Enzyme / pH dual-responsive drug-release hydrogel as well as preparation method and application thereof

Abstract

The invention discloses enzyme / pH dual-responsive water-release hydrogel as well as a preparation method and application thereof.According to the enzyme / pH dual-responsive water-release hydrogel and the preparation method and application thereof, lignin which is wide in source and environmentally friendly is subjected to methacrylic acid treatment, lignin with pH response is obtained, and drugs can be released in an alkaline environment, so that premature leakage of the drugs is avoided, and the curative effect of the drugs is powerfully improved; the hyaluronic acid hydrogel disclosed by the invention is a biocompatible and degradable material similar to an extracellular matrix, can be degraded with hyaluronidase secreted by bacteria to release internal nano drug-loaded particles, has specific responsiveness, and can be degraded in a proper place, so that drugs can be controllably released; the bioavailability and the targeting property of the medicine are favorably improved. The enzyme / pH dual-responsive drug-release hydrogel disclosed by the invention has a relatively strong antibacterial property on escherichia coli and staphylococcus aureus, can effectively promote healing of bacterial infected wounds, especially infected wounds in a weakly alkaline environment, and has a satisfactory treatment effect clinically.

Description

Technical Field

[0001] The invention relates to the field of hydrogels, and more specifically to an enzyme / pH dual-responsive drug-releasing hydrogel and a preparation method and application thereof. Background Art

[0002] The skin is an important physical barrier against external stimuli, but it is susceptible to severe damage, such as scalds and burns. When the skin is damaged, it is often accompanied by bacterial infection. Bacterial infection is the second leading cause of death worldwide and is usually treated with antibiotics. However, excessive use of antibiotics can lead to bacterial resistance and reduce the effectiveness of treatment. In recent years, the incorporation of drugs into drug delivery systems to form controllable drug delivery systems has received great attention. This strategy can improve the targeting and activity of antibiotics, reduce side effects, and can inhibit resistance mechanisms by maintaining high and sustained local drug concentrations.

[0003] Some clinical research reports indicate that due to the presence of enzymes and bacteria, the pH value of infected wounds is between 7 and 9, while normal skin is weakly acidic. Due to individual differences and the severity of infection, the pH value of the microenvironment will fluctuate within a certain range, and the binding instability between the drug and the carrier will continue to release the drug even under physiological conditions without bacterial attack. Therefore, a multi-stimulus responsive material can be designed as a drug carrier to construct an "on-demand" drug delivery platform so that the drug can only be released when multiple environmental conditions are met, such as a specific pH value, temperature or enzyme.

[0004] Lignin is the second most abundant biopolymer on Earth after cellulose. It has abundant functional groups such as carboxyl, phenolic hydroxyl, and thiol, and can be chemically modified to control the heterogeneity of the structure. Lignin nanoparticles have the characteristics of high drug loading efficiency, high bioavailability, low cytotoxicity, no hemolytic activity, and sustained drug release. Due to these remarkable properties, researchers have shown great interest in the preparation of lignin nanoparticles and their use in high value-added applications. Lignin nanoparticles as drug carriers and their applications are currently a widely studied area.

[0005] Hyaluronic acid is a negatively charged natural polysaccharide present in the extracellular matrix component of connective tissue. It is biodegradable, non-cytotoxic, has high water absorption and retention capacity, and has considerable activity in cell attachment, proliferation and differentiation. Many bacteria can produce hyaluronidase during their growth and reproduction, so hyaluronic acid can be used as a capping agent to target bacteria and respond to hyaluronidase in the bacterial microenvironment. Summary of the invention

[0006] The first object of the present invention is to provide an enzyme / pH dual responsive drug-releasing hydrogel to solve the technical problem that the existing technology only has responsive characteristics to a single infection environment and does not have dual responses to enzymes and pH.

[0007] The second object of the present invention is to provide a method for preparing an enzyme / pH dual responsive drug-releasing hydrogel.

[0008] The second object of the present invention is to provide an application of an enzyme / pH dual responsive drug-releasing hydrogel.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides an enzyme / pH dual-responsive drug-releasing hydrogel, wherein the dual-responsive hydrogel comprises a pH-responsive nanocarrier and an enzyme-responsive hydrogel, wherein the pH-responsive nanocarrier is composed of methyl methacrylate-modified lignin, and the enzyme-responsive hydrogel is formed by amidation of hyaluronic acid and polyethyleneimine.

[0011] The second aspect of the present invention provides a method for preparing an enzyme / pH dual responsive drug-releasing hydrogel, comprising the following steps:

[0012] S1. Preparation of methacrylated lignin: Under nitrogen atmosphere and a certain temperature, a certain amount of lignin is added into a lithium chloride solution and completely dissolved, and an organic base catalyst and methacrylic anhydride are added at 50° C. The methacrylated lignin is prepared after washing and purification.

[0013] S2. Preparation of pH-responsive nanocarriers: 200 mg of methacrylate lignin, 10 mg of hydrophobic agent and 20 mg of initiator were dissolved in 2 g of chloroform. 20 ml of surfactant aqueous solution was added and stirred at 1000 rpm for 30 min at 25 °C. Then ultrasonic treatment was performed for 3 min under ice cooling (ultrasonication for 20 s, pause for 10 s) to prevent solvent evaporation and polymerization caused by heating. Then 5 ml of a solution consisting of 1.0 wt% SDS, a certain amount of antibiotics and 10 mg of N, N'-methylenebisacrylamide was dripped in. After that, cross-linking polymerization was carried out for 12 h under gentle stirring at 60 °C. Finally, it was dialyzed with water for 24 hours for purification (MWCO = 3500 Da), and stored at 4 °C after freeze-drying.

[0014] S3. Preparation of enzyme / pH dual-responsive drug-releasing hydrogel: Use a certain amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl radicals of hyaluronic acid, stir with drug-loaded nanoparticles and polyethyleneimine for a period of time to form a hydrogel formed by amidation of hyaluronic acid and polyethyleneimine.

[0015] Furthermore, the dissolution temperature of the lignin in step S1 is 60-90°C, preferably 90°C.

[0016] Furthermore, the mass of the lignin in step S1 is 1-5 g.

[0017] Furthermore, in the lithium chloride solution used to dissolve lignin in step S1, the mass percentage concentration of lithium chloride is 2-5wt%, preferably 4wt%.

[0018] Furthermore, the organic base catalyst in step S1 is at least one of diethylamine, diisopropylethylamine, dimethylamine, triethylamine, trimethylamine and triethyltetramine, preferably triethylamine.

[0019] Furthermore, the washing solvent in step S1 is one or a combination of isopropanol or chloroform.

[0020] Furthermore, the hydrophobic agent in step S2 is one of hexadecane, olive oil or palm oil, preferably hexadecane.

[0021] Furthermore, the initiator in step S2 is 2,2-azobis(2-methylpropionitrile).

[0022] Furthermore, the concentration of the surfactant in step S2 is 0.1-2 wt%, preferably 1.0 wt%.

[0023] Furthermore, the surfactant in step S2 is one of SDS, Lutensol AT25 or lecithin, preferably SDS.

[0024] Furthermore, the antibiotic in step S2 is one or two of vancomycin, azithromycin, rifampicin, mupirocin, amoxicillin, enrofloxacin, and fusidic acid.

[0025] Furthermore, the mass of the antibiotic added in step S2 is 100 mg.

[0026] Furthermore, in step S3, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 6:4 to 9:1.

[0027] Furthermore, the mass percentage of polyethyleneimine in step S3 is 1-5%, preferably 2%.

[0028] Furthermore, the stirring time in step S3 is 6 to 12 hours.

[0029] The third aspect of the present invention provides an application of an enzyme / pH dual-responsive drug-releasing hydrogel, characterized in that the drug-releasing hydrogel can be used to kill Escherichia coli or Staphylococcus aureus.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention obtains pH-responsive lignin by methacrylating lignin, which is widely available and environmentally friendly. The lignin can release drugs in an alkaline environment to avoid premature leakage of the drugs, thereby greatly improving the efficacy of the drugs.

[0032] (2) The hyaluronic acid hydrogel of the present invention is a biocompatible and degradable material similar to the extracellular matrix, which can be degraded by hyaluronidase secreted by bacteria to release the nano drug-loaded particles inside. It has specific responsiveness and can be degraded in appropriate places, thereby releasing drugs in a controllable manner, which helps to improve the bioavailability and targeting of drugs.

[0033] (3) The enzyme / pH dual-responsive drug-releasing hydrogel of the present invention has strong antibacterial properties against Escherichia coli and Staphylococcus aureus, and can effectively promote the healing of bacterially infected wounds, especially infected wounds in a weakly alkaline environment. It has a satisfactory therapeutic effect in clinical practice and is of great significance in enhancing drug delivery efficiency and reducing side effects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 1 It is a schematic diagram of the responsive drug release of the hydrogel prepared by the present invention.

[0036] Figure 2 This is the cumulative release curve of the hydrogel prepared in Example 4 after degradation with or without hyaluronidase.

[0037] Figure 3 This is the cumulative release curve of the hydrogel prepared in Example 4 at different pH values.

[0038] Figure 4 This is a comparison chart of the antibacterial properties of the hydrogels prepared in Example 4 and Comparative Example 1.

[0039] Figure 5 The photos are flat plate coating photos of the hydrogels prepared in Example 4 (right picture) and Comparative Example 1 (left picture) after antibacterial treatment. DETAILED DESCRIPTION

[0040] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments, not all of the embodiments. These embodiments are used to illustrate the present invention, not to limit the present invention.

[0041] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the embodiments of the present invention belong. In the event of a conflict, this specification takes precedence.

[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0043] Embodiment 1:

[0044] S1. Dissolve 2 wt% lithium chloride in dimethyl sulfoxide, add 1 g lignin to the solution at 60 ° C under nitrogen atmosphere. After complete dissolution, add 1 mL 10 mmol diethylamine, stir at 50 ° C for 15 min, add 3 ml methacrylic anhydride to the solution, and react at 50 ° C for 20 h. Wash with isopropanol by centrifugation at 5000 rpm for 3 times. Further purify the product by dissolving in chloroform and centrifuging in isopropanol, and dry in an oven at 60 ° C to obtain a brown solid.

[0045] S2. Dissolve 200 mg of methyl methacrylate lignin, 10 mg of olive oil and 20 mg of 2,2-azobis(2-methylpropionitrile) in 2 g of chloroform. Add the solution to 20 mL of 0.1 wt% SDS aqueous solution and stir at 1000 rpm for 30 min at 25 °C. Then ultrasonicate for 3 min (ultrasonicate for 20 s, pause for 10 s) under ice cooling to prevent solvent evaporation and polymerization caused by heating. Then drop 5 ml of a solution consisting of 1.0 wt% SDS, 100 mg of vancomycin and 10 mg of N,N'-methylenebisacrylamide. Then carry out cross-linking polymerization at 60 °C with gentle stirring for 12 h. Finally, dialysis with water for 24 h to purify (MWCO = 3500 Da), freeze-dry and store at 4 °C.

[0046] S3. Dissolve 25mg EDC and 3.6mg NHS in 2mL H2O, add 1mL 10mg / mL hyaluronic acid solution and 1mL 10mg / mL nanocarrier, mix well, and incubate on a shaker for 30min (37°C, 300r / min) to activate the carboxyl free radicals of hyaluronic acid. Subsequently, add 2mL 1% polyethyleneimine to the above solution, stir well and continue incubation for 6h to form a hydrogel formed by amidation of hyaluronic acid and polyethyleneimine, add 10mL H2O and wash three times to effectively remove excess nanocarriers, and store at 4°C for use.

[0047] Embodiment 2:

[0048] S1. Dissolve 4 wt% lithium chloride in dimethyl sulfoxide, add 3 g lignin to the solution at 75 ° C under nitrogen atmosphere. After complete dissolution, add 1 mL 10 mmol diisopropylethylamine at 50 ° C, stir for 15 min, add 3 ml methacrylic anhydride to the solution, and react at 50 ° C for 20 h. Wash with isopropanol by centrifugation at 5000 rpm for 3 times. Further purify the product by dissolving in chloroform and centrifuging in isopropanol, and dry in an oven at 60 ° C to obtain a brown solid.

[0049] S2. Dissolve 200 mg of methyl methacrylate lignin, 10 mg of hexadecane and 20 mg of 2,2-azobis(2-methylpropionitrile) in 2 g of chloroform. Continue to add 20 mL of 1.0 wt% Lutensol AT25 aqueous solution and stir at 1000 rpm for 30 min at 25 °C. Then ultrasonically treat for 3 min (ultrasonication for 20 s, pause for 10 s) under ice cooling to prevent solvent evaporation and polymerization caused by heating. Then drop 5 ml of a solution consisting of 1.0 wt% SDS, 100 mg of antibiotics and 10 mg of N,N'-methylenebisacrylamide. Then carry out cross-linking polymerization for 12 h at 60 °C with gentle stirring. Finally, dialysis with water for 24 hours to purify (MWCO = 3500 Da), freeze-dry and store at 4 °C.

[0050] S3. Dissolve 25mg EDC and 10.7mg NHS in 2mL H2O, add 1mL 10mg / mL hyaluronic acid solution and 1mL 10mg / mL nanocarrier, mix well, and incubate on a shaker for 30min (37°C, 300r / min) to activate the carboxyl free radicals of hyaluronic acid. Subsequently, add 2mL 3% polyethyleneimine to the above solution, stir well and continue incubation for 10h to form a hydrogel formed by amidation of hyaluronic acid and polyethyleneimine, add 10mL H2O and wash three times to effectively remove excess nanocarriers, and store at 4°C for use.

[0051] Embodiment 3:

[0052] S1. Dissolve 5wt% lithium chloride in dimethyl sulfoxide, add 5g lignin to the solution at 90℃ under nitrogen atmosphere. After complete dissolution, add 1mL 10mmol trimethylamine, stir at 50℃ for 15min, add 3ml methacrylic anhydride to the solution, and react at 50℃ for 20h. Wash with isopropanol by centrifugation at 5000rpm for 3 times. Further purify the product by dissolving in chloroform and centrifuging in isopropanol, and dry in an oven at 60℃ to obtain a brown solid.

[0053] S2. Dissolve 200 mg of methyl methacrylate lignin, 10 mg of palm oil and 20 mg of 2,2-azobis(2-methylpropionitrile) in 2 g of chloroform. Add the solution to 20 mL of 2 wt% lecithin aqueous solution and stir at 1000 rpm for 30 min at 25 °C. Then ultrasonicate for 3 min (ultrasonicate for 20 s, pause for 10 s) under ice cooling to prevent solvent evaporation and polymerization caused by heating. Then drop 5 ml of a solution consisting of 1.0 wt% SDS, 100 mg of vancomycin and 10 mg of N,N'-methylenebisacrylamide. Then carry out cross-linking polymerization at 60 °C with gentle stirring for 12 h. Finally, dialysis with water for 24 h to purify (MWCO = 3500 Da), freeze-dry and store at 4 °C.

[0054] S3. Dissolve 24mg EDC and 6mg NHS in 2mL H2O, add 1mL 10mg / mL hyaluronic acid solution and 1mL 10mg / mL nanocarrier, mix well, and incubate on a shaker for 30min (37°C, 300r / min) to activate the carboxyl free radicals of hyaluronic acid. Subsequently, add 2mL 5% polyethyleneimine to the above solution, stir well and continue incubation for 12h to form a hydrogel formed by amidation of hyaluronic acid and polyethyleneimine, add 10mL H2O and wash three times to effectively remove excess nanocarriers, and store at 4°C for use.

[0055] Embodiment 4:

[0056] S1. Dissolve 4 wt% lithium chloride in dimethyl sulfoxide, add 2 g lignin to the solution at 90 ° C under nitrogen atmosphere. After complete dissolution, add 1 mL 10 mmol triethylamine, stir at 50 ° C for 15 min, add 3 ml methacrylic anhydride to the solution, and react at 50 ° C for 20 h. Wash with isopropanol at 5000 rpm for 3 times. Further purify the product by dissolving in chloroform and centrifuging in isopropanol, and dry in an oven at 60 ° C to obtain a brown solid.

[0057] S2. Dissolve 200 mg of methyl methacrylate lignin, 10 mg of hexadecane and 20 mg of 2,2-azobis(2-methylpropionitrile) in 2 g of chloroform. Add the solution to 20 mL of 1.0 wt% SDS aqueous solution and stir at 1000 rpm for 30 min at 25 °C. Then ultrasonically treat for 3 min (ultrasonication for 20 s, pause for 10 s) under ice cooling to prevent solvent evaporation and polymerization caused by heating. Then drop 5 ml of a solution consisting of 1.0 wt% SDS, 100 mg of vancomycin and 10 mg of N,N'-methylenebisacrylamide. Then carry out cross-linking polymerization for 12 h at 60 °C with gentle stirring. Finally, dialysis with water for 24 hours to purify (MWCO = 3500 Da), freeze-dry and store at 4 °C.

[0058] S3. Dissolve 25mg EDC and 3.6mg NHS in 2mL H2O, add 1mL 10mg / mL hyaluronic acid solution and 1mL 10mg / mL nanocarrier, mix well, and incubate on a shaker for 30min (37°C, 300r / min) to activate the carboxyl free radicals of hyaluronic acid. Subsequently, add 2mL 2% polyethyleneimine to the above solution, stir well and continue incubation for 12h to form a hydrogel formed by amidation of hyaluronic acid and polyethyleneimine, add 10mL H2O and wash three times to effectively remove excess nanocarriers, and store at 4°C for use.

[0059] Comparative Example 1: This embodiment differs from Embodiment 4 in that no antibiotics are added in step S2, and the other steps and parameters are the same as those in Embodiment 4.

[0060] Determination of encapsulation efficiency and drug loading rate:

[0061] During the synthesis process of Examples 1 to 4, the supernatant was collected and the OD value at 280 nm was measured; encapsulation efficiency = (amount of antibiotic added - antibiotic mass in supernatant) / antibiotic added; drug loading rate = (amount of antibiotic added - antibiotic mass in supernatant) / hydrogel mass. The results are shown in Table 1.

[0062] Table 1 Encapsulation efficiency and drug loading rate of enzyme / pH dual responsive drug release hydrogel

[0063]

[0064] In vitro antibiotic release experiment:

[0065] Weigh 5 mg of the sample prepared in Example 4, add or not add 150 U / mL hyaluronidase, immerse in 8 mL pH = 7.0 PBS solution, another group first add hyaluronidase degradation for half an hour, then add 8 mL pH = 5.5, 7.0, 8.5 PBS solution, place in an air shaker, adjust the temperature to 37 ° C, and the speed is 120 rpm / min. Take out 2 mL of solution in the system at different set time points for measurement, and supplement with an equal volume of PBS, and use a visible spectrophotometer to measure the absorbance of the collected solution at 280 nm. Record the total drug release time, and draw a drug release curve according to the time and cumulative release amount.

[0066] In vitro antibacterial test:

[0067] Take 1mL of bacterial solution from the Escherichia coli stock solution, add it to a shaking tube containing 4ml LB liquid culture medium, then put it into a constant temperature shaker, adjust the temperature to 37°C, and culture at a speed of 120r / min overnight. Use an enzyme marker to measure the absorbance value at 600nm, dilute the bacterial solution to an OD of 0.5, and add 1mL of the sample prepared in Example 4 and Comparative Example 1. Incubate in a constant temperature shaker, set to 37°C, and the speed is 120r / min. Take out 200μL of liquid at different preset time points and place it in a 96-well plate, use an enzyme marker to measure the absorbance value at 600nm, and draw a bacterial growth curve. After incubation for 24h, take out 200μL of liquid for agar plate coating, place it in a biochemical incubator and continue to culture for 24h, and take pictures to observe the colony.

[0068] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0070] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A method for preparing an enzyme / pH dual-responsive drug-releasing hydrogel, characterized in that: The following steps are involved: S1. Preparation of methacrylated lignin: 1-5 g of lignin was completely dissolved in a 2-5 wt% lithium chloride solution at a temperature of 60-90°C and a nitrogen atmosphere, 1 mL of 10 mmol of triethylamine was added at 50°C, stirred for 15 min, 3 ml of methacrylic anhydride was added to the solution, and reacted at 50°C for 20 h; washed with isopropanol by centrifugation at 5000 rpm for 3 times; the product was further purified by dissolving in chloroform and centrifuging in isopropanol, and dried in an oven at 60°C to obtain a brown solid; S2. Preparation of pH-responsive nanocarriers: 200 mg of methyl methacrylate lignin, 10 mg of hydrophobic agent and 20 mg of initiator were dissolved in 2 g of chloroform; 20 ml of surfactant aqueous solution was added, and stirred at 1000 rpm for 30 min at 25 °C; then ultrasonic treatment was performed for 3 min under ice cooling to prevent solvent evaporation and polymerization caused by heating; then 5 ml of a solution consisting of 0.1-2 wt% SDS, 25-200 mg of antibiotics and 10 mg of N,N'-methylenebisacrylamide was added dropwise; then cross-linking polymerization was performed at 60 °C with gentle stirring for 12 h; finally, it was purified by dialyzing with water for 24 h, and stored at 4 °C after freeze-drying; S3. Preparation of enzyme / pH dual-responsive drug-releasing hydrogel: Add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a mass ratio of 6:4 to 9:1 into the solution, 1mL of 10mg / mL hyaluronic acid solution and 1mL of 10mg / mL nanocarriers, mix well, and incubate on a shaker for 30 minutes to activate the carboxyl free radicals of hyaluronic acid. Subsequently, add 2mL of 1-5% polyethyleneimine to the above solution, stir well and continue incubation for 6 hours to form a hydrogel formed by the amidation of hyaluronic acid and polyethyleneimine. Add 10mL of H2O and wash three times to effectively remove excess nanocarriers, and store at 4°C for use.

2. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S1, the dissolution temperature of the lignin is 60-90°C, preferably 90°C; the mass of the lignin is 1-5g.

3. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S1, the mass percentage concentration of lithium chloride in the lithium chloride solution used to dissolve lignin is 2-5wt%, preferably 4wt%.

4. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S1, the organic base catalyst is at least one of diethylamine, diisopropylethylamine, dimethylamine, triethylamine, trimethylamine and triethyltetramine, preferably triethylamine; and the washing solvent is one or a combination of isopropanol or chloroform.

5. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S2, the hydrophobic agent is one of hexadecane, olive oil or palm oil, preferably hexadecane; and the initiator is 2,2-azobis(2-methylpropionitrile).

6. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S2, the concentration of the surfactant is 0.1-2 wt%, preferably 1.0 wt%; the surfactant is one of SDS, Lutensol AT25 or lecithin.

7. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S2, the antibiotic is one or two of vancomycin, azithromycin, rifampicin, mupirocin, amoxicillin, enrofloxacin, and fusidic acid; and the mass of the added antibiotic is 100 mg.

8. The method for preparing an enzyme / pH dual responsive drug release hydrogel according to claim 1, characterized in that: In step S3, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 6:4-9:1; the mass percentage of polyethyleneimine is 1-5%, preferably 2%.

9. The enzyme / pH dual responsive drug release hydrogel prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The dual-responsive hydrogel comprises a pH-responsive nanocarrier and an enzyme-responsive hydrogel; the pH-responsive nanocarrier is composed of lignin modified by methacrylic acid, the enzyme-responsive hydrogel is formed by amidation of hyaluronic acid and polyethyleneimine, and the loaded drug is an antibiotic.

10. Application of an enzyme / pH dual responsive drug release hydrogel, characterized in that: The drug-releasing hydrogel is used for killing Escherichia coli or Staphylococcus aureus.

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