Modified antibacterial composite biological dressing as well as preparation method and application thereof

Abstract

The invention discloses a modified antibacterial composite biological dressing as well as a preparation method and application thereof, and belongs to the technical field of biological dressings. The biological dressing is formed by compounding a modified antibacterial layer, release paper and an isolating layer, the release paper is adhered to one side of the modified antibacterial layer, and the isolating layer is adhered to the other side of the modified antibacterial layer; the modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified double-enzyme carrier and an acetic acid aqueous solution as raw materials; the modified double-enzyme carrier is prepared by aminating SiO2 microspheres and co-immobilizing the aminated SiO2 microspheres with glucose oxidase and horse radish peroxidase. The biological dressing prepared by the invention has good air permeability and antibacterial property, is free of cytotoxicity, and can be applied to postoperative wound care.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological dressings, and in particular relates to a modified antibacterial composite biological dressing and a preparation method and application thereof. Background Art

[0002] Wound care is a crucial component of healthcare. Effective wound care not only alleviates patient pain but also promotes wound healing and reduces complications. Composite biological dressings have been a research hotspot in recent years. Combining the advantages of multiple biomaterials, they can better mimic the microenvironment of human tissue, providing more favorable conditions for wound healing.

[0003] Antibiotics possess potent antimicrobial activity, effectively inhibiting or killing a wide range of bacteria. Antibiotic-containing dressings achieve this antimicrobial effect by releasing antibiotics. However, with the widespread use of antibiotics, bacterial resistance is becoming increasingly serious. Many bacteria have developed resistance to common antibiotics, limiting the use of antibiotic-containing dressings. Furthermore, the use of antibiotics can cause adverse reactions, such as allergic reactions and intestinal dysbiosis.

[0004] Existing composite biological dressings still have some deficiencies in terms of antibacterial properties and bioactivity, which require further improvement and optimization. In addition, with the increasing severity of antibiotic resistance, the development of new antibacterial dressings has important clinical significance and social value.

[0005] Therefore, the present invention develops a modified antibacterial composite biological dressing to solve the technical problems in the prior art that traditional dressings contain silver ions, antibiotics, etc., which are prone to drug resistance, are cytotoxic at high concentrations, affect wound healing, and cannot dynamically adjust the bactericidal intensity according to the wound microenvironment, resulting in low antibacterial efficiency and excessive stimulation of the wound surface. Summary of the Invention

[0006] The purpose of the present invention is to provide a modified antibacterial composite biological dressing and its preparation method and application, which are used to solve the technical problems in the prior art that traditional dressings contain silver ions, antibiotics, etc., which are prone to drug resistance, are cytotoxic at high concentrations, affect wound healing, cannot dynamically adjust the bactericidal intensity according to the wound microenvironment, and lead to low antibacterial efficiency and excessive stimulation of the wound surface.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A modified antibacterial composite biological dressing is composited by a modified antibacterial layer, a release paper, and an isolation layer. The release paper is adhered to one side of the modified antibacterial layer, and the isolation layer is adhered to the other side of the modified antibacterial layer. The modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified dual-enzyme carrier, and an acetic acid aqueous solution. The modified dual-enzyme carrier is prepared by amino-modifying SiO2 microspheres and co-immobilizing them with glucose oxidase and horseradish peroxidase. The isolation layer is a polyurethane film or non-woven fabric.

[0008] Furthermore, the method for preparing the modified antibacterial layer comprises the following steps: (1) Dissolve chitosan quaternary ammonium salt, sodium alginate, and gelatin in an acetic acid aqueous solution, then add the modified dual enzyme carrier, stir in the dark, and let stand to obtain a biological dressing solution; (2) The biological dressing liquid is heated and stirred to react to obtain a cross-linking liquid; the surface of the polytetrafluoroethylene mold is coated with a dimethyl silicone oil ethanol solution, dried to form an anti-adhesive film, and the cross-linking liquid is poured into the polytetrafluoroethylene mold for casting and pre-cross-linking, preliminary molding, static cross-linking, curing and shaping, demolding, and sterilization to obtain a modified antibacterial layer.

[0009] Preferably, in step (1), the amount ratio of chitosan quaternary ammonium salt, sodium alginate, gelatin, modified dual enzyme carrier and acetic acid aqueous solution is (3.5-3.8) g: (1.5-1.6) g: (2-2.15) g: (0.5-0.6) g: 100 mL, wherein the concentration of the acetic acid aqueous solution is 2vt%, and the pH value is 2.9-3.1; the stirring speed is 150-200 rpm, the stirring time is 5-8 min, the stirring temperature is 28-32 ° C, and the standing time is 5-10 min.

[0010] Preferably, the heating temperature in step (2) is 36-38°C, the stirring reaction speed is 50-55rpm, the stirring reaction time is 55-65min, the concentration of the dimethyl silicone oil ethanol solution is 0.1-0.12wt%, the drying temperature is 55-60°C, the drying time is 20-25min, the casting thickness is 1.9-2.1mm, the pre-crosslinking temperature is 36.5-37.5°C, the pre-crosslinking humidity is 78-82%, the pre-crosslinking time is 60-65min, the static crosslinking time is 1-2h, and the curing molding is gradient curing. First, the mold is moved into a cold storage at 3.5-4.5°C and left to stand for 2-2.5h; then the mold is transferred to a constant humidity box at 20-25°C and a humidity of 40-42%, and left to stand for 4-4.5h.

[0011] Furthermore, the preparation method of the modified dual-enzyme carrier comprises the following steps: S1. Add the ethyl orthosilicate solution dropwise into the cetyltrimethylammonium bromide solution, heat in a water bath, adjust the pH to 10.5, stir to react, centrifuge, and wash to obtain SiO2 microspheres; S2. Dispersing SiO2 microspheres in ethanol, adding aminopropyltriethoxysilane solution, and reflux reaction to obtain amino-modified mesoporous SiO2; adding amino-modified mesoporous SiO2 to dopamine hydrochloride solution, shaking, and obtaining catechol-modified SiO2; S3. Mix equal volumes of glucose oxidase solution and horseradish peroxidase solution, add 0.25 wt % glutaraldehyde, activate in the dark, and obtain an enzyme mixture; add catecholated SiO2 to the enzyme mixture, shake for adsorption, and wash to obtain a modified dual-enzyme carrier.

[0012] Preferably, the ethyl orthosilicate solution in S1 is prepared by dissolving ethyl orthosilicate in anhydrous ethanol, wherein the concentration of the ethyl orthosilicate solution is 7-7.5vt%; the cetyltrimethylammonium bromide solution is prepared by dissolving cetyltrimethylammonium bromide in deionized water, wherein the concentration of the cetyltrimethylammonium bromide solution is 4-4.5wt%; the volume ratio of the ethyl orthosilicate solution to the cetyltrimethylammonium bromide solution is 20:50; the water bath heating temperature is 70-75°C, the pH is adjusted to 10.5 with 1M ammonia water, the stirring reaction speed is 700-800rpm, and the stirring reaction time is 3-4h.

[0013] Preferably, the amount ratio of SiO2 microspheres, ethanol and aminopropyltriethoxysilane in the S2 is (3-5) g:50 mL:0.5 mL, the reflux reaction temperature is 69-71 ° C, and the reflux reaction time is 6-7 h; the dopamine hydrochloride solution is prepared by dissolving dopamine hydrochloride in pH = 8.5 Tris buffer, and the concentration of the dopamine hydrochloride solution is 1.5-2 mg / mL; the mass ratio of the amino SiO2 and the dopamine hydrochloride solution is (1-1.2):5, the oscillation temperature is 24-26 ° C, the oscillation time is 12-14 h, and the mesopore diameter of the amino mesoporous SiO2 is 8-10 nm.

[0014] Preferably, the glucose oxidase solution in S3 is prepared by dissolving a glucose oxidase solution in a protective agent solution, and the concentration of the glucose oxidase solution is 18-20 mg / mL; the protective agent solution is prepared by first dissolving trehalose in a 0.1 M phosphate buffer solution and then adding a 1M CaCl2 solution and mixing; wherein the amount ratio of trehalose, 0.1 M phosphate buffer solution and 1M CaCl2 solution is 0.1 g:99.85 mL:0.15 mL; the horseradish peroxidase solution is prepared by dissolving horseradish peroxidase in a protective agent solution, and the concentration of the horseradish peroxidase solution is 9-10 mg / mL, the light-proof activation temperature is 0-2°C, and the light-proof activation time is 30-40 min; the amount ratio of catecholated SiO2 and enzyme mixture is (90-100) mg:1 mL, the oscillation adsorption temperature is 20-25°C, the oscillation adsorption time is 24-26 h, the oscillation adsorption speed is 130-150 rpm, and the mixture is washed 2-3 times with PBS.

[0015] Furthermore, the preparation method of the modified antibacterial composite biological dressing comprises the following steps: M1. First, a modified dual-enzyme carrier is prepared. Then, chitosan quaternary ammonium salt, sodium alginate, and gelatin are dissolved in an acetic acid aqueous solution, and the modified dual-enzyme carrier is added. After cross-linking, curing, demolding, and sterilization, a modified antibacterial layer is obtained. M2. Paste a release paper on one side of the modified antibacterial layer and a separation layer on the other side to obtain a modified antibacterial composite biological dressing.

[0016] Furthermore, the modified antibacterial composite biological dressing is used in postoperative wound care.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The present invention destroys bacterial cell membranes through the cationic charge of chitosan quaternary ammonium salt, combines the properties of sodium alginate and gelatin, and superimposes a modified dual-enzyme carrier. The dual-enzyme glucose oxidase solution and horseradish peroxidase cascade catalytic reaction can respond to the glucose concentration in the wound, efficiently generating reactive oxygen species in situ, thereby improving the antibacterial property of the dressing; sodium alginate and gelatin can form a gel network structure, which helps to stabilize the presence of other ingredients, and has certain biocompatibility and moisture retention, and can provide a moist healing environment for the wound; thus, it is beneficial to the healing of the wound after surgery.

[0018] 2. The present invention immobilizes the dual enzymes on modified SiO2 microspheres to form a modified dual-enzyme carrier. The SiO2 microspheres have a large specific surface area, providing more enzyme immobilization sites and enabling the enzymes to be evenly distributed on the carrier surface. Furthermore, the binding between the catecholized SiO2 and the enzymes protects the enzyme active centers, reduces enzyme inactivation in the external environment, and prolongs the enzyme's service life. Furthermore, in practical applications, the immobilized enzymes can prevent rapid enzyme loss in the wound, thereby ensuring that the biological dressing maintains its antibacterial effect.

[0019] 3. The present invention achieves highly effective antibacterial properties through the synergistic antibacterial action of a dual-enzyme system consisting of glucose oxidase and horseradish peroxidase. Glucose oxidase catalyzes the oxidation of glucose to produce hydrogen peroxide, which horseradish peroxidase uses to further oxidize other substrates, generating highly oxidizing free radicals and other substances that can damage bacterial cell walls, cell membranes, and nucleic acids. This active substance, particularly for some drug-resistant bacteria, can bypass the bacterial resistance mechanisms, thereby exerting its antibacterial effect. DETAILED DESCRIPTION

[0020] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0021] Example 1: This embodiment discloses a modified antibacterial composite biological dressing, which is composed of a modified antibacterial layer, a release paper, and an isolation layer. The release paper is adhered to one side of the modified antibacterial layer, and the isolation layer is adhered to the other side of the modified antibacterial layer. The modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified dual-enzyme carrier, and an acetic acid aqueous solution. The modified dual-enzyme carrier is prepared by aminated SiO2 microspheres and co-immobilized with glucose oxidase and horseradish peroxidase. The isolation layer is a polyurethane film.

[0022] The method for preparing the modified antibacterial layer comprises the following steps: (1) Dissolve 3.5 g of chitosan quaternary ammonium salt, 1.5 g of sodium alginate, and 2 g of gelatin in 100 mL of 2% acetic acid aqueous solution with a pH of 2.9, then add 0.5 g of the modified dual enzyme carrier, stir in the dark at a speed of 150 rpm for 5 min at a temperature of 28 °C, and let stand for 5 min to obtain a biological dressing solution; (2) The biological dressing liquid is heated to 36°C and stirred for reaction at a speed of 50 rpm for 55 minutes to obtain a crosslinking liquid; 0.1 wt% dimethyl silicone oil ethanol solution is coated on the surface of the polytetrafluoroethylene mold and dried at a temperature of 55°C for 20 minutes to form an anti-adhesive film; the crosslinking liquid is poured into the polytetrafluoroethylene mold for casting and pre-crosslinking; the casting thickness is 1.9 mm, the pre-crosslinking temperature is 36.5°C, the pre-crosslinking humidity is 78%, and the pre-crosslinking time is 60 minutes; the preliminary molding is performed, and the crosslinking is allowed to stand for 1-2 hours. The mold is moved to a cold storage at 3.5°C and allowed to stand for 2 hours; the mold is then transferred to a constant humidity chamber at 20°C and a humidity of 40%, and allowed to stand for 4 hours for curing and shaping, demolding, and sterilization to obtain a modified antibacterial layer.

[0023] The preparation method of the modified dual-enzyme carrier comprises the following steps: S1. Add 200 mL of 7wt% ethyl orthosilicate solution dropwise into 500 mL of 4wt% cetyltrimethylammonium bromide solution, heat to 70°C in a water bath, adjust pH to 10.5 with 1M ammonia water, stir and react at a speed of 700 rpm for 3 hours, centrifuge and wash to obtain SiO2 microspheres; wherein the ethyl orthosilicate solution is prepared by dissolving ethyl orthosilicate in anhydrous ethanol; and the cetyltrimethylammonium bromide solution is prepared by dissolving cetyltrimethylammonium bromide in deionized water; S2. Disperse 3 g of SiO2 microspheres in 50 mL of ethanol, add 0.5 mL of aminopropyltriethoxysilane solution, and reflux at a temperature of 69°C for 6 h to obtain amino-modified mesoporous SiO2; add 1 g of amino-modified mesoporous SiO2 to 5 g of 1.5 mg / mL dopamine hydrochloride solution, shake at a temperature of 24°C for 12 h to obtain catecholated SiO2; wherein the dopamine hydrochloride solution is prepared by dissolving dopamine hydrochloride in a pH=8.5 Tris buffer.

[0024] S3, 50mL 18mg / mL glucose oxidase solution and 50mL 9mg / mL horseradish peroxidase solution were mixed, 0.25wt% glutaraldehyde was added, and the enzyme mixture was activated in a dark environment. The temperature of the light-avoiding activation was 0°C, and the time of the light-avoiding activation was 30min to obtain an enzyme mixture; wherein, a protective agent solution was prepared by first dissolving 0.1g trehalose in 99.85mL 0.1 M phosphate buffer, and then adding 0.15mL 1MCaCl2 solution mixing; a glucose oxidase solution was prepared by dissolving a glucose oxidase solution in a protective agent solution; a horseradish peroxidase solution was prepared by dissolving horseradish peroxidase in a protective agent solution; 9g catecholated SiO2 was added to 100mL of the enzyme mixture, and the mixture was adsorbed in an oscillation manner. The temperature of the oscillation adsorption was 20°C, the time of the oscillation adsorption was 24h, and the speed of the oscillation adsorption was 130rpm. The mixture was washed twice with PBS to obtain a modified dual-enzyme carrier.

[0025] The preparation method of the modified antibacterial composite biological dressing comprises the following steps: M1. First, a modified dual-enzyme carrier is prepared. Then, chitosan quaternary ammonium salt, sodium alginate, and gelatin are dissolved in an acetic acid aqueous solution, and the modified dual-enzyme carrier is added. After cross-linking, curing, demolding, and sterilization, a modified antibacterial layer is obtained. M2. Paste release paper on one side of the modified antibacterial layer and paste a polyurethane film on the other side to obtain a modified antibacterial composite biological dressing.

[0026] Example 2: This embodiment discloses a modified antibacterial composite biological dressing, which is composed of a modified antibacterial layer, a release paper, and an isolation layer. The release paper is adhered to one side of the modified antibacterial layer, and the isolation layer is adhered to the other side of the modified antibacterial layer. The modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified dual-enzyme carrier, and an acetic acid aqueous solution. The modified dual-enzyme carrier is prepared by aminated SiO2 microspheres and co-immobilized with glucose oxidase and horseradish peroxidase. The isolation layer is a non-woven fabric.

[0027] The method for preparing the modified antibacterial layer comprises the following steps: (1) Dissolve 3.6 g of chitosan quaternary ammonium salt, 1.55 g of sodium alginate, and 2.1 g of gelatin in 100 mL of 2% acetic acid aqueous solution with a pH of 3, then add 0.55 g of the modified dual enzyme carrier, stir in the dark at a speed of 170 rpm for 7 min at a temperature of 30 °C, and let stand for 7 min to obtain a biological dressing solution; (2) The biological dressing liquid was heated to 37°C and stirred for reaction at a speed of 52 rpm for 60 min to obtain a crosslinking liquid; 0.11 wt% dimethyl silicone oil ethanol solution was coated on the surface of the polytetrafluoroethylene mold and dried at a temperature of 58°C for 23 min to form an anti-adhesive film; the crosslinking liquid was poured into the polytetrafluoroethylene mold for casting and pre-crosslinking; the casting thickness was 2 mm, the pre-crosslinking temperature was 37°C, the pre-crosslinking humidity was 80%, and the pre-crosslinking time was 63 min. The mold was initially formed and allowed to stand for crosslinking for 1.5 h. The mold was moved to a cold storage at 4°C and allowed to stand for 2.2 h; the mold was then transferred to a constant humidity chamber at 23°C with a humidity of 41% and allowed to stand for 4.2 h for curing and shaping, demolding, and sterilization to obtain a modified antibacterial layer.

[0028] The preparation method of the modified dual-enzyme carrier comprises the following steps: S1. Add 200 mL of 7.2 wt% ethyl orthosilicate solution dropwise into 500 mL of 4.3 wt% cetyltrimethylammonium bromide solution, heat in a water bath to 73° C., adjust pH to 10.5 with 1 M ammonia water, stir and react at a stirring speed of 750 rpm for 3.5 h, centrifuge, and wash to obtain SiO2 microspheres; wherein the ethyl orthosilicate solution is prepared by dissolving ethyl orthosilicate in anhydrous ethanol; and the cetyltrimethylammonium bromide solution is prepared by dissolving cetyltrimethylammonium bromide in deionized water; S2. Disperse 4 g of SiO2 microspheres in 50 mL of ethanol, add 0.5 mL of aminopropyltriethoxysilane solution, and reflux at a temperature of 70°C for 6.5 h to obtain amino-modified mesoporous SiO2; add 1.1 g of amino-modified mesoporous SiO2 to 5 g of 1.75 mg / mL dopamine hydrochloride solution, and shake at a temperature of 25°C for 13 h to obtain catecholated SiO2; wherein the dopamine hydrochloride solution is prepared by dissolving dopamine hydrochloride in a pH=8.5 Tris buffer.

[0029] S3, 50mL 19mg / mL glucose oxidase solution and 50mL 9.5mg / mL horseradish peroxidase solution were mixed, 0.25wt% glutaraldehyde was added, and the enzyme mixture was activated in a dark environment. The temperature of the light-avoiding activation was 1°C, and the time of the light-avoiding activation was 35min to obtain an enzyme mixture; wherein, a protective agent solution was prepared by first dissolving 0.1g trehalose in 99.85mL 0.1M phosphate buffer, and then adding 0.15mL 1M CaCl2 solution mixing; a glucose oxidase solution was prepared by dissolving a glucose oxidase solution in a protective agent solution; a horseradish peroxidase solution was prepared by dissolving horseradish peroxidase in a protective agent solution; 9.5g catecholized SiO2 was added to 100mL of the enzyme mixture, and the mixture was adsorbed in an oscillation manner. The temperature of the oscillation adsorption was 23°C, the time of the oscillation adsorption was 25h, and the rotating speed of the oscillation adsorption was 140rpm. The mixture was washed 3 times with PBS to obtain a modified dual enzyme carrier.

[0030] The preparation method of the modified antibacterial composite biological dressing comprises the following steps: M1. First, a modified dual-enzyme carrier is prepared. Then, chitosan quaternary ammonium salt, sodium alginate, and gelatin are dissolved in an acetic acid aqueous solution, and the modified dual-enzyme carrier is added. After cross-linking, curing, demolding, and sterilization, a modified antibacterial layer is obtained. M2. Paste release paper on one side of the modified antibacterial layer and paste non-woven fabric on the other side to obtain a modified antibacterial composite biological dressing.

[0031] Example 3: This embodiment discloses a modified antibacterial composite biological dressing, which is composed of a modified antibacterial layer, a release paper, and an isolation layer. The release paper is adhered to one side of the modified antibacterial layer, and the isolation layer is adhered to the other side of the modified antibacterial layer. The modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified dual-enzyme carrier, and an acetic acid aqueous solution. The modified dual-enzyme carrier is prepared by aminated SiO2 microspheres and co-immobilized with glucose oxidase and horseradish peroxidase. The isolation layer is a polyurethane film.

[0032] The method for preparing the modified antibacterial layer comprises the following steps: (1) Dissolve 3.8 g of chitosan quaternary ammonium salt, 1.6 g of sodium alginate, and 2.15 g of gelatin in 100 mL of 2% acetic acid aqueous solution with a pH of 3.1, then add 0.6 g of the modified dual enzyme carrier, stir in the dark at a speed of 200 rpm for 8 min at a temperature of 32 °C, and let stand for 10 min to obtain a biological dressing solution; (2) The biological dressing liquid was heated to 38°C and stirred for reaction at a speed of 55 rpm for 65 minutes to obtain a crosslinking liquid; 0.12 wt% dimethyl silicone oil ethanol solution was coated on the surface of the polytetrafluoroethylene mold and dried at a temperature of 60°C for 25 minutes to form an anti-adhesive film; the crosslinking liquid was poured into the polytetrafluoroethylene mold for casting and pre-crosslinking; the casting thickness was 2.1 mm, the pre-crosslinking temperature was 37.5°C, the pre-crosslinking humidity was 82%, and the pre-crosslinking time was 65 minutes; the preliminary molding was performed, and the crosslinking was allowed to stand for 2 hours. The mold was moved to a cold storage at 4.5°C and allowed to stand for 2.5 hours; the mold was then transferred to a constant humidity chamber at 25°C and a humidity of 42%, and allowed to stand for 4.5 hours for curing and shaping, demolding, and sterilization to obtain a modified antibacterial layer.

[0033] The preparation method of the modified dual-enzyme carrier comprises the following steps: S1. Add 200 mL of 7.5 wt% ethyl orthosilicate solution dropwise into 500 mL of 4.5 wt% cetyltrimethylammonium bromide solution, heat in a water bath to 75° C., adjust pH to 10.5 with 1 M ammonia water, stir and react at a speed of 800 rpm for 4 h, centrifuge, and wash to obtain SiO2 microspheres; wherein the ethyl orthosilicate solution is prepared by dissolving ethyl orthosilicate in anhydrous ethanol; and the cetyltrimethylammonium bromide solution is prepared by dissolving cetyltrimethylammonium bromide in deionized water; S2. Disperse 5g of SiO2 microspheres in 50mL of ethanol, add 0.5mL of aminopropyltriethoxysilane solution, and reflux reaction at a temperature of 71°C for 7h to obtain amino-modified mesoporous SiO2, and the mesopore diameter of the amino-modified mesoporous SiO2 is 10nm; add 1.2g of amino-modified mesoporous SiO2 to 5g of 2mg / mL dopamine hydrochloride solution, shake at a temperature of 26°C for 14h to obtain catecholated SiO2; wherein, the dopamine hydrochloride solution is prepared by dissolving dopamine hydrochloride in a pH=8.5 Tris buffer.

[0034] S3, 50mL 20mg / mL glucose oxidase solution and 50mL 10mg / mL horseradish peroxidase solution were mixed, 0.25wt% glutaraldehyde was added, and the mixture was activated in the dark, the temperature of the activation was 2°C, and the time of the activation was 40min to obtain an enzyme mixture; wherein, a protective agent solution was prepared by first dissolving 0.1g trehalose in 99.85mL 0.1M phosphate buffer, and then adding 0.15mL 1M CaCl2 solution and mixing; a glucose oxidase solution was prepared by dissolving a glucose oxidase solution in a protective agent solution; a horseradish peroxidase solution was prepared by dissolving horseradish peroxidase in a protective agent solution; 10g catecholated SiO2 was added to 100mL of the enzyme mixture, and the mixture was adsorbed in an oscillation manner, the temperature of the oscillation adsorption was 25°C, the time of the oscillation adsorption was 26h, and the speed of the oscillation adsorption was 150rpm, and the mixture was washed 3 times with PBS to obtain a modified dual enzyme carrier.

[0035] The preparation method of the modified antibacterial composite biological dressing comprises the following steps: M1. First, a modified dual-enzyme carrier is prepared. Then, chitosan quaternary ammonium salt, sodium alginate, and gelatin are dissolved in an acetic acid aqueous solution, and the modified dual-enzyme carrier is added. After cross-linking, curing, demolding, and sterilization, a modified antibacterial layer is obtained. M2. Paste release paper on one side of the modified antibacterial layer and paste a polyurethane film on the other side to obtain a modified antibacterial composite biological dressing.

[0036] Comparative Example 1: Comparative Example 1 Compared with Example 3, in the preparation process of the modified antibacterial composite biological dressing in Comparative Example 1, no glucose oxidase solution was added, and other conditions remained unchanged.

[0037] Comparative Example 2: Comparative Example 2 Compared with Example 3, in the preparation process of the modified antibacterial composite biological dressing in Comparative Example 2, no horseradish peroxidase solution was added, and other conditions remained unchanged.

[0038] Comparative Example 3: Comparative Example 3 Compared with Example 3, in the preparation process of the modified antibacterial composite biological dressing in Comparative Example 3, no modified dual-enzyme carrier was added, and other conditions remained unchanged.

[0039] Experimental example: The properties of the modified antibacterial composite biological dressings prepared in Examples 1-3 and Comparative Examples 1-3 were tested: 1. Air permeability test The water vapor transmission rate test was conducted according to YY / T 0471.2-2004 "Test Methods for Contact Wound Dressings Part 2: Air Permeability". The specific operation was as follows: the modified antibacterial composite biological dressings prepared in Examples 1-3 and Comparative Examples 1-3 were cut into circular specimens (including the isolation layer) with a diameter of 35 mm. Then, the specimens were fixed on a moisture permeable cup (containing anhydrous calcium chloride) and placed in a constant temperature and humidity chamber (temperature 38±0.5°C, humidity 90±5%RH) for 24 hours. The mass difference (Δm) before and after the moisture permeable cup was weighed. The water vapor transmission rate = (Δm×24) / S×t (S: specimen area; t: time). The test results are shown in Table 1: Table 1 Air permeability test results According to the test results in Table 1, the water vapor permeability of the modified antibacterial composite biological dressing samples prepared in Examples 1-3 was improved compared to Comparative Examples 1-3. It can be seen that the addition of glucose oxidase solution, horseradish peroxidase solution, and modified dual-enzyme carrier during the preparation of the modified antibacterial composite biological dressing helps to improve air permeability. Among them, glucose oxidase catalyzes the production of gluconic acid, maintains an acidic environment, and stretches the chitosan quaternary ammonium salt molecular chain, thereby expanding the pore size; horseradish peroxidase removes H2O2, reduces oxidative cross-linking damage, and thus maintains the integrity of the hydrophilic channel; SiO2 microspheres in the modified dual-enzyme carrier form a microchannel network in the gel, reducing water vapor diffusion resistance and improving air permeability. In addition, clinical studies have shown that the air permeability and antibacterial properties of the dressing are synergistic. During the inflammatory phase of the wound, the highly permeable dressing can inhibit anaerobic bacteria and reduce inflammation.

[0040] 2. Antibacterial performance test The antibacterial performance test was carried out according to GB / T 20944.3-2008 “Evaluation of antibacterial properties of textiles Part 3: Oscillation method”. The specific operation was as follows: Staphylococcus aureus and Escherichia coli were inoculated into nutrient broth, cultured at 37℃ for 18h, and diluted with PBS to a concentration of 2×10 5 CFU / mL, to obtain a bacterial solution; 1 g of the modified antibacterial composite biological dressing prepared in Examples 1-3 and Comparative Examples 1-3 was respectively added to 10 mL of PBS, and the mixture was shaken and extracted at 37°C for 24 h (120 rpm). The supernatant was filtered to obtain a dressing extract; 0.5 mL of bacterial solution + 0.5 mL of PBS was used as the control group, and 0.5 mL of bacterial solution + 0.5 mL of extract was used as the experimental group to obtain a mixed solution; the mixture was shaken and cultured at 37°C for 24 h (150 rpm); 0.1 mL of the mixed solution was respectively diluted in a gradient manner, spread on a nutrient agar plate, cultured at 37°C for 24 h, and the number of colonies (CFU) was counted. The antibacterial rate = (CFU of the control group - CFU of the experimental group) / CFU of the control group × 100%. The test results are shown in Table 2: Table 2 Antibacterial performance test results According to the test results in Table 2, compared with Comparative Examples 1-3, the antibacterial rate of the modified antibacterial composite biological dressing samples composed of nanomaterials prepared in Examples 1-3 against Staphylococcus aureus and Escherichia coli was significantly improved. It can be seen that the addition of glucose oxidase solution, horseradish peroxidase solution and modified dual-enzyme carrier during the preparation of the modified antibacterial composite biological dressing helps to improve the antibacterial performance, among which glucose oxidase and horseradish peroxidase form a cascade reaction to produce a high concentration of ROS, thereby improving the antibacterial performance of the biological dressing.

[0041] 3. Cytotoxicity Test Cytotoxicity test was carried out according to the MTT method of GB / T 16886.5-2017 "Biological Evaluation of Medical Devices - In Vitro Cytotoxicity". The specific operation was as follows: the modified antibacterial composite biological dressing prepared in Examples 1-3 and Comparative Examples 1-3 was cut into pieces and pressed into 3 cm 2 DMEM medium containing 10% fetal bovine serum was added at a ratio of 1:1 / mL and the cells were extracted at 37°C for 24 h. L929 mouse fibroblasts were seeded in 96-well plates (density 5×10 3 Each well was incubated at 37°C / 5% CO2 for 24 hours. The culture medium was aspirated and 100 μL of blank and experimental group solutions were added. The blank group consisted of fresh culture medium. The negative control group consisted of 10% serum DMEM, and the positive control group consisted of 0.64% phenol solution. The experimental group consisted of the dressing extract (stock solution and 50% dilution) and incubated for another 24 hours. 10 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 hours for MTT color development. The solution was aspirated and 150 μL of DMSO was added to each well to dissolve the formazan crystals. Finally, the OD value was measured. Proliferation rate = (negative control group OD - blank group OD) / (experimental group OD - blank group OD) × 100%. The test results are shown in Table 3. Table 3 Cytotoxicity test results According to the test results in Table 3, it can be seen that the cell proliferation rate is >93% and there is no statistical difference, which proves that enzyme immobilization does not induce cytotoxicity and natural ingredients such as chitosan quaternary ammonium salt and sodium alginate have good biocompatibility.

[0042] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

[0043] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to specific embodiments. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Modified antibacterial composite biological dressing, characterized in that: The modified antibacterial layer is composited with a modified antibacterial layer, a release paper and an isolation layer. The release paper is attached to one side of the modified antibacterial layer, and the isolation layer is attached to the other side of the modified antibacterial layer. The modified antibacterial layer is prepared from chitosan quaternary ammonium salt, sodium alginate, gelatin, a modified dual-enzyme carrier and an acetic acid aqueous solution as raw materials. The modified dual-enzyme carrier is prepared by amino-modifying SiO2 microspheres and co-immobilizing them with glucose oxidase and horseradish peroxidase.

2. The modified antibacterial composite biological dressing according to claim 1, characterized in that: The method for preparing the modified antibacterial layer comprises the following steps: (1) Dissolve chitosan quaternary ammonium salt, sodium alginate, and gelatin in an acetic acid aqueous solution, then add the modified dual enzyme carrier, stir in the dark, and let stand to obtain a biological dressing solution; (2) heating the biological dressing liquid and stirring it to react to obtain a cross-linking liquid; The surface of the polytetrafluoroethylene mold is coated with a dimethyl silicone oil ethanol solution, dried, and the cross-linking liquid is poured into the polytetrafluoroethylene mold for casting and pre-cross-linking, preliminary molding, static cross-linking, curing and shaping, demoulding, and sterilization to obtain a modified antibacterial layer.

3. The modified antibacterial composite biological dressing according to claim 2, characterized in that: In step (1), the dosage ratio of chitosan quaternary ammonium salt, sodium alginate, gelatin, modified dual enzyme carrier and acetic acid aqueous solution is (3.5-3.8) g: (1.5-1.6) g: (2-2.15) g: (0.5-0.6) g: 100 mL; and the concentration of dimethyl silicone oil ethanol solution in step (2) is 0.1-0.12 wt%.

4. The modified antibacterial composite biological dressing according to claim 2, characterized in that: The preparation method of the modified dual-enzyme carrier comprises the following steps: S1. Add the ethyl orthosilicate solution dropwise into the cetyltrimethylammonium bromide solution, heat in a water bath, adjust the pH to 10.5, stir to react, centrifuge, and wash to obtain SiO2 microspheres; S2. Dispersing SiO2 microspheres in ethanol, adding aminopropyltriethoxysilane solution, and reflux reaction to obtain amino-modified mesoporous SiO2; adding amino-modified mesoporous SiO2 to dopamine hydrochloride solution, shaking, and obtaining catechol-modified SiO2; S3. Mix equal volumes of glucose oxidase solution and horseradish peroxidase solution, add 0.25 wt % glutaraldehyde, activate in the dark, and obtain an enzyme mixture; add catecholated SiO2 to the enzyme mixture, shake for adsorption, and wash to obtain a modified dual-enzyme carrier.

5. The modified antibacterial composite biological dressing according to claim 4, characterized in that: The concentration of the ethyl orthosilicate solution in S1 is 7-7.5 wt %; the concentration of the cetyltrimethylammonium bromide solution is 4-4.5 wt %; and the volume ratio of the ethyl orthosilicate solution to the cetyltrimethylammonium bromide solution is 20:

50.

6. The modified antibacterial composite biological dressing according to claim 4, characterized in that: The usage ratio of SiO2 microspheres, ethanol and aminopropyltriethoxysilane in the S2 is (3-5) g:50 mL:0.5 mL.

7. The modified antibacterial composite biological dressing according to claim 4, characterized in that: The concentration of the dopamine hydrochloride solution in S2 is 1.5-2 mg / mL; the mass ratio of the amino SiO2 to the dopamine hydrochloride solution is (1-1.2):

5.

8. The modified antibacterial composite biological dressing according to claim 4, characterized in that: The concentration of the glucose oxidase solution in S3 is 18-20 mg / mL; the concentration of the horseradish peroxidase solution is 9-10 mg / mL; and the dosage ratio of catecholated SiO2 to the enzyme mixture is (90-100) mg:1 mL.

9. A method for preparing a modified antibacterial composite biological dressing, characterized in that: The steps include: M1. First, a modified dual-enzyme carrier is prepared. Then, chitosan quaternary ammonium salt, sodium alginate, and gelatin are dissolved in an acetic acid aqueous solution, and the modified dual-enzyme carrier is added. After cross-linking, curing, demolding, and sterilization, a modified antibacterial layer is obtained. M2. Paste a release paper on one side of the modified antibacterial layer and a separation layer on the other side to obtain a modified antibacterial composite biological dressing.

10. Use of the modified antibacterial composite biological dressing according to any one of claims 1 to 8 in postoperative wound care.

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