A composition with pH-responsive release of antibacterial polypeptides, preparation method and application thereof

Abstract

The application relates to the field of biological medicine, in particular to a composition with pH response release antibacterial polypeptide, a preparation method and application thereof. The composition comprises: a decellularized matrix material, an activator, a grafting agent and antibacterial polypeptide. The decellularized matrix and the antibacterial polypeptide are connected through an imine bond, the imine bond is hydrolyzed under acidic conditions, the antibacterial polypeptide is released, and the imine bond is reconnected under a neutral environment; the process is dynamically reversible, so that the antibacterial polypeptide is released in a responsive manner. The dressing can promote the repair and regeneration of a wound, and simultaneously inhibit the growth and proliferation of bacteria, and has great application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a composition with pH response release antibacterial polypeptide, preparation method and application thereof. BACKGROUND

[0002] For wound infection, there are several strategies in the clinic, antibiotics are recognized as the best treatment for bacterial infection, but the increase of drug-resistant microorganisms threatens the effectiveness of antibiotics, bacterial drug resistance has become an urgent public safety problem that needs to be solved, it is estimated that by 2050, 10 million people will die from sepsis and other diseases caused by bacterial infection every year, at the same time, high-dose antibiotics have great harm to the liver and kidney of the human body, affecting the health of the human body. The second is to use metal ions with antibacterial effect, such as silver ion, copper ion, zinc ion, etc., metal ions can destroy the cell membrane and nucleic acid material of bacteria, interfere with the normal metabolism of bacteria, so as to achieve the effect of killing and inhibiting bacteria, however, metal ions will also affect the growth and proliferation of normal cells while killing bacteria, containing certain cytotoxicity, which affects the healing of the wound. The third is to use dressings with antibacterial effect, such dressings usually contain antibacterial drugs or metal ions, which can inhibit the adhesion and growth of bacteria on the wound surface while promoting wound healing, but the combination of such dressings and antibacterial components is usually a simple physical effect, which cannot control the controllable and on-demand release of drugs, and it is difficult to avoid the negative effects of drugs or ions on wound healing.

[0003] In recent years, as a kind of biological material with natural structure and active ingredients, acellular matrix is widely used as wound dressing. Acellular matrix contains rich collagen, adhesion protein, fibronectin and growth factor and other active ingredients, which can provide suitable microenvironment and signal molecules to promote cell migration, proliferation and differentiation, so as to accelerate wound healing. However, acellular matrix material lacks antibacterial effect and is easy to be contaminated and degraded by bacteria, so it cannot be applied when the wound is in an infected state, which limits its use in clinic, so it is necessary to modify their surface to endow them with antibacterial ability. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the present application provides a composition with pH response release antibacterial polypeptide, preparation method and application thereof, which realizes the controllable release of antibacterial peptides according to the change of the microenvironment of the wound.

[0005] The technical scheme adopted by the present application to solve its technical problems is:

[0006] The first object of the present application is to provide a composition with pH response release antibacterial polypeptide, comprising: acellular matrix, activator, grafting agent and antibacterial polypeptide.

[0007] Further, the grafting agent is NH2-PEG-CHO.

[0008] Further, the activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and / or N-hydroxysuccinimide solution.

[0009] Further, the decellularized matrix is one of decellularized dermal matrix, decellularized pericardial matrix, decellularized corneal matrix, amniotic membrane matrix, porcine small intestinal submucosa matrix, decellularized bladder membrane matrix, decellularized fat matrix, and decellularized peritoneal membrane matrix.

[0010] Further, the decellularized matrix is prepared by a virus inactivation, deimmunization, and freeze-drying molding process.

[0011] Further, the antibacterial peptide is one or more of Cecropin A, Bactenecin, Melittin MLT, Cecropin B, CysHHC10, tetrapeptide ARG-Gly-Phe-Phe, Crotalicidin, Drosomycin, and Temporin L.

[0012] The second object of the present application provides a preparation method of a composition with PH-responsive release of antibacterial peptides.

[0013] Further, the specific preparation method is:

[0014] S1. preparing a decellularized matrix material by a virus inactivation, deimmunization, and freeze-drying molding process;

[0015] S2. activating the decellularized matrix material in step S1 using an activating agent;

[0016] S3. treating the decellularized matrix in step S2 with a grafting agent;

[0017] S4. dissolving the antibacterial peptide in pure water, placing the decellularized matrix treated with the grafting agent in step S3 into the solution of the antibacterial peptide, and grafting the antibacterial peptide to the surface of the decellularized matrix membrane.

[0018] Further, the activating agent in step S2 is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and / or N-hydroxysuccinimide (NHS) solution, the decellularized matrix prepared in step S1 is placed in a 0.1 mol 2-(N-morpholino)ethanesulfonic acid buffer solution, the pH value of the buffer solution is between 4.5 and 7.2, the carboxyl group on the decellularized matrix material is activated using the activating agent, the reaction temperature is between 4 and 30℃, and the reaction time is 0.5-24h.

[0019] Further, the grafting agent in S3 is NH2-PEG-CHO, and the NH2-PEG-CHO solution is added at a concentration of 10-100 mg / ml, and the reaction is carried out for 12-72 h under slight stirring, so that Schiff reaction occurs between the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO, and the PEG is grafted to the surface of the decellularized matrix membrane.

[0020] A third object of the present application provides the use of the above-mentioned composition as a wound dressing.

[0021] The present application has the following beneficial effects:

[0022] The decellularized matrix and the antibacterial polypeptide are connected by an imine bond, the imine bond is hydrolyzed under acidic conditions to release the antibacterial polypeptide, and the imine bond is reconnected under a neutral environment, and this process is dynamically reversible, thereby realizing the responsive release of the antibacterial polypeptide. When microorganisms colonize and grow in the wound, anaerobic glycolysis of sugar occurs to produce a large amount of lactic acid component, thereby reducing the pH value of the microenvironment of the wound. The dressing contacts the acidic hydrogen ions, the imine bond is broken, the antibacterial peptide is released into the wound environment, and the antibacterial and bacteriostatic effects are exerted. When the microorganisms in the wound are reduced, the pH gradually returns to the normal neutral state, the antibacterial polypeptide is reconnected with the dressing, and the solubility of the antibacterial polypeptide in the wound environment is reduced, thereby reducing the side effects of the antibacterial polypeptide on the wound. This dressing can promote the repair and regeneration of the wound while inhibiting the growth and proliferation of bacteria, and has great application potential. BRIEF DESCRIPTION OF DRAWINGS

[0023] The present application is further described below in conjunction with the drawings and examples.

[0024] Figure 1 is a fluorescence graph for detecting the grafting of the antibacterial polypeptide by fluorescence characterization in the present application;

[0025] Figure 2 is an analysis graph for detecting the grafting of the antibacterial polypeptide by fluorescence characterization in the present application;

[0026] Figure 3 is a release curve graph of the antibacterial polypeptide in a neutral environment detected by Coomassie brilliant blue in the present application;

[0027] Figure 4 is a release curve graph of the antibacterial polypeptide in an acidic environment detected by Coomassie brilliant blue in the present application;

[0028] Figure 5 is a cytotoxicity analysis graph of the cells modified by the antibacterial polypeptide detected by CCK-8 reagent in the present application. DETAILED DESCRIPTION

[0029] In the present application, various common chemical reagents used in the examples are commercially available and can be purchased through conventional channels.

[0030] The application provides a composition with pH-responsive release of antibacterial polypeptide, a preparation method and application thereof, and realizes controllable release of the antibacterial peptide according to the change of the microenvironment of a wound.

[0031] Preparation of the decellularized matrix: the decellularized matrix material is prepared through a virus inactivation, deimmunogenicity and freeze-drying forming process.

[0032] The main component of the decellularized matrix is collagen, and the collagen molecular chain contains a large number of carboxyl groups. The application activates the carboxyl groups on the collagen molecular chain by using an activator, and the activated carboxyl groups and polyethylene glycol (PEG) grafted with amino groups undergo Schiff reaction to generate an amide bond, and the product of the application is prepared by reacting the amide bond with a grafting agent. Therefore, the decellularized matrix material can be a decellularized dermal matrix, a decellularized pericardial membrane matrix, a decellularized corneal matrix, an amniotic membrane matrix, a pig small intestinal submucosa matrix, a decellularized bladder membrane matrix, a decellularized fat matrix, a decellularized peritoneum and the like.

[0033] The decellularized matrix modification method is as follows:

[0034] 1. Prepare a decellularized matrix film with a size of 5cm*5cm, and then put it into a 50ml 0.1mol 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH value of 4.5-7.2, activate the carboxyl groups on the decellularized matrix material by using a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and / or N-hydroxysuccinimide (NHS) solution system, the solubility of EDC is 2-10mM, the solubility of NHS is 2-10mM, the reaction temperature is 4-30℃, and the reaction time is 0.5-24h.

[0035] 2. After the decellularized matrix is activated, NH2-PEG-CHO (Mw=2000D) is added with a solubility of 10-100mg / ml, and the decellularized material is slightly stirred and reacted for 12-72h to make the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO undergo Schiff reaction, and the PEG is grafted to the surface of the decellularized matrix film through an amide bond.

[0036] 3. Dissolve the antibacterial polypeptide in pure water, adjust the pH of the solution to 6.5-7.5 by using an acid-base reagent, put the decellularized matrix film grafted with NH2-PEG-CHO into the antibacterial polypeptide solution, and react at 4-30℃ for 0.5-24h to make the amino groups in the antibacterial peptide and the aldehyde groups react to generate an imine bond, and graft the antibacterial polypeptide to the surface of the decellularized matrix film.

[0037] The antibacterial peptide can be Cecropin A, Bactenecin, Melittin MLT, Cecropin B, CysHHC10, tetrapeptide ARG-Gly-Phe-Phe, Crotalicidin, Drosomycin, Temporin L, etc.

[0038] The specific preparation method is as follows:

[0039] S1. Prepare the acellular matrix material by a virus inactivation, deimmunization and freeze-drying forming process;

[0040] S2. Put the acellular matrix prepared in step S1 into a 0.1 mol 2-(N-morpholino) ethanesulfonic acid buffer solution, the pH value of the buffer solution is between 4.5-7.2, and the carboxyl groups on the acellular matrix material are activated by using an activating agent, and the reaction temperature is between 4-30℃;

[0041] S3. Add NH2-PEG-CHO (Mw=2000D) with a solubility of 10-100 mg / ml, and slightly stir for 12-72 h to allow Schiff reaction between the carboxyl groups on the acellular material and the amino groups of NH2-PEG-CHO, so as to graft PEG to the surface of the acellular matrix membrane;

[0042] S4. Dissolve the antibacterial peptide in pure water with a solubility of 0.1 mg / ml-50 mg / ml, and a pH of 6.5-7.5, and put the acellular matrix grafted with PEG in step S3 into the antibacterial peptide solution, and react at 4-30℃ for 2-24 h to allow the amino groups in the antibacterial peptide to react with the aldehyde groups, so as to graft the antibacterial peptide to the surface of the acellular matrix membrane.

[0043] The preparation of the acellular matrix material in step S1 by a virus inactivation, deimmunization and freeze-drying forming process is a prior art.

[0044] In order to facilitate the understanding of those skilled in the art, the present application will be further described below in combination with examples, and the content mentioned in the embodiments is not a limitation of the application.

[0045] Example 1:

[0046] A preparation method of a composition with pH-responsive release of antibacterial peptides is as follows:

[0047] S1. Preparation of antibacterial acellular porcine small intestinal submucosa matrix;

[0048] S2. The pig small intestinal submucosa matrix treated in step S1 is placed in 50 ml of 0.1 mol 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, the pH value of the buffer solution is between 4.5, and the carboxyl groups on the decellularized matrix material are activated by using a 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution system, the solubility of EDC is 2 mM, the solubility of NHS is 2 mM, the reaction temperature is 4°C, and the reaction time is 6 h;

[0049] S3. NH2-PEG-CHO (Mw = 2000D) is added at a solubility of 10 mg / ml, and the reaction is carried out for 72 h under slight stirring, so that Schiff reaction occurs between the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO, and PEG is grafted to the surface of the decellularized matrix membrane;

[0050] S4. Cecropin B antibacterial peptide is dissolved in pure water at a solubility of 0.1 mg / ml, the pH of the solution is adjusted to 6.5 by using an acid-base reagent, the decellularized matrix membrane grafted with NH2-PEG-CHO is placed in the antibacterial peptide solution, and the reaction is carried out at 4°C for 24 h, so that the amino groups in the antibacterial peptide react with the aldehyde groups, the antibacterial peptide is grafted to the surface of the decellularized matrix membrane, and SIS-C (a composition with pH-responsive release of antibacterial polypeptide) is obtained.

[0051] Example 2:

[0052] A preparation method of a composition with pH-responsive release of antibacterial polypeptide is as follows:

[0053] S1. Preparation of antibacterial decellularized dermal matrix;

[0054] S2. The decellularized dermal matrix treated in step S1 is placed in 50 parts of deionized water, the pH value is 7.2, EDC and NHS are added, the carboxyl groups on the decellularized matrix material are activated, the solubility of EDC is 10 mM, the solubility of NHS is 10 mM, the reaction temperature is 30°C, and the reaction time is 4 h;

[0055] S3. NH2-PEG-CHO (Mw = 2000D) is added at a solubility of 100 mg / ml, and the reaction is carried out for 12 h under slight stirring, so that Schiff reaction occurs between the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO, and PEG is grafted to the surface of the decellularized matrix membrane;

[0056] S4. Dissolve the MLT melittin peptide in pure water with a concentration of 50 mg / ml, adjust the pH of the solution to 7.5 by using hydrochloric acid and NaOH, put the above-mentioned acellular dermal matrix powder grafted with NH2-PEG-CHO into the antibacterial peptide solution, react at 15°C for 24 h to allow the amino group in the antibacterial peptide to react with the aldehyde group, graft the antibacterial peptide to the surface of the acellular matrix membrane, obtain the acellular dermal matrix powder grafted with MLT melittin peptide, and obtain ADM-M (a composition with pH-responsive release of antibacterial polypeptides).

[0057] Example 3:

[0058] A preparation method of a composition with pH-responsive release of antibacterial polypeptides is as follows:

[0059] S1. Preparation of antibacterial bovine type I collagen dressing;

[0060] S2. Dissolve 1 g of cleaned and disinfected bovine Achilles tendon in 100 ml of 3% acetic acid solution, add 0.1 g of pepsin, shake at 15°C for 48 h, filter and dialyze the filtrate to obtain a pure collagen solution,

[0061] S3. Add NH2-PEG-CHO (Mw = 2000D) with a concentration of 50 mg / ml, add EDC and NHS, the concentration of EDC is 5 mg / ml, and the concentration of NHS is 5 mg / ml, gently stir, and react for 6 h to allow Schiff reaction between the carboxyl group on the acellular material and the amino group of NH2-PEG-CHO, and graft PEG to the molecular chain of collagen;

[0062] S4. Put the gel into a solution with a pH of 6.5, add Melittin antibacterial peptide with a concentration of 25 mg / ml, and react for 12 h to obtain Melittin grafted collagen gel dressing, and obtain collagen gel with pH-responsive release of antibacterial polypeptides.

[0063] Example 4:

[0064] A preparation method of a composition with pH-responsive release of antibacterial polypeptides is as follows:

[0065] S1. Preparation of antibacterial acellular bladder matrix membrane;

[0066] S2. Put the acellular bladder matrix membrane obtained in step S1 into 50 ml of MES solution with a pH of 6.2, add EDC and NHS catalysts, and react for 12 h.

[0067] S3. Add NH2-PEG-CHO (Mw=2000D) at a concentration of 70mg / ml, stir gently, and react for 50h to allow the Schiff reaction to occur between the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO, thereby grafting PEG onto the surface of the decellularized matrix membrane.

[0068] S4. The modified decellularized bladder matrix membrane was added to a 30 mg / ml Perinerin antimicrobial peptide solution and reacted for 20 h to obtain a Perinerin-modified decellularized bladder matrix membrane, which was then lyophilized for later use to obtain a composition that releases antimicrobial peptides in response to pH.

[0069] Example 5:

[0070] The preparation method of a composition that releases antimicrobial peptides in response to pH is as follows:

[0071] S1. Preparation of antibacterial decellularized pericardial matrix;

[0072] S2. Place the decellularized pericardium obtained in step S1 into 50 ml of MES solution with a pH of 6.5, add EDC and NHS catalysts, both with a solubility of 3 mM, and react for 30 h.

[0073] S3. Add NH2-PEG-CHO (Mw=2000D) at a concentration of 80mg / ml, stir gently, and react for 60h to allow the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO to undergo a Schiff reaction, thereby grafting PEG onto the surface of the decellularized matrix membrane.

[0074] S4. The modified decellularized pericardium was added to a 10 mg / ml solution of Mytichitin-A antimicrobial peptide and reacted for 20 h to obtain Mytichitin-A modified decellularized pericardium. The pericardium was then lyophilized for later use to obtain a composition that releases antimicrobial peptides in response to pH.

[0075] Example 6: Grafting effect test performance test:

[0076] The grafting performance of compositions SIS-C, ADM-M, and the comparative example SIS prepared in Examples 1-2 was tested. The comparative example SIS was a decellularized matrix without grafted antimicrobial peptides. The distribution of peptides on the decellularized matrix material was tested by fluorescence reaction. Fluorescein can bind to the amino group of the antimicrobial peptide to produce a fluorescence reaction. The grafting of antimicrobial peptides on the surface of the decellularized matrix was observed by using the fluorescent probe fluorescein to bind to the amino group of the antimicrobial peptide to produce fluorescence.

[0077] The experimental process is: the fluorescein is dissolved in DMF solution to obtain a 10wt% fluorescein solution, 10ul of the solution is added on the surface of the unmodified and modified decellularized matrix, and the reaction is carried out in the dark for 10-15min, then a small amount of anhydrous ethanol is used to wash away the unreacted solution, and the confocal microscope is used for observation, and the fluorescence intensity is analyzed by ImageJ software, and the confocal results and fluorescence intensity analysis results are shown.

[0078] As shown in Figures 1-2 The results show that the fluorescence intensity value of the decellularized matrix grafted with antibacterial peptides is significantly different from that of the control group, indicating that the antibacterial peptides of example 1-2 are successfully grafted onto the surface of the decellularized matrix.

[0079] Example 7 in vitro release

[0080] Coomassie brilliant blue can stain protein and is widely used for quantitative analysis of protein, the absorbance value of its solution is proportional to the content of protein, and protein with a solubility of 0-1000ug / ml can be determined, the reaction process is sensitive, the data is accurate, and the operation is simple. When coomassie blue is in free state, it is red, and it is blue after combining with protein, and the maximum absorption peak wavelength is 595nm. The content of antibacterial peptide in the solution can be quantitatively analyzed by testing the absorbance value of the solution at 595nm by ultraviolet-visible spectrophotometer.

[0081] The SIS without grafting antibacterial peptides and SIS-C and ADM-M are put into 12-hole plates, 1ml PBS buffer solution or 1ml MES buffer solution with pH of 5.5 is added in the three materials, and the culture is carried out in a constant temperature incubator with temperature of 37℃. 40ul of release solution is taken out at 1h, 2h, 4h, 8h, 12h, 24h, 2d, 3d and 5d, coomassie blue solution is added for reaction for 5min, the absorbance value (i.e. OD595nm) of antibacterial peptide standard solution at 595nm is determined by ultraviolet-visible spectrophotometer, the solubility of antibacterial peptide is obtained through the standard curve, and the release curve of antibacterial peptide is drawn.

[0082] As shown in Figures 3-4 The analysis shows that the release rate of antibacterial peptide is obviously higher in acidic condition than in neutral environment, indicating that the acidic environment is beneficial to the release of antibacterial peptide.

[0083] Example 8, cytotoxicity detection:

[0084] The fibroblasts are cultured on the decellularized matrix materials of SIS, SIS-C and ADM-M, the proliferation of fibroblasts in vitro is quantitatively characterized by CCK-8 reagent, and the cytotoxicity of the decellularized matrix material grafted with antibacterial peptides is evaluated. The test results are shown in Figure 5As shown: from the figure can be seen that compared with SIS, SIS-C and ADM-M can significantly promote the proliferation of fibroblasts in vitro, indicating that the antibacterial peptide modified decellularized matrix material does not have cytotoxicity.

[0085] Example 9, antibacterial effect:

[0086] The standard bacteria strains Escherichia coli and Staphylococcus aureus were selected, and were cultured in PBS buffer and nutrient conditions respectively to form neutral and acidic microenvironments, and were placed in a 37°C constant temperature incubator for 48h. The bacteria liquid after culture was diluted to a concentration of 10 6 CFU / ml, the bacteria liquid cultured in PBS and nutrient conditions was added to the 12-well plate respectively, and the SIS, SIS-C and ADM-M samples were placed in the constant temperature incubator for culture. 100μl of bacteria liquid was taken at 3, 5, 7 and 10 days respectively to detect the absorbance at 600nm, and the antibacterial rate was calculated by the formula antibacterial rate = (OD 对照组 -OD 实验组 ) / OD 对照组 *100%, and the antibacterial rate of the material was calculated.

[0087] The results are shown in Tables 1 and 2 below: compared with unmodified SIS, SIS-C and ADM-M have obvious antibacterial effect in neutral and acidic environments, and the antibacterial rate in acidic conditions is significantly higher than that in neutral environment, indicating that more antibacterial peptides are released in acidic environment, proving that the release of antibacterial peptides is pH-responsive.

[0088] Table 1. Antibacterial rate of samples in neutral environment

[0089]

[0090] Table 2. Antibacterial rate of samples in acidic environment

[0091]

[0092] The above examples are the preferred implementation of the present application, in addition to this, the present application can be realized in other ways, without departing from the concept of the present application, any obvious substitution within the protection scope of the present application.

Claims

1. A composite material exhibiting pH-responsive release of antibacterial peptides, characterized in that, The composite material is a product formed by grafting antimicrobial peptides onto the surface of a decellularized matrix material via imine bonds; The imine bond is formed in the following manner: After the decellularized matrix material is activated by the activator, it is linked with the grafting agent NH2-PEG-CHO through amide bonds, so that PEG is grafted onto the surface of the decellularized matrix material. The amino group in the antimicrobial peptide reacts with the aldehyde group of the grafting agent NH2-PEG-CHO to form an imine bond, thereby grafting the antimicrobial peptide onto the surface of the decellularized matrix material. The activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and / or N-hydroxysuccinimide.

2. The composite material with pH-responsive release of antibacterial peptides according to claim 1, characterized in that, The decellularized matrix is ​​one of the following: decellularized dermal matrix, decellularized pericardial matrix, decellularized corneal matrix, amniotic matrix, porcine small intestinal submucosa matrix, decellularized bladder membrane matrix, decellularized adipose matrix, and decellularized peritoneum.

3. The composite material with pH-responsive release of antibacterial peptides according to claim 1, characterized in that, The decellularized matrix was prepared through virus inactivation, deimmunogenicity removal, and freeze-drying.

4. The composite material with pH-responsive release of antibacterial peptides according to claim 1, characterized in that, The antimicrobial polypeptide is one or more of the following: cecropin A, Bactenecin, bee venom peptide MLT, Cecropin B, CysHHC10, tetrapeptide ARG-Gly-Phe-Phe, Crotalicidin, Drosophila antimicrobial peptide, and Temporin L.

5. A method for preparing a composite material that releases antibacterial peptides in response to pH, used to prepare the composite material according to any one of claims 1-4, characterized in that... : S1. Decellularized matrix materials were prepared by virus inactivation, deimmunogenicity removal, and freeze-drying molding processes; S2. Activate the decellularized matrix material from step S1 using an activator; S3. Treat the decellularized matrix from step S2 with a grafting agent; S4. Dissolve the antimicrobial peptide in pure water, and place the decellularized matrix treated with the grafting agent in S3 into the solution of the antimicrobial peptide to graft the antimicrobial peptide onto the surface of the decellularized matrix membrane.

6. The method for preparing the composite material with pH-responsive release of antibacterial peptides according to claim 5, characterized in that, In step S2, the activator is a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and / or N-hydroxysuccinimide (NHS). The decellularized matrix prepared in step S1 is placed in a 0.1 mol 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH value between 4.5 and 7.

2. The activator is used to activate the carboxyl groups on the decellularized matrix material, and the reaction temperature is between 4 and 30°C.

7. The method for preparing the composite material with pH-responsive release of antibacterial peptides according to claim 5, characterized in that, The grafting agent in S3 is NH2-PEG-CHO. The concentration of NH2-PEG-CHO is 10-100 mg / ml. After gentle stirring, the reaction is carried out for 12-72 hours to allow the carboxyl groups on the decellularized material and the amino groups of NH2-PEG-CHO to undergo a Schiff reaction, thereby grafting PEG onto the surface of the decellularized matrix membrane.

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