CN116271206A - Gallium porphyrin complex/silk fibroin antibacterial dressing and preparation method thereof - Google Patents
Gallium porphyrin complex/silk fibroin antibacterial dressing and preparation method thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0004—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing inorganic materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0085—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/009—Materials resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
Traditional medical dressings used in clinic to pack or treat wounds often contain antibiotics and cotton substrates, and the problem of resistance to antibiotics and the non-degradability of cotton substrates severely increase pain and care costs for patients. Aiming at the problems, the invention provides a gallium porphyrin complex/silk fibroin antibacterial dressing which takes silk fibroin freeze-dried gel as a dressing substrate and adds gallium porphyrin complex as antibacterial components, and provides a method for preparing the antibacterial dressing by crosslinking, fully freezing and freeze-drying gallium porphyrin complex/silk fibroin hydrogel. The invention has good biocompatibility and degradability, and the dressing has higher antibacterial activity, antibacterial rate and antibacterial stability, and can further avoid inflammation of wounds caused by infection.
Description
Technical Field
The invention relates to the field of sanitary materials and preparation thereof, in particular to an antibacterial dressing and a preparation method thereof.
The background technology is as follows:
the multi-drug resistant bacteria mainly refer to bacteria which simultaneously show drug resistance to three or more antibacterial drugs used clinically. Most nosocomial infections are caused by multiple resistant bacteria. The strains are widely distributed and spread quickly, and bring serious challenges to clinical medical care and control of nosocomial infections. Therefore, there is an increasing demand for developing novel antibacterial materials which are not easily resistant to drugs.
Traditional medical dressings used in clinic to pack or treat wounds often contain antibiotics and cotton substrates, and the problem of resistance to antibiotics and the non-degradability of cotton substrates severely increase pain and care costs for patients.
Metal ion antibacterial agents are the most promising antibacterial materials at present, however, the metal ion antibacterial agents widely used at present have potential safety hazards, and limit the development of the field. Among them, gallium ion is a metal antibacterial agent which is emerging in recent years, and compared with other metal antibacterial agents, gallium ion has the most remarkable advantages of good biological safety and effectiveness for various multi-drug resistant bacteria, but the application thereof is still being explored.
The gallium porphyrin complex is used as a novel metal antibacterial material, and the application in the field of medical materials is still blank.
Disclosure of Invention
In order to solve the defects and the blank existing in the prior art, the invention provides a gallium porphyrin complex/silk fibroin antibacterial dressing: the silk fibroin freeze-dried gel is used as a dressing substrate, and a gallium porphyrin complex is added as an antibacterial component to prepare the silk fibroin gel; wherein the tyrosine of the gallium porphyrin complex oxidized Silk Fibroin (SF) forms stable cross-linking, the silk fibroin in the gallium porphyrin complex/silk fibroin antibacterial dressing is a porous framework structure, and the silk fibroin comprises a beta-sheet structure.
The invention also provides a preparation method of the gallium porphyrin complex/silk fibroin antibacterial dressing, which comprises the following steps:
step 1, silk fibroin extraction: degumming and dissolving silkworm cocoons, dialyzing, performing reverse dialysis by using polyethylene glycol, and concentrating to obtain a silk fibroin aqueous solution.
Step 2, preparing a high antibacterial activity gallium porphyrin complex: adding porphyrin micromolecules and gallium chloride into an anhydrous pyridine solvent, introducing nitrogen for reaction under the protection of heating, cooling to room temperature after the reaction is finished to obtain a dark red solution, removing the solvent, and collecting a precipitate; washing with a washing liquid and drying;
step 3, hydrogel preparation: uniformly dispersing a gallium porphyrin complex and silk fibroin in a solution, wherein the gallium porphyrin complex oxidizes tyrosine of the silk fibroin to form stable crosslinking;
step 4, preparation of the antibacterial dressing: placing the gallium porphyrin complex/silk fibroin complex in an ultralow temperature refrigerator, fully freezing, and freeze-drying; and (3) placing the obtained gallium porphyrin complex/silk fibroin hydrogel at the temperature of-80 ℃ for full freezing, so that silk fibroin molecules in the system form a porous framework structure, and the solvent water is solidified into solid ice to support the stability of the framework structure. And then freeze-drying the mixture to obtain the composite material. The gallium porphyrin complex/silk fibroin complex is freeze-dried, ice sublimates into water vapor in the freeze-drying process and is discharged out of the system, and a porous framework structure can be formed at the moment, and the porous structure can absorb wound exudates and accommodate cell and tissue adsorption growth.
And 5, sterilizing the composite material at high temperature and high pressure. And (3) sterilizing the composite material at high temperature and high pressure. Inducing silk fibroin to generate an additional beta-sheet structure, effectively enhancing the mechanical structure of the silk fibroin and obtaining the final gallium porphyrin complex/silk fibroin antibacterial dressing.
Preferably, in the step 1: degumming silkworm cocoons, putting the silkworm cocoons into Na2CO3 solution, heating and dissolving the silkworm cocoons at 65 ℃, and putting the obtained solution into a dialysis bag for dialysis by deionized water. Then, the mixture was subjected to reverse dialysis with a 10.0wt% polyethylene glycol (PEG) solution and allowed to stand. Centrifuging to remove undissolved residues, concentrating to obtain aqueous solution of silk fibroin, and storing in refrigerator at 4deg.C;
preferably, in the step 2, the porphyrin small molecule is tetra-p-methoxyphenyl porphyrin.
Preferably, in the step 2, porphyrin small molecules and gallium chloride are added into anhydrous pyridine solvent, and nitrogen is introduced for protection, heating and reflux reaction. After the reaction is finished, cooling to room temperature to obtain a dark red solution, spin-drying, collecting precipitate, washing with a small amount of precooled ethanol, washing the crude product with chloroform, filtering, and drying in a vacuum dryer overnight to obtain dark purple powder.
In the step 3: different proportions of the gallium porphyrin complex are added into the silk fibroin aqueous solution to obtain different hydrogel prefabricated liquids, the prefabricated liquids are subjected to UV irradiation or standing at room temperature for a long time to obtain covalent crosslinked hydrogel through crosslinking of silk fibroin tyrosine residues by the gallium porphyrin complex, and different antibacterial properties of the dressing can be achieved due to different gallium porphyrin complex contents.
Preferably, the adding amount of the gallium porphyrin complex in the step 3 is 1g-6.5g, and the mass volume ratio of the gallium porphyrin complex to the hydrogel solution is 3% -18%.
Preferably, the adding amount of the gallium porphyrin complex in the step 3 is 2.5g-4g, and the mass volume ratio of the gallium porphyrin complex to the hydrogel solution is 7% -12%.
The beneficial effects are that:
1. silk fibroin has good biocompatibility and degradability, and the degradation products are amino acids, so that the healing of wounds can be accelerated.
2. The gallium porphyrin complex and the silk fibroin are mutually crosslinked, so that the antibacterial activity, the antibacterial rate and the antibacterial stability of the dressing are improved, inflammation of wounds caused by infection can be further avoided, and the limitation of the existing dressing is remarkably improved.
3. The antibacterial dressing has the advantages of good biological safety, simple and rapid preparation method and low cost, and is suitable for batch preparation for clinic.
The specific embodiment is as follows:
example 1:
preparation of gallium porphyrin complex/silk fibroin antibacterial dressing:
1. silk extract: 10g of silkworm cocoon is degummed, placed in 0.02mol/L Na2CO3 solution, placed in a 65 ℃ oven for standing and dissolving for 5h, the obtained solution is placed in a dialysis bag and dialyzed with deionized water for 3 days, deionized water is replaced every 12h, and then 10.0wt% polyethylene glycol (PEG) solution is used for reverse dialysis and standing for 12h. Centrifuging at 8000rpm for 40min, removing undissolved residues, concentrating to obtain aqueous solution of silk fibroin, and storing in refrigerator at 4deg.C;
2. preparing a gallium porphyrin complex with high antibacterial activity: a certain amount of tetra-p-methoxyphenyl porphyrin (1.0 mmol) and gallium chloride (0.5 mmol) are weighed and placed in a 100mL round bottom flask, 30mL of anhydrous pyridine solvent is added, and nitrogen is introduced for protection, heating and reflux reaction are carried out for 8h. After the reaction is finished, cooling to room temperature to obtain a dark red solution, spin-drying, collecting precipitate, washing with a small amount of precooled ethanol, washing the crude product with chloroform, filtering, and drying in a vacuum dryer overnight to obtain dark purple powder.
3. Preparation of hydrogel: 2.5g of a gallium porphyrin complex was added to an aqueous silk fibroin solution to obtain a 6% (w/v) hydrogel preparation, which was subjected to ultraviolet irradiation to crosslink SF tyrosine residues via the gallium porphyrin complex to obtain a covalently crosslinked hydrogel.
4. Preparation of an antibacterial dressing: and (3) placing the obtained gallium porphyrin complex/silk fibroin hydrogel at the temperature of-80 ℃ for full freezing, so that silk fibroin molecules in the system form a porous framework structure, and the solvent water is solidified into solid ice to support the stability of the framework structure. And then freeze-drying the mixture to obtain the composite material.
5. And (3) sterilizing the composite material at high temperature and high pressure, inducing the silk fibroin to generate an additional beta-sheet structure, and effectively enhancing the mechanical structure of the silk fibroin to obtain the final gallium porphyrin complex/silk fibroin antibacterial dressing.
Examples 2 to 4, comparative example 5:
the difference from example 1 is that: the added gallium porphyrin complexes have different mass, so that the gallium porphyrin complex contents with different proportions are formed. The addition mass is shown in table 1:
example 6: the difference from example 1 is that: and thirdly, standing at room temperature to obtain the pure physical crosslinked hydrogel. The relevant test data for example 6 were not significantly different from example 1.
Performance test:
cell compatibility test: the effect of the antimicrobial dressing of example 1-example 4, comparative example 5 on proliferation of M.phi.RAW 264.7, fibroblasts (RS-1) and vascular endothelial cells (HUVEC) in culture for 1,3,7 days was examined using MTT to determine the biocompatibility of the hydrogels. Cell viability calculated from absorbance change after blank correction is shown in table 2. With the increase of the content of the gallium porphyrin complex added in the preparation process of the antibacterial dressing, the cell compatibility of the material is not changed significantly. I.e. the material has no significant inhibition on the growth of cells.
Blood compatibility test: hemolysis experiments were performed on gallium porphyrin complex/silk fibroin antimicrobial dressings to determine the haemocompatibility of the dressing. Fresh rabbit blood was prepared as a suspension of erythrocytes at a concentration of 10.0% (v/v). Next, examples 1 to 5 were each placed in a 1mL centrifuge tube containing 1mL of red blood cell suspension and incubated for 2 hours at 37℃in a constant temperature and humidity incubator. Likewise, red blood cell suspensions diluted with PBS and ultrapure water were used as negative and positive controls, respectively. Finally, the above-mentioned red blood cell suspension after incubation for 2 hours was centrifuged at 3500rpm for 10 minutes, and the supernatant was collected and its absorbance at 540nm was measured with an ultraviolet-visible spectrophotometer to determine the release of hemoglobin. The relevant data are shown in table 3.
The data demonstrate that either the silk fibroin dressing (comparative example 5) or the dressing crosslinked with the gallium porphyrin complex and silk fibroin exhibited lower hemolysis rates reflecting better blood compatibility.
Antibacterial activity detection:
antibacterial activity of the gallium porphyrin complex/silk fibroin antibacterial dressing was studied, including inhibition of growth of escherichia coli, staphylococcus aureus, and pseudomonas aeruginosa.
1. And (3) detection of a bacteriostasis zone: the filter paper sheet method is adopted, and the night bacteria are firstly selected, diluted and cultured to the logarithmic phase. 100 mu L of bacterial liquid is uniformly smeared on a non-resistant solid flat plate, then the cylindrical antibacterial dressings of examples 1-4 and comparative example 5 with the diameter of 1cm are added, and the mixture is placed in a 37 ℃ incubator for 20 hours, and the diameter of the area of the bacteria growth inhibition is measured through a ruler.
2. Antibacterial rate and antibacterial stability detection: the antibacterial activity of the nanofibers of examples 1-6 was evaluated using an oscillation method and the antibacterial rate was calculated using a viable bacteria plate count method. The samples were washed 30 times according to the standard AATCC 61-2010 washing method, and the antibacterial activity of the modified samples was evaluated by an oscillation method, and the antibacterial rate was calculated by a viable bacteria plate count method.
As the content of the gallium porphyrin complex is increased, the size of the bacteriostasis zone is obviously increased. Has good antibacterial effect on three kinds of common infectious bacteria in hospitals. The antibacterial rate and antibacterial stability of the composition are obviously improved compared with those of comparative examples.
In vivo safety test:
establishing a mouse subcutaneous implantation model, and verifying the safety, degradability and inflammatory reaction of the gallium porphyrin complex/silk fibroin antibacterial dressing in vivo: after the mice were anesthetized, the antimicrobial dressing of examples 1-5 was back implanted, sacrificed 3,7, 14, 28 days later, the material and surrounding tissue paraffin were removed and sectioned. H & E staining, observe dressing degradation and inflammatory response.
No inflammatory and foreign body reactions were observed in all control and test groups, and thus the gallium porphyrin complex/silk fibroin antimicrobial dressing prepared by the crosslinking treatment was found to be biocompatible for mouse subcutaneously transplanted tissue.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (9)
1. A gallium porphyrin/silk fibroin antibacterial dressing, characterized in that: the gallium porphyrin complex/silk fibroin antibacterial dressing is prepared by taking silk fibroin freeze-dried gel as a dressing substrate and adding gallium porphyrin complex as an antibacterial component, tyrosine of the gallium porphyrin complex oxidized silk fibroin forms stable crosslinking, silk fibroin in the gallium porphyrin complex/silk fibroin antibacterial dressing is of a porous framework structure, and the silk fibroin comprises a beta-sheet structure.
2. A method for producing a gallium porphyrin/silk fibroin antimicrobial dressing according to claim 1, characterized in that: the method comprises the following steps:
step 1, silk fibroin extraction;
step 2, preparing a high antibacterial activity gallium porphyrin complex;
step 3, preparing gallium porphyrin complex/silk fibroin hydrogel;
step 4, preparing an antibacterial dressing;
step 5, sterilizing the composite material at high temperature and high pressure;
in the step 3, the gallium porphyrin complex and the silk fibroin are uniformly dispersed and in the solution, and tyrosine of the gallium porphyrin oxidized silk fibroin forms stable crosslinking; in the step 4, the gallium porphyrin complex/silk fibroin complex is placed in an ultralow temperature refrigerator to be sufficiently frozen and then is frozen and dried.
3. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 2, wherein the method comprises the following steps: the silk fibroin extraction method in the step 1 is as follows: degumming and dissolving silkworm cocoons, dialyzing, performing reverse dialysis by using polyethylene glycol, and concentrating to obtain a silk fibroin aqueous solution.
4. A method for making a gallium porphyrin/silk fibroin antimicrobial dressing according to claim 3, wherein: the silk fibroin extraction method comprises the following specific steps: degumming silkworm cocoons, putting the silkworm cocoons into Na2CO3 solution, heating and dissolving the silkworm cocoons at 65 ℃, and putting the obtained solution into a dialysis bag for dialysis by deionized water. Then, reversely dialyzing and standing by using 10.0 weight percent of polyethylene glycol (PEG) solution; centrifuging to remove undissolved residues, and concentrating to obtain silk fibroin aqueous solution.
5. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 2, wherein the method comprises the following steps: the preparation process in the step 2 is as follows: adding porphyrin micromolecules and gallium chloride into an anhydrous pyridine solvent, introducing nitrogen for reaction under the protection of heating, cooling to room temperature after the reaction is finished to obtain a dark red solution, removing the solvent, and collecting a precipitate; washing with the washing solution, and drying.
6. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 5, wherein the method comprises the following steps: the porphyrin small molecule is tetra-p-methoxyphenyl porphyrin.
7. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 2, wherein the method comprises the following steps: in the step 3, the method for forming stable cross-linking by the gallium porphyrin complex and the silk fibroin comprises the following steps: the two are crosslinked by exposure to ultraviolet light or by standing at room temperature.
8. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 2, wherein the method comprises the following steps: in the step 3, the adding amount of the gallium porphyrin complex is 1g-6.5g, and the mass volume ratio of the gallium porphyrin complex to the silk fibroin hydrogel solution is 3% -18%.
9. The method for manufacturing the gallium porphyrin/silk fibroin antibacterial dressing according to claim 7, wherein the method comprises the following steps: in the step 3, the adding amount of the gallium porphyrin complex is 2.5g-4g, and the mass volume ratio of the gallium porphyrin complex to the silk fibroin hydrogel solution is 7% -12%.
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