboronic acid (PBA) and 0.83g (0.3mmol) of chlorine to the solution 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM), after being completely dissolved, adjust the solution with 1mol/L hydrochloric acid solution pH value to 6.5, stirred at room temperature for 72 hours, then transferred to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyzed with deionized water for 3 days at room temperature, and obtained the dialysate obtained after freeze-drying with a desktop lyophilizer to obtain the Phenylborated sodium alginate (SA-PBA polymer conjugate), the product is stored in a desiccator for subsequent use;

S2. Preparation of acellular porcine small intestinal submucosa matrix material (SIS) freeze-dried powder

S21. Remove the dirt in the fresh pig small intestine, clean it, scrape off the small intestine tissue with a knife until it becomes translucent to obtain the pig small intestine mucosa, and cut the processed pig small intestine mucosa into 2cm×2cm size, then immerse it in a phosphate buffer solution (PBS buffer solution) containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid (EDTA), and stir at a speed of 300rpm at 40°C for 7h on a magnetic stirrer, Centrifuge at a speed of 8000rpm and collect the precipitate;

S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether (Triton-X-100) and 25 mmol/L ethylenediaminetetraacetic acid (EDTA) for 25 hours, centrifuge and Collect the precipitate and wash the precipitate several times with PBS buffer solution;

S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing 30 U/mL deoxyribonuclease I (DNase I) and 10 mmol/L magnesium chloride (MgCl ₂ ), and stir at 40° C. for 25 hours to completely remove the Cells, centrifuge and collect the pellet, wash the pellet several times with PBS buffer solution;

S24. Soak and sterilize the precipitate obtained in step S23 with a 4% ethanol solution with a peracetic acid concentration of 0.1% for 2 hours, centrifuge and collect the precipitate, wash with PBS buffer solution several times, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal submucosa tissue ;

S25. Put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into a 0.5 mol/L acetic acid solution according to the ratio of using 15 mg of pepsin per 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, and stirred for 25 hours , use filter cloth to filter to remove larger particles, then transfer the filtrate to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyze with deionized water for 3 days at room temperature, freeze-dry the filtrate, and obtain the decellularized porcine small intestinal submucosa matrix Material (SIS) lyophilized powder;

S3. Preparation of self-healing hydrogel wound dressing (SA-PBA/AMP/SIS hydrogel) loaded with antimicrobial peptides

Weigh 0.05 g of SA-PBA obtained in step S1 and dissolve it in 1 mL of PBS buffer solution with pH=8.0 to obtain solution A with a mass concentration of SA-PBA of 5%; weigh a predetermined amount of decellularized porcine small intestinal mucosa obtained in step S2 The lyophilized powder of the lower matrix material was dissolved in PBS buffer solution, and a solution B with a mass concentration of SIS of 5% was configured to obtain a solution B; cecropin (AMP) powder was dispersed into solution B, and the dispersed mass concentration of the antimicrobial peptide was 0.1 %, then solution A and solution B were uniformly mixed according to the mass ratio of 1:1.4 to obtain the antimicrobial peptide-loaded self-healing hydrogel wound dressing (SA-PBA/SIS hydrogel).

Example 7

Embodiment 7 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which differs from Example 4 in that the prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides (SA - The final concentration of cecropin (AMP) in PBA/AMP/SIS hydrogel) was 0.05 mg/mL.

Example 8

Embodiment 8 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which differs from Example 4 in that the prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides (SA - The final concentration of cecropin (AMP) in PBA/AMP/SIS hydrogel) was 0.1 mg/mL.

Example 9

Embodiment 9 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which differs from Example 4 in that the prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides (SA - The final concentration of cecropin (AMP) in PBA/AMP/SIS hydrogel) was 0.2 mg/mL.

Example 10

Embodiment 10 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which differs from Example 4 in that the prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides (SA - The final concentration of cecropin (AMP) in PBA/AMP/SIS hydrogel) was 0.8 mg/mL.

Example 11

Embodiment 10 of the present invention provides a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, which differs from Example 4 in that the prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides (SA - The final concentration of cecropin (AMP) in PBA/AMP/SIS hydrogel) was 1.0 mg/mL.

Comparative example 1

Comparative example 1 provides a kind of preparation method of self-healing hydrogel wound dressing, and it comprises following specific operation steps:

Preparation of S1, phenylborated sodium alginate (SA-PBA)

Weigh 1g of sodium alginate (SA), dissolve it in 20mL of pure water, then add 0.38g (0.25mmol) of 3-aminomethylphenylboronic acid (PBA) and 0.83g (0.3mmol) of chlorine to the solution 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM), after being completely dissolved, adjust the solution with 1mol/L hydrochloric acid solution pH value to 6.5, stirred at room temperature for 72 hours, then transferred to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyzed with deionized water for 3 days at room temperature, and obtained the dialysate obtained after freeze-drying with a desktop lyophilizer to obtain the Phenylborated sodium alginate (SA-PBA polymer conjugate), the product is stored in a desiccator for subsequent use;

S2. Preparation of acellular porcine small intestinal submucosa matrix material (SIS) freeze-dried powder

S21. Remove the dirt in the fresh pig small intestine, clean it, scrape off the small intestine tissue with a knife until it becomes translucent to obtain the pig small intestine mucosa, and cut the processed pig small intestine mucosa into 2cm×2cm size, then immerse it in a phosphate buffer solution (PBS buffer solution) containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid (EDTA), and stir at a speed of 300rpm at 40°C for 7h on a magnetic stirrer, Centrifuge at a speed of 8000rpm and collect the precipitate;

S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether (Triton-X-100) and 25 mmol/L ethylenediaminetetraacetic acid (EDTA) for 25 hours, centrifuge and Collect the precipitate and wash the precipitate several times with PBS buffer solution;

S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing 30 U/mL deoxyribonuclease I (DNase I) and 10 mmol/L magnesium chloride (MgCl ₂ ), and stir at 40° C. for 25 hours to completely remove the Cells, centrifuge and collect the pellet, wash the pellet several times with PBS buffer solution;

S24. Soak and sterilize the precipitate obtained in step S23 with a 4% ethanol solution with a peracetic acid concentration of 0.1% for 2 hours, centrifuge and collect the precipitate, wash with PBS buffer solution several times, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal submucosa tissue ;

S25. Put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into a 0.5 mol/L acetic acid solution according to the ratio of using 15 mg of pepsin per 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, and stirred for 25 hours , use filter cloth to filter to remove larger particles, then transfer the filtrate to a dialysis bag with a molecular weight cut-off of 6-8 kDa, dialyze with deionized water for 3 days at room temperature, freeze-dry the filtrate, and obtain the decellularized porcine small intestinal submucosa matrix Material (SIS) lyophilized powder;

S3, preparation of self-healing hydrogel wound dressing (SA-PBA/SIS hydrogel)

Weigh 0.05 g of SA-PBA obtained in step S1 and dissolve it in 1 mL of PBS buffer solution with pH=7.4 to obtain solution A with a mass concentration of SA-PBA of 5%; weigh a predetermined amount of decellularized porcine small intestinal mucosa obtained in step S2 The lyophilized powder of the lower matrix material (SIS) was dissolved in PBS buffer solution, and a solution B with a mass concentration of SIS of 5% was prepared; the solution A and solution B were mixed evenly according to the mass ratio of 1:1, and the mass of SA-PBA was obtained. A self-healing hydrogel wound dressing (2.5% SA-PBA/2.5% SIS hydrogel) with a concentration and a mass concentration of SIS of 2.5%.

Comparative example 2

Comparative example 2 provides a kind of preparation method of self-healing hydrogel wound dressing, and its difference is: the prepared SA-PBA mass concentration is 2.5%, SIS mass concentration is 1.5% self-healing hydrogel wound dressing (2.5 % SA-PBA/1.5% SIS hydrogel).

Comparative example 3

Comparative example 3 provides a kind of preparation method of self-healing hydrogel wound dressing, and its difference is: the prepared SA-PBA mass concentration is 2.5%, SIS mass concentration is 3.5% self-healing hydrogel wound dressing (2.5 % SA-PBA/1.5% SIS hydrogel).

The self-healing performance of the 2.5% SA-PBA/2.5% SIS hydrogel prepared in Comparative Example 1 was tested. Specifically, please refer to Figure 1. Figure 1 is a schematic diagram of the self-healing performance of the self-healing hydrogel wound dressing described in Comparative Example 1. As shown in the figure, the technical solution of the present invention adopts linear hydrophilic polysaccharide and decellularized The porcine small intestinal submucosa matrix material is cross-linked with phenylboronic acid to form a composite hydrogel matrix, based on the esterification reaction between phenylboronic acid and linear hydrophilic polysaccharides and the amino functional group and carboxyl functional group contained in the decellularized porcine small intestinal submucosa matrix material , through the reaction cross-linked into a gel to obtain a hydrogel that can be used as a wound dressing. Through the formation of dynamic covalent bonds, the crosslinking reaction makes full use of the reversibility and balance of the dynamic covalent bonds themselves, so that the obtained hydrogel matrix has good swelling properties and can exhibit self-healing ability under certain conditions. .

The SA and SA-PBA prepared in Example 4 were subjected to proton nuclear magnetic resonance spectrum analysis. Specifically, in order to use deuterated heavy water as a solvent, weigh 3 to 5 mg SA and SA-PBA and dissolve them respectively. After the solution is completely dissolved and clear, put it into a clean nuclear magnetic tube, and use a nuclear magnetic resonance spectrometer to perform nuclear magnetic resonance at room temperature. Structural determination, and adopt MestReNova software to carry out pattern analysis, please refer to Fig. 2, Fig. 2 is the nuclear magnetic hydrogen spectrogram of SA and SA-PBA prepared in the embodiment 4, find out from the figure, SA-PBA is in chemical shift ppm= The absorption peak appeared in the interval of 7-8, which was attributed to the hydrogen on the benzene ring in the structure of phenylboronic acid, which proved that the synthesis of SA-PBA was successful.

A rheological test was carried out on the self-healing hydrogel wound dressing prepared in Comparative Examples 1-3. Specifically, rheological measurements were performed with a stainless steel parallel-plate rotor with a diameter of 25 mm. Wherein, G' represents the elastic modulus of the sample, and G " represents the viscous modulus of the sample. After the SA-PBA and SIS solution are mixed into a gel in the mold, the obtained hydrogel is taken out from the mold, and Perform dynamic strain scanning from 0.1 to 10rad/s at room temperature, determine the linear viscoelastic range of the hydrogel, record the change curves of elastic modulus (G') and viscous modulus (G"), and obtain Figure 3, which is 2.5%SA-PBA/1.5%SIS, 2.5%SA-PBA/2.5%SIS, 2.5%SA-PBA/3.5%SIS three groups of self-healing hydrogel wound dressings prepared in comparative example 1,2,3 The relationship diagram of elastic modulus (G') and viscous modulus (G") with angular frequency. When the elastic modulus is greater than the viscous modulus, the hydrogel behaves as a gel state. It can be seen from the figure that when the water After the gel is gelled, G' is always greater than G" with the increase of the angular frequency, indicating that the hydrogel can be in a stable gel state, and the elastic modulus of the hydrogel in the 2.5% SA-PBA/2.5% SIS group To be significantly larger than the other two groups, showing better strength.

The in vitro release experiment of AMP was carried out on the antimicrobial peptide-loaded self-healing hydrogel wound dressing prepared in Example 4 of the present invention. Specifically, 2.0 mg of AMP standard substance was accurately weighed and placed in a 10 mL volumetric flask, dissolved in PBS with pH=7.4 to the mark and shaken evenly, and the obtained solution was AMP mother solution. Then the AMP mother solution was diluted with PBS into solutions with concentrations of 10 μg/mL, 30 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL. Use a UV spectrophotometer to detect the absorbance of the solution at 221 nm, and make a standard curve according to the relationship between absorbance and concentration. The AMP-loaded hydrogel prepared according to Example 4 was placed in 10 mL of PBS with a pH of 7.4 and shaken on a shaker at 37°C. Start timing, take 1mL of supernatant in EP tube at the pre-set time points, i.e. 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 hours, and then add the same amount of PBS For the buffer solution, a subsequent release experiment was carried out under the same conditions, and the absorbance of AMP in the supernatant taken out was detected by a UV spectrophotometer, and the concentration was calculated according to the standard curve, and the cumulative release percentage of AMP was calculated. Each sample is done in parallel three times, and the results are represented by mean and standard deviation, record relevant data, and make the in vitro release curve of AMP in SA-PBA/AMP/SIS hydrogel as shown in Figure 4, and Figure 4 is an embodiment 4. The prepared self-healing hydrogel wound dressing loaded with antimicrobial peptides is shown in the in vitro release curve of AMP. It can be seen from the figure that AMP can be continuously released from the hydrogel within 4 days, playing a long-term antibacterial effect .

In vitro antibacterial tests were carried out on the hydrogels prepared in Comparative Example 1 and Examples 4, 7-11 of the present invention. Specifically, the antimicrobial properties of the hydrogels were evaluated with Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The bacterial optical density value (Optical Density, OD value for short) in the antibacterial test was adjusted to 0.1. The hydrogel sample was co-cultured with the bacterial suspension in a biochemical incubator at 37°C, and after 12 hours, the OD value of the blended bacterial solution was measured. Dilute 100 μL of the bacterial suspension and inoculate it on an LB agar plate, and count the number of cultivable colonies after cultivating at 37°C for 24 hours as a control sample, using the following formula

AR(%)=( _Nc - _Ns )/ _Nc ×100

Calculate the antibacterial rate (AR), where Nc is the average number of colonies of the control sample, Ns is the average number of bacterial colonies of the hydrogel sample, and the relevant data are shown in Table 1:

Table 1: In vitro antibacterial test results of different hydrogels

As can be seen from the table, the antibacterial rate of the hydrogel without AMP (comparative example 1) is negative, which may be the reason that the pure hydrogel material provides nutrients for bacteria, and after adding AMP, the hydrogel Show antibacterial property, when AMP concentration in the hydrogel is 0.5mg/mL (embodiment 4), its antibacterial rate to escherichia coli and Staphylococcus aureus reaches 96.53% and 93.22% respectively, shows that under this concentration, water The gel showed good antibacterial effect.

In summary, the antimicrobial peptide-loaded self-healing hydrogel wound dressings described in Examples 1 to 3 of the present invention are composed of linear hydrophilic polysaccharides and acellular porcine small intestinal submucosa matrix material cross-linked with phenylboronic acid The hydrogel matrix is based on the esterification reaction between phenylboronic acid (PBA) and linear hydrophilic polysaccharides and the amino functional groups and carboxyl functional groups contained in the matrix material of the acellular porcine small intestine submucosa. Hydrogels for wound dressings. Through the formation of dynamic covalent bonds, the crosslinking reaction makes full use of the reversibility and balance of the dynamic covalent bonds themselves, so that the obtained hydrogel matrix has good swelling properties and can exhibit self-healing ability under certain conditions. , thus avoiding that the existing hydrogel wound dressings are easily broken or broken due to exposure to external tension or tissue activities after use, which not only leads to deterioration or even loss of their own performance, but also further causes foreign bacteria to invade and cause wound infection At the same time, the dynamic covalent bond can also endow the hydrogel matrix with suitable softness, which will not cause foreign body sensation to the wound during use, and can be easily self-created and exfoliated after the wound is healed, without causing any damage to the wound surface. Secondary damage.

In addition, in the present invention, by selecting linear hydrophilic polysaccharides from natural sources and acellular porcine small intestinal submucosa matrix material, the prepared hydrogel material has good biocompatibility, and the decellularized porcine small intestinal submucosa matrix material (SIS), as an extracellular matrix (ECM), has less immunogenicity, and most of its components are type I and type III collagen, which can provide a certain mechanical strength, and at the same time, a variety of cytokines are retained in SIS, Such as basic fibroblast growth factor (bFGF), transforming growth factor (TGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc., these components play an important role in tissue remodeling and wound healing, and are used in Wound dressing can effectively promote wound repair.

Further, antimicrobial peptide materials are loaded in the composite hydrogel matrix. Compared with inorganic nano-antibacterial particles and antibiotics, antimicrobial peptides (AMPs) are not easy to produce drug resistance because of their antibacterial mechanism and antibiotics. AMPs have attracted much attention due to their broad-spectrum antibacterial, antifungal, antiviral and immune enhancing effects. Antimicrobial peptides have excellent antibacterial performance and are not easy to produce drug resistance, and when loaded on the composite hydrogel matrix, they can also participate in the formation of dynamic covalent bonds, thereby showing the characteristics of slow release, which can play a role of long-acting antibacterial effect.

The preparation method of the antimicrobial peptide-loaded self-healing hydrogel wound dressing described in Examples 4 to 11 of the present invention first uses phenylboronic acid to modify the linear hydrophilic polysaccharide, endows it with a phenylboronic acid structure, and further combines with the self-made The amino and carboxyl functional groups rich in the acellular porcine small intestinal submucosa matrix material react to achieve the purpose of cross-linking to form dynamic covalent bonds. The preparation method is simple to operate, and has low requirements on equipment conditions and temperature conditions, and low energy consumption. The preparation cost is low, and it is convenient for large-scale clinical application.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims (5)

1. a preparation method of a self-healing hydrogel wound dressing loaded with antimicrobial peptides, characterized in that, comprising the following specific steps: S1. Preparation of phenylborated linear hydrophilic polysaccharide Dissolve linear hydrophilic polysaccharide powder in pure water, then add 3-aminomethylphenylboronic acid and 4-(4,6-dimethoxy-1,3,5-triazine chloride -2-yl)-4-methylmorpholine, adjust the pH value of the solution to 6.0-7.0 after being completely dissolved, transfer it to a dialysis bag after stirring at room temperature, and dialyze with deionized water for a predetermined time under predetermined temperature conditions, The obtained dialysate is freeze-dried to obtain the phenylborated linear hydrophilic polysaccharide; Wherein, the linear hydrophilic polysaccharide is a polysaccharide derived from natural sources and contains a carboxyl functional group, which is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, and heparin; The molar ratio of 3-aminomethylphenylboronic acid and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride is 1:1～ 1:1.5; S2. Preparation of lyophilized powder of acellular porcine small intestinal submucosa matrix material S21. Cut the cleaned and treated pig small intestinal mucosa into small pieces and immerse in PBS buffer solution containing 0.25wt% trypsin and 1mmol/L ethylenediaminetetraacetic acid, stir at a predetermined temperature for a predetermined time, centrifuge and collect the precipitate ; S22. Add the precipitate obtained in step S21 to a PBS buffer solution containing 1 wt% polyethylene glycol octylphenyl ether and 25 mmol/L ethylenediaminetetraacetic acid for a predetermined time, centrifuge and collect the precipitate, and wash the precipitate with PBS buffer solution several times; S23. Immerse the precipitate obtained in step S22 into a PBS buffer solution containing 30U/mL DNase I and 10mmol/L magnesium chloride, stir at a predetermined temperature for a predetermined time, to completely remove the cells in the tissue, centrifuge and collect the precipitate , using PBS buffer solution to wash the precipitate several times; S24. Soak and sterilize the precipitate obtained in step S23 in 4% ethanol solution with a peracetic acid concentration of 0.1% for 1.5-2 hours, centrifuge and collect the precipitate, wash several times with PBS buffer solution, and freeze-dry to obtain freeze-dried decellularized porcine small intestinal mucosa lower level organization; S25. According to the ratio of using 15 mg of pepsin per 100 mg of freeze-dried decellularized porcine small intestinal submucosa tissue, put the freeze-dried decellularized porcine small intestinal submucosa tissue and pepsin obtained in step S24 into the acetic acid solution, stir for 22-26 hours, and use a filter cloth Filter to remove larger particles, then transfer the filtrate to a dialysis bag, dialyze with deionized water for a predetermined time under predetermined temperature conditions, and freeze-dry the filtrate to obtain a decellularized porcine small intestine submucosa matrix material freeze-dried powder; S3. Preparation of self-healing hydrogel wound dressings loaded with antimicrobial peptides Weighing a predetermined amount of phenylborated linear hydrophilic polysaccharide powder obtained in step S1 and dissolving it in PBS buffer solution to obtain solution A; weighing a predetermined amount of lyophilized powder of acellular porcine small intestinal submucosa matrix material obtained in step S2 and dissolving it in PBS In the buffer solution, solution B is obtained; the cecropin antimicrobial peptide powder is dispersed into the step solution B, and then solution A and solution B are uniformly mixed to obtain the antimicrobial peptide-loaded self-healing hydrogel wound dressing, and the cecropin The concentration of antimicrobial peptides in the self-healing hydrogel wound dressing loaded with antimicrobial peptides is 0.05-1.0 mg/L. 2. The preparation method of the self-healing hydrogel wound dressing loaded with antimicrobial peptide according to claim 1, characterized in that: in step S3, the solvents of solution A and solution B are PBS buffer solution, and its pH range is 7.0 ~8.0, the mass concentration of phenylborated linear hydrophilic polysaccharide in solution A is 5%, the mass concentration of decellularized porcine small intestinal submucosa matrix material in solution B is 5%, and the mass ratio of solution A to solution B is 1 :0.6～1:1.4. 3. The preparation method of the antimicrobial peptide-loaded self-healing hydrogel wound dressing according to claim 1, characterized in that: the mass concentration of the antimicrobial peptide in solution B in step S2 is 0.01-0.1%. 4. The preparation method of the antimicrobial peptide-loaded self-healing hydrogel wound dressing according to claim 1, characterized in that: in both step S1 and step S2, a molecular weight cut-off of 6-8 kDa is used for dialysis, and the dialysis time is 2 ~3d. 5. The preparation method of the antimicrobial peptide-loaded self-healing hydrogel wound dressing according to claim 1, characterized in that: in step S21, stirring at 35-40°C for 5-7 hours; in step S22, the precipitation treatment time 23-25h; in step S23, stirring at 35-40°C for 23-25h; in step S24, stirring for 23-25h.

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