A cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel, its preparation method and application

Abstract

The present invention discloses a cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel, its preparation method and application. Phenol groups and 5-methylfurfuryl groups are grafted onto natural polysaccharides, and then they are compounded with a quaternary ammonium salt cross-linking agent QMal containing maleimide groups at the ends. Using uric acid as a substrate, under the cascade catalysis of urate oxidase and horseradish peroxidase, phenolic cross-linking, i.e., the first cross-linking, is first achieved. At the same time, the maleimide groups in the quaternary ammonium salt cross-linking agent QMal and the 5-methylfurfuryl groups grafted onto natural polysaccharides achieve the second cross-linking through the Diels-Alder reaction, and finally a double-cross-linked multifunctional natural polysaccharide hydrogel is obtained. The quaternary ammonium salt groups in the natural polysaccharide hydrogel enhance the antibacterial property of the natural polysaccharide hydrogel, the phenolic groups endow the natural polysaccharide hydrogel with excellent antioxidant property, and allantoin, a by-product generated by the catalysis of uric acid by urate oxidase, also has the effects of anti-inflammatory and accelerating wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical multifunctional hydrogel materials, and particularly relates to a cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Wound healing is the inherent ability of biological tissues to self-repair after injury. However, in cases of infection caused by bacteria such as Escherichia coli and Staphylococcus aureus, serious complications may occur. Wound infection triggers the body's immune response, causes inflammation, slows down the healing process, and even requires medical intervention, resulting in increased medical costs. If not treated promptly, the infection may worsen and even lead to sepsis. Therefore, in order to accelerate the entire wound healing process, the use of wound dressings is indispensable. Among various types of wound dressings, hydrogel dressings have unique advantages. Their porous structure is conducive to the transmission of nutrients, can also absorb exudate at the wound site, keep the wound moist, and have shape adaptability, etc. As a good carrier, hydrogels can load antibacterial drugs or transition metal oxides, etc., to achieve specific functions to promote wound healing, but this method will produce side effects such as drug resistance. Traditional free radical polymerization and cross-linking to prepare hydrogels use initiators with certain toxicity and relatively harsh preparation conditions, which are not conducive to subsequent biomedical applications. Therefore, it is of great significance in clinical applications to prepare hydrogel wound dressings with inherent antibacterial, antioxidant, and adhesive properties by a mild method. Summary of the Invention

[0003] The purpose of the present invention is to provide a cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel and a preparation method thereof in view of the deficiencies of existing hydrogel materials such as those loaded with antibacterial drugs. This method first grafts phenol groups and 5-methylfurfuryl groups onto natural polysaccharides, and then composites them with a quaternary ammonium salt cross-linking agent QMal containing maleimide groups at the end. Using uric acid as a substrate, under the cascade catalytic action of uricase and horseradish peroxidase, phenolic cross-linking, i.e., the first cross-linking, is first achieved. At the same time, the maleimide groups in the quaternary ammonium salt cross-linking agent QMal and the 5-methylfurfuryl groups grafted onto the natural polysaccharides achieve the second cross-linking through the Diels-Alder reaction, thereby obtaining a double-cross-linked multifunctional natural polysaccharide hydrogel. Among them, the quaternary ammonium salt groups enhance the antibacterial property of the natural polysaccharide hydrogel, and the phenol groups endow the natural polysaccharide hydrogel with excellent antioxidant properties; in addition, allantoin, a by-product generated by the catalytic oxidation of uric acid by uricase, also has the effects of anti-inflammatory and accelerating wound healing. Therefore, the prepared cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel can be used as a potential medical wound dressing.

[0004] The present invention adopts the following technical solution to solve the above technical problems. A preparation method of a cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel is characterized in that the specific steps are as follows:

[0005] Step S1: Dissolve natural polysaccharide containing amino groups or natural polysaccharide containing carboxyl groups in deionized water to obtain a natural polysaccharide solution containing amino groups or a natural polysaccharide solution containing carboxyl groups, where the natural polysaccharide containing amino groups is chitosan, carboxymethyl chitosan, carboxyethyl chitosan or hydroxyethyl chitosan, and the natural polysaccharide containing carboxyl groups is sodium hyaluronate, sodium alginate or chondroitin sulfate;

[0006] Step S2: Add p-hydroxybenzaldehyde to the natural polysaccharide solution containing amino groups obtained in Step S1, stir and react at room temperature, then add sodium borohydride to the reaction solution and continue to react. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain amino polysaccharide grafted with phenol groups; or add tyramine or tyrosine to the natural polysaccharide solution containing carboxyl groups obtained in Step S1, and graft the phenol groups onto the carboxyl polysaccharide by amidation reaction under the action of a catalyst, where the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain carboxyl polysaccharide grafted with phenol groups;

[0007] Step S3: Dissolve the amino polysaccharide grafted with phenol groups obtained in Step S2 in deionized water, add 5-methylfurfural and stir and react at room temperature, then add sodium borohydride to the reaction solution and continue to react. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain amino polysaccharide grafted with phenol groups and 5-methylfurfuryl groups; or dissolve the carboxyl polysaccharide grafted with phenol groups obtained in Step S2 in deionized water, add 5-methylfurfurylamine, and graft the 5-methylfurfuryl groups onto the carboxyl polysaccharide by amidation reaction under the action of a catalyst, where the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain carboxyl polysaccharide grafted with phenol groups and 5-methylfurfuryl groups;

[0008] Step S4: Dissolve 4-bromomethylbenzaldehyde and N,N,N',N'-tetramethyl-1,3-propanediamine in N,N-dimethylformamide, stir and react at 30~80 °C. After the reaction is completed, precipitate the reaction solution with ether or tetrahydrofuran to obtain a quaternary ammonium salt intermediate product;

[0009] Step S5: Dissolve the quaternary ammonium salt intermediate product obtained in Step S4 in deionized water, and then add 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride to react to obtain a quaternary ammonium salt crosslinking agent QMal solution. The structural formula of this quaternary ammonium salt crosslinking agent QMal is ;

[0010] Step S6: Using an aqueous uric acid solution as a solvent, dissolve the polysaccharide with grafted phenol groups and 5-methylfurfuryl groups or the polysaccharide with grafted phenol groups and 5-methylfurfuryl carboxyl groups obtained in step S3 to obtain a polysaccharide solution with grafted phenol groups and 5-methylfurfuryl groups or a polysaccharide solution with grafted phenol groups and 5-methylfurfuryl carboxyl groups. Then, mix it evenly with the quaternary ammonium salt crosslinking agent QMal solution obtained in step S5. Subsequently, add an uricase solution and a horseradish peroxidase solution in sequence, mix evenly to obtain a gelation precursor solution, and let the gelation precursor solution stand at room temperature to obtain a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

[0011] Further defined, the concentration of the natural polysaccharide solution containing amino groups or the natural polysaccharide solution containing carboxyl groups in step S1 is 20 - 100 mg / mL.

[0012] Further defined, in step S2, the molar ratio of p-hydroxybenzaldehyde to the amino groups in the natural polysaccharide containing amino groups is 0.2 - 2.0:1, and the molar ratio of sodium borohydride to p-hydroxybenzaldehyde is 0.2 - 1.0:1; in step S2, the molar ratio of tyramine or tyrosine to the carboxyl groups in the natural polysaccharide containing carboxyl groups is 0.2 - 2.0:1, and the molar ratio of the catalyst to the carboxyl groups in the natural polysaccharide containing carboxyl groups is 0.2 - 5:1.

[0013] Further defined, in step S3, the molar ratio of 5-methylfurfural to the amino groups in the polysaccharide with grafted phenol groups is 0.2 - 2.0:1, and the molar ratio of sodium borohydride to 5-methylfurfural is 0.2 - 1.0:1; in step S3, the molar ratio of 5-methylfurfurylamine to the carboxyl groups in the polysaccharide with grafted phenol groups is 0.2 - 2.0:1, and the molar ratio of the catalyst to the carboxyl groups in the polysaccharide with grafted phenol groups is 0.2 - 5:1.

[0014] Further defined, in step S4, the molar ratio of 4-bromomethylbenzaldehyde to N,N,N’,N’-tetramethyl-1,3-propanediamine is 2.0 - 4.0:1.

[0015] Further defined, the concentration of the quaternary ammonium salt crosslinking agent QMal solution in step S5 is 5 - 150 mg / mL.

[0016] Further defined, the concentration of the aqueous uric acid solution in step S6 is 1 mg / mL, and the gelation precursor solution stands at 25 - 40 °C for 2 - 720 min to obtain a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

[0017] Further defined, the concentration of the chitosan solution grafted with phenol groups and 5-methylfurfuryl or the carboxymethyl chitosan solution grafted with phenol groups and 5-methylfurfuryl in step S6 is 30-100 mg / mL, the volume ratio of the urate oxidase solution to the chitosan solution grafted with phenol groups and 5-methylfurfuryl or the carboxymethyl chitosan solution grafted with phenol groups and 5-methylfurfuryl is 0.1-0.5:1, the concentration of the urate oxidase solution is 1-10 mg / mL, the volume ratio of the horseradish peroxidase solution to the chitosan solution grafted with phenol groups and 5-methylfurfuryl or the carboxymethyl chitosan solution grafted with phenol groups and 5-methylfurfuryl is 0.02-0.06:1, and the concentration of the horseradish peroxidase solution is 2-20 mg / mL.

[0018] The cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel described in the present invention is characterized by being prepared based on the above method.

[0019] Application of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel described in the present invention in the preparation of biomedical materials.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: The cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel material provided by the present invention adopts a mild enzyme-catalyzed cross-linking strategy, avoiding the harsh reaction conditions of traditional free radical polymerization and cross-linking; at the same time, it contains quaternary ammonium salt groups with antibacterial properties and phenolic groups with antioxidant properties, avoiding the disadvantages such as drug resistance brought by traditional hydrogel materials by loading antibacterial and anti-inflammatory drugs, having a long-term effect on promoting wound healing, and the gel components used are natural polysaccharide polymers, having good biocompatibility and degradability. Description of the Drawings

[0021] Figure 1 It is a scanning electron micrograph of the freeze-dried cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel prepared in Example 1;

[0022] Figure 2 It is the antibacterial property test result of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel prepared in Example 1;

[0023] Figure 3 It is the antioxidant property test result of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel prepared in Example 1;

[0024] Figure 4 It is the HE staining map of the wound tissue on the 14th day of the full-thickness skin wound experiment of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel prepared in Example 1 in mice. Detailed Embodiments

[0025] The above content of the present invention will be further described in detail through the following examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0026] Example 1

[0027] Step S1: Dissolve 1 g of carboxymethyl chitosan in 100 mL of deionized water to obtain a carboxymethyl chitosan solution.

[0028] Step S2: Add 0.6 g of p-hydroxybenzaldehyde to the carboxymethyl chitosan solution obtained in Step S1, stir and react at room temperature for 4 h, then add 0.1 g of sodium borohydride to the reaction solution and continue to react for 12 h. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water for three days, and then freeze-dry to obtain carboxymethyl chitosan containing phenol groups.

[0029] Step S3: Dissolve 0.5 g of the carboxymethyl chitosan grafted with phenol groups obtained in Step S2 in 50 mL of deionized water, add 0.14 g of 5-methylfurfural, stir and react at room temperature for 1 h, then add 0.05 g of sodium borohydride to the reaction solution and continue to react for 12 h. After the reaction is completed, load the reaction solution into a dialysis bag, put it into deionized water for dialysis for three days, and then freeze-dry to obtain carboxymethyl chitosan grafted with phenol groups and 5-methylfurfuryl groups.

[0030] Step S4: Dissolve 0.2 g of 4-bromomethylbenzaldehyde and 0.4 g of N,N,N’,N’-tetramethyl-1,3-propanediamine in N,N-dimethylformamide, stir and react at 60 °C for 12 h. After the reaction is completed, precipitate the reaction solution with ether to obtain a quaternary ammonium salt intermediate product.

[0031] Step S5: Dissolve 60 mg of the quaternary ammonium salt intermediate product obtained in Step S4 in 1 mL of deionized water, and then add 40 mg of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride and react for 1 h to obtain a quaternary ammonium salt crosslinking agent QMal solution.

[0032] Step S6: Use a 1 mg / mL uric acid aqueous solution as a solvent, dissolve 50 mg of the carboxymethyl chitosan grafted with phenol groups and 5-methylfurfuryl groups obtained in Step S3 in 1 mL of uric acid aqueous solution, then mix it with the quaternary ammonium salt crosslinking agent QMal solution obtained in Step S5 in an equivalent amount of 5-methylfurfuryl groups and maleimide groups, and then add 150 μL of uricase aqueous solution (1 mg / mL) and 50 μL of horseradish peroxidase aqueous solution (2 mg / mL) and mix well. The resulting gel precursor solution is allowed to stand at 37 °C for 300 min to obtain a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

[0033] Example 2

[0034] Step S1: Dissolve 1 g of sodium hyaluronate in 50 mL of deionized water to obtain a sodium hyaluronate solution;

[0035] Step S2: Add 0.5 g of tyramine and 1.0 g of 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride to the sodium hyaluronate solution obtained in Step S1, stir and react at room temperature for 12 h. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water for three days, and then lyophilize to obtain sodium hyaluronate grafted with phenol groups;

[0036] Step S3: Dissolve 0.5 g of the sodium hyaluronate grafted with phenol groups obtained in Step S2 in 50 mL of deionized water, add 0.15 g of 5-methylfurfurylamine and 0.5 g of 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride, stir and react at room temperature for 12 h. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water for three days, and then lyophilize to obtain sodium hyaluronate grafted with phenol groups and 5-methylfurfuryl groups;

[0037] Step S4: Dissolve 0.4 g of 4-bromomethylbenzaldehyde and 0.4 g of N,N,N’,N’-tetramethyl-1,3-propanediamine in N,N-dimethylformamide, stir and react at 40 °C for 24 h. After the reaction is completed, precipitate the reaction solution with ether to obtain a quaternary ammonium salt intermediate product;

[0038] Step S5: Dissolve 60 mg of the quaternary ammonium salt intermediate product obtained in Step S4 in 1 mL of deionized water, add 40 mg of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride and react for 1 h to obtain a quaternary ammonium salt crosslinker QMal solution;

[0039] Step S6: Use a 1 mg / mL uric acid aqueous solution as a solvent, dissolve 60 mg of the sodium hyaluronate grafted with phenol groups and 5-methylfurfuryl groups obtained in Step S3 in 1 mL of the uric acid aqueous solution, then mix it with the quaternary ammonium salt crosslinker QMal solution obtained in Step S5 in an equivalent amount of 5-methylfurfuryl groups and maleimide groups, then add 160 μL of an uricase aqueous solution (1 mg / mL) and 40 μL of a horseradish peroxidase aqueous solution (2 mg / mL), and after mixing evenly, the resulting gel precursor solution is allowed to stand at 37 °C for 600 min to obtain a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

[0040] To illustrate the various properties of the cascade enzyme-catalyzed crosslinked chitosan hydrogel provided by the present invention, the cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in Example 1 was tested, and the test results are shown in Figures 1 to 4 .

[0041] Figure 1Scanning electron micrograph of the freeze-dried cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in Example 1. As can be observed from the figure, a three-dimensional through-network skeleton structure is present.

[0042] Figure 2 Antibacterial performance test results of the cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in Example 1. As shown in the figure, compared with the blank control group, the prepared cascade enzyme-catalyzed crosslinked chitosan hydrogel dressing exhibits excellent antibacterial performance against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus.

[0043] Figure 3 Antioxidant test results of the cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in Example 1. As shown in the figure, the prepared cascade enzyme-catalyzed crosslinked chitosan hydrogel dressing shows good scavenging ability for hydroxyl radicals.

[0044] Figure 4 HE staining image of the wound tissue on the 14th day of the full-thickness skin wound experiment in mice using the cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in Example 1. Compared with the blank control group, the group with the prepared cascade enzyme-catalyzed crosslinked chitosan hydrogel dressing has more pores, blood vessels, and other relatively complete skin tissues and appendages. The newly formed epidermis is close to the thickness of the normal skin epidermis, demonstrating that the prepared multifunctional hydrogel dressing has good wound-healing promotion performance.

[0045] In summary, the cascade enzyme-catalyzed crosslinked chitosan hydrogel prepared in the present invention exhibits good antibacterial, antioxidant and other properties, and in the full-thickness skin wound experiment in mice, it can promote the regeneration of relatively mature skin tissues and their appendages, thus accelerating wound healing.

[0046] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A preparation method of a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel, characterized in that The specific steps are as follows: Step S1: Dissolve natural polysaccharide containing amino groups or natural polysaccharide containing carboxyl groups in deionized water to obtain a natural polysaccharide solution containing amino groups or a natural polysaccharide solution containing carboxyl groups, wherein the natural polysaccharide containing amino groups is chitosan, carboxymethyl chitosan, carboxyethyl chitosan or hydroxyethyl chitosan, and the natural polysaccharide containing carboxyl groups is sodium hyaluronate, sodium alginate or chondroitin sulfate; Step S2: Add p-hydroxybenzaldehyde to the natural polysaccharide solution containing amino groups obtained in Step S1, stir and react at room temperature, then add sodium borohydride to the reaction solution to continue the reaction. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain amino polysaccharide grafted with phenol groups; or add tyramine or tyrosine to the natural polysaccharide solution containing carboxyl groups obtained in Step S1, and graft the phenol groups onto the carboxyl polysaccharide by amidation reaction under the action of a catalyst, wherein the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain carboxyl polysaccharide grafted with phenol groups; Step S3: Dissolve the amino polysaccharide grafted with phenol groups obtained in Step S2 in deionized water, add 5-methylfurfural and stir and react at room temperature, then add sodium borohydride to the reaction solution to continue the reaction. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain amino polysaccharide grafted with phenol groups and 5-methylfurfuryl groups; or dissolve the carboxyl polysaccharide grafted with phenol groups obtained in Step S2 in deionized water, add 5-methylfurfurylamine, and graft the 5-methylfurfuryl groups onto the carboxyl polysaccharide by amidation reaction under the action of a catalyst, wherein the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 4-(4,6-dimethoxytriazin)-4-methylmorpholine hydrochloride. After the reaction is completed, load the reaction solution into a dialysis bag, dialyze in deionized water and then freeze-dry to obtain carboxyl polysaccharide grafted with phenol groups and 5-methylfurfuryl groups; Step S4: Dissolve 4-bromomethylbenzaldehyde and N,N,N’,N’-tetramethyl-1,3-propanediamine in N,N-dimethylformamide, stir and react at 30~80 °C. After the reaction is completed, precipitate the reaction solution with ether or tetrahydrofuran to obtain a quaternary ammonium salt intermediate product; Step S5: Dissolve the quaternary ammonium salt intermediate obtained in Step S4 in deionized water, and then add 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride for reaction to obtain a quaternary ammonium salt crosslinking agent QMal solution. The structural formula of the quaternary ammonium salt crosslinking agent QMal is ; Step S6: Using an aqueous uric acid solution as a solvent, dissolve the amino polysaccharide grafted with phenol groups and 5-methylfurfuryl groups or the carboxyl polysaccharide grafted with phenol groups and 5-methylfurfuryl groups obtained in Step S3 to obtain an amino polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups or a carboxyl polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups. Then mix it evenly with the quaternary ammonium salt crosslinking agent QMal solution obtained in Step S5, and then sequentially add uricase solution and horseradish peroxidase solution, mix evenly to obtain a gelation precursor solution, and let the gelation precursor solution stand at room temperature to obtain a cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

2. The preparation method of the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel according to claim 1, characterized in that: In step S1, the concentration of the natural polysaccharide solution containing amino groups or the natural polysaccharide solution containing carboxyl groups is 20-100 mg / mL.

3. The preparation method of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel according to claim 1, characterized in that: In step S2, the molar ratio of p-hydroxybenzaldehyde to the amino groups in the natural polysaccharide containing amino groups is 0.2-2.0:1, and the molar ratio of sodium borohydride to p-hydroxybenzaldehyde is 0.2-1.0:1; in step S2, the molar ratio of tyramine or tyrosine to the carboxyl groups in the natural polysaccharide containing carboxyl groups is 0.2-2.0:1, and the molar ratio of the catalyst to the carboxyl groups in the natural polysaccharide containing carboxyl groups is 0.2-5:

1.

4. The preparation method of the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel according to claim 1, wherein: In step S3, the molar ratio of 5-methylfurfural to the amino groups in the amino polysaccharide grafted with phenol groups is 0.2-2.0:1, and the molar ratio of sodium borohydride to 5-methylfurfural is 0.2-1.0:1; in step S3, the molar ratio of 5-methylfurfurylamine to the carboxyl groups in the carboxyl polysaccharide grafted with phenol groups is 0.2-2.0:1, and the molar ratio of the catalyst to the carboxyl groups in the carboxyl polysaccharide grafted with phenol groups is 0.2-5:

1.

5. The preparation method of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel according to claim 1, wherein: In step S4, the molar ratio of 4-bromomethylbenzaldehyde to N,N,N',N'-tetramethyl-1,3-propanediamine is 2.0-4.0:

1.

6. The preparation method of the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel according to claim 1, wherein: In step S5, the concentration of the quaternary ammonium salt crosslinker QMal solution is 5-150 mg / mL.

7. The preparation method of the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel according to claim 1, characterized in that: In step S6, the concentration of the uric acid aqueous solution is 1 mg / mL, and the gelation precursor solution is allowed to stand at 25-40 °C for 2-720 min to obtain the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel.

8. The preparation method of the cascade enzyme-catalyzed cross-linked natural polysaccharide hydrogel according to claim 1, wherein: In step S6, the concentration of the amino polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups or the carboxyl polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups is 30-100 mg / mL, the volume ratio of the uricase solution to the amino polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups or the carboxyl polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups is 0.1-0.5:1, the concentration of this uricase solution is 1-10 mg / mL, the volume ratio of the horseradish peroxidase solution to the amino polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups or the carboxyl polysaccharide solution grafted with phenol groups and 5-methylfurfuryl groups is 0.02-0.06:1, and the concentration of this horseradish peroxidase solution is 2-20 mg / mL.

9. A cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel, characterized in that: The cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel is prepared by the method according to any one of claims 1-8.

10. Use of the cascade enzyme-catalyzed crosslinked natural polysaccharide hydrogel according to claim 9 in the preparation of biomedical materials.

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