An injectable enzyme-crosslinked polysaccharide hydrogel, its preparation method and its application

Abstract

This invention belongs to the field of biomedical materials technology, specifically disclosing an injectable enzymatically cross-linked polysaccharide hydrogel, its preparation method, and its applications. The hydrogel is formed by cross-linking a mixture of polysaccharide, modified polysaccharide, and enzyme, with the following specific weight proportions: polysaccharide 0.5-20%, modified polysaccharide 0.5-20%, enzyme 0.001-0.1%, and the remainder being water. The injectable enzymatically cross-linked polysaccharide hydrogel provided by this invention has high water content and a rich three-dimensional porous structure, providing favorable conditions for cell infiltration, proliferation, and differentiation, as well as angiogenesis in granulation tissue. Furthermore, the modified chitosan imparts hemostatic properties to the hydrogel, and the grafted phenol groups provide antioxidant effects. The injectable enzymatically cross-linked polysaccharide hydrogel provided by this invention is simple to prepare, has good reproducibility, and is not limited by precise operations or instruments. This hydrogel is suitable for the treatment of various types of wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and specifically relates to an injectable enzymatically cross-linked polysaccharide hydrogel, its preparation method, and its application. Background Technology

[0002] The skin, the largest organ in the human body, is a vital protective barrier for maintaining blood balance and plays a crucial role in defending against external attacks. However, injuries such as cuts and burns are unavoidable in daily life, and damage to the integrity or function of skin tissue significantly reduces patients' quality of life. The skin wound healing mechanism is highly complex, involving hemostasis, inflammation, proliferation, wound remodeling, and scar tissue formation. For wound management, especially chronic wounds (such as infected wounds, burn lesions, and diabetic foot ulcers), traditional dressings (such as gauze and cotton) are no longer sufficient for clinical needs. Therefore, various biomaterials have emerged, such as electrospun scaffolds, foams, sponges, and membranes.

[0003] An ideal skin wound dressing needs to possess good tissue compatibility and moisturizing properties, be able to absorb tissue exudate, and also have certain mechanical strength, tissue adhesion, and apparent microstructure to stably exist on the wound surface, prevent external contamination, inhibit bacterial growth, and promote cell adhesion, proliferation, and differentiation. Based on these characteristics, hydrogels have stood out from numerous candidate materials, becoming the most promising skin wound dressings. However, the cross-linking method has a significant impact on the structure and function of hydrogels. Common cross-linking methods are mainly divided into physical cross-linking and chemical cross-linking. For example, ultraviolet light-mediated cross-linking, due to the addition of photoinitiators, can make the hydrogel exhibit certain cytotoxicity.

[0004] Therefore, enzyme-catalyzed, mild cross-linking reactions have become a simple and effective method for preparing bio-friendly hydrogels. Most of the enzymes involved in cross-linking are those that catalyze naturally occurring reactions in the human body and function effectively at neutral pH, in aqueous environments, and at moderate temperatures. Therefore, exploring novel enzyme-catalyzed cross-linked hydrogels is of great significance and holds promise for providing innovative pathways for further applications in tissue engineering. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides an injectable enzyme-linked hydrogel, its preparation method, and its application, which has excellent biocompatibility and overcomes the disadvantages of existing hydrogels, such as toxic side effects caused by crosslinking agents.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an injectable enzymatically crosslinked hydrogel, wherein the hydrogel is formed by crosslinking a mixture of polysaccharide, modified polysaccharide and enzyme, wherein the polysaccharide is a polysaccharide containing galactose side chains, and the modified polysaccharide is a polysaccharide with amino groups, and the specific weight ratios are as follows: 0.5-20% polysaccharide, 0.5-20% modified polysaccharide, 0.001-0.1% enzyme, and the remainder is water.

[0008] Preferably, the polysaccharide includes one or more of mannan, arabinogalactan, arabinoxylan, xyloglucan, and ferula polysaccharide.

[0009] Preferably, the modified polysaccharide is modified chitosan (CPA) grafted with arginine and phenol groups.

[0010] Preferably, the enzymes include galactose oxidase Gox and horseradish peroxidase HRP.

[0011] Secondly, the present invention provides a method for preparing injectable enzymatically catalyzed polysaccharide hydrogels, characterized by comprising the following steps:

[0012] (1) Extracting polysaccharides from polysaccharide raw materials;

[0013] (2) Modified polysaccharides were synthesized in one pot using polysaccharides and grafts as raw materials;

[0014] (3) Mixed polysaccharides, modified polysaccharides and enzyme solutions.

[0015] Preferably, in step (1), 10g of raw material is dissolved in 1000mL of deionized water, the temperature is gradually raised to 40℃, and the mixture is continuously stirred. Then, impurities are removed by centrifugation. The reaction time in the process is 1-48h.

[0016] Preferably, step (2) includes the following steps:

[0017] (1) At room temperature, 1g of an amino polysaccharide is dissolved in an acidic solution to obtain solution A;

[0018] (2) At room temperature, 1 g of 3-(4-hydroxyphenyl)propionic acid was dissolved in an ethanol solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS were added. The mixture was stirred until homogeneous, the pH was adjusted, and the reaction was activated for 0.5 h to obtain solution B.

[0019] (3) At room temperature, 2.11g of L-arginine was dissolved in acetic acid solution, EDC and NHS were added, the mixture was stirred until homogeneous, the pH was adjusted, and the reaction was activated for 2 hours to obtain solution C.

[0020] (4) Add solutions B and C dropwise to solution A in sequence, stir to mix them thoroughly, adjust the pH and couple the reaction for 24 hours; after the reaction is complete, dialyze to remove the reactants and byproducts, and freeze dry to obtain the modified polysaccharide.

[0021] Preferably, step (3) includes the following steps:

[0022] (1) Dissolve the polysaccharide, modified polysaccharide and enzyme;

[0023] (2) Mix the polysaccharide, modified polysaccharide and enzyme in the proportions specified in claim 1 and stir continuously until a gel is formed.

[0024] Preferably, the solvent used in the process is one or more of water, phosphate buffer, Green's solution, and glycerol.

[0025] Secondly, the present invention provides the application of injectable enzymatically catalyzed polysaccharide hydrogels in the preparation of medical wound dressings.

[0026] Preferably, the application includes dressings for mechanically damaged wounds and ulcerative wounds.

[0027] In a specific implementation, the hydrogel is formed by mixing and stirring xylo-glucan (XG), modified chitosan (CPA) grafted with arginine and phenol groups, galactose oxidase (Gox), and horseradish peroxidase (HRP).

[0028] In this process, under the action of Gox, the galactose group in the XG structure is oxidized to generate aldehyde group and releases H2O2. Under the action of H2O2 and HRP, the phenol group on the modified chitosan undergoes covalent cross-linking. The aldehyde group in XG and the amino group on the modified chitosan form a dynamic Schiff base bond, thereby forming a double cross-linked and dynamic covalent network, which endows the hydrogel with self-healing function and can be injected for use.

[0029] The purpose of this invention is to provide the application of injectable enzymatic polysaccharide hydrogels in the preparation of medical wound dressings; the applications include dressings for mechanically damaged wounds and ulcerous wounds.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) This invention provides a novel enzymatic crosslinking method for preparing hydrogels in a one-pot process. The substrate is mixed evenly and then stirred magnetically to form a gel. No heating or other conditions are required, and no additional crosslinking agents or chemical components, such as H2O2, are added. The preparation is simple and beneficial to improving the biocompatibility of hydrogels.

[0032] (2) The injectable enzymatic polysaccharide hydrogel provided by this invention has high water content and a rich three-dimensional porous structure, which can provide favorable conditions for cell infiltration, proliferation and differentiation, as well as blood vessel germination in granulation tissue. On the other hand, the modified chitosan endows the hydrogel with hemostatic properties, and the grafted phenol groups can play an antioxidant role. In addition, the hydrogel has good biocompatibility. A series of in vitro biological evaluations, including cytotoxicity experiments, have proven that the hydrogel has good biocompatibility and is non-irritating to human skin tissue. The injectable enzymatic polysaccharide hydrogel provided by this invention is simple to prepare, has good reproducibility, and is not limited by delicate operation or instruments. This hydrogel is suitable for the treatment of various wounds. Attached Figure Description

[0033] The invention will now be further described with reference to the accompanying drawings.

[0034] Figure 1 This is a schematic diagram of the gelation time in Example 3 of the present invention;

[0035] Figure 2 This is a cross-sectional scanning electron microscope image of Embodiment 3 of the present invention;

[0036] Figure 3 The rheological performance characterization diagrams for Embodiment 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention are respectively the rheometer time scan result, rheometer strain scan result, rheometer "three-platform" scan result, and rheometer strain alternating scan result.

[0037] Figure 4 These are illustrations of the antioxidant effects of Embodiment 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention.

[0038] Figure 5 Cell compatibility characterization of Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention;

[0039] Figure 6 This is a diagram illustrating the hemostatic performance of Embodiment 3 of the present invention;

[0040] Figure 7 The images shown are typical photographs of wound healing on days 0, 3, 7, 14, and 21, and statistical images of wound contraction area for each group, as shown in Example 3 of this invention. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the embodiments.

[0042] Example 1: Preparation of xylo-glucan (XG)

[0043] 10g of tamarind powder was dissolved in 1000mL of deionized water, and the solution was gradually heated to 40℃ and stirred continuously for 24 hours to obtain a homogeneous solution. Then, the solution was centrifuged at 8000 rpm for 10 minutes to remove insoluble impurities. The supernatant was retained after removing the impurities. The supernatant was frozen at -80℃ and then freeze-dried. The spongy solid obtained after freeze-drying was xyloglucan, named XG.

[0044] Example 2: Preparation of Modified Chitosan (CPA)

[0045] At room temperature, 1g of chitosan (CS) is dissolved in 100ml of 1% (v / v) acetic acid solution and magnetically stirred until completely dissolved to obtain a 1wt% CS solution, which is solution A.

[0046] At room temperature, 1 g of 3-(4-hydroxyphenyl)propionic acid was dissolved in 20 ml of 50% (v / v) ethanol solution, and 0.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.3 g of N-hydroxysuccinimide (NHS) were added. The mixture was stirred until homogeneous, and the pH was adjusted to around 5 using 1 M HCl. The reaction was activated at 25 °C for 0.5 h to obtain solution B.

[0047] At room temperature, 2.11 g of L-arginine was dissolved in a 1% (v / v) acetic acid solution, 3.49 g of EDC and 1.15 g of NHS were added, and the mixture was stirred until homogeneous. The pH was adjusted to around 5 using 1 M HCl, and the reaction was activated at 25 °C for 2 h to obtain solution C.

[0048] Solution B and solution C were added dropwise to solution A in sequence, and stirred to mix thoroughly. The pH was adjusted to 5-6 using 1M HCl, and the temperature was 25℃. The coupling reaction was carried out for 24 hours. After the reaction was completed, the reactants and byproducts were removed by dialysis, and the chitosan (CPA) modified and grafted with arginine and phenol groups was obtained by freeze drying.

[0049] Example 3: Xylglucan / modified chitosan enzymatic polysaccharide hydrogel

[0050] Prepare a 3 wt% xyloglucan solution in deionized water; prepare a 4 wt% modified chitosan solution in deionized water; prepare a 16 U / ml Gox enzyme solution and a 48 U / ml HRP enzyme solution. Mix 2 mL of xyloglucan solution, 1 mL of modified chitosan solution, 0.25 mL of deionized water, 0.5 mL of Gox enzyme solution, and 0.25 mL of HRP enzyme solution, and stir magnetically for a certain period of time to gel. Name this hydrogel XG3 / CPA2 hydrogel.

[0051] Specifically, XG, CPA, Gox, and HRP were dissolved in deionized water to prepare solutions of 3wt%, 2wt%, 16U / ml, and 48U / ml, respectively; the components were added to brown sample vials in proportion and formed into gels under magnetic stirring.

[0052] Example 4

[0053] An injectable enzymatically catalyzed polysaccharide hydrogel is provided, wherein the hydrogel is formed by cross-linking a mixture of xyloglucan, modified chitosan grafted with phenol groups and arginine, and Gox and HRP enzymes, with the following specific weight proportions: xyloglucan 0.5-20%, modified chitosan 0.5-20%, Gox enzyme 0.001-0.1%, HRP enzyme 0.001-0.1%, and the remainder being water; wherein the modified chitosan is bonded to xyloglucan through Schiff base bonds, and the phenol groups in the modified chitosan structure are bonded through enzymatic reactions to form C-C or CO covalent bonds.

[0054] The aforementioned injectable enzymatic polysaccharide hydrogel can be used in the preparation of medical wound dressings, including dressings for mechanically damaged wounds and ulcerous wounds.

[0055] Example 5

[0056] An injectable enzymatically catalyzed polysaccharide hydrogel is disclosed, comprising a mixture of polysaccharide, modified polysaccharide, and enzyme, cross-linked together. The specific weight percentages are as follows: 0.5-20% polysaccharide, 0.5-20% modified polysaccharide, 0.001-0.1% enzyme, with the remainder being water. The polysaccharide is a polysaccharide containing galactose side chains, and the modified polysaccharide is a polysaccharide containing amino groups. The polysaccharide includes one or more of mannan, arabinogalactan, arabinoxylan, xyloglucan, and *Ferula africana* polysaccharide; the modified polysaccharide is chitosan (CPA) grafted with arginine and phenol groups; and the enzyme is galactose oxidase (Gox) and horseradish peroxidase (HRP).

[0057] Comparative Example 1: Preparation of xyloglucan / modified chitosan enzymatic polysaccharide hydrogel

[0058] Prepare a 4 wt% xyloglucan solution in deionized water; prepare a 4 wt% modified chitosan solution in deionized water; prepare a 16 U / ml Gox enzyme solution and a 48 U / ml HRP enzyme solution. Mix 2 mL of xyloglucan solution, 1 mL of modified chitosan solution, 0.25 mL of deionized water, 0.5 mL of Gox enzyme solution, and 0.25 mL of HRP enzyme solution, and stir magnetically for a certain period of time to gel. Name this hydrogel XG4 / CPA2 hydrogel.

[0059] Comparative Example 2: Preparation of xyloglucan / modified chitosan enzymatic polysaccharide hydrogel

[0060] Prepare a 4 wt% xyloglucan solution in deionized water; prepare a 4 wt% modified chitosan solution in deionized water; prepare a 16 U / ml Gox enzyme solution and a 48 U / ml HRP enzyme solution. Mix 2 mL of xyloglucan solution, 0.5 mL of modified chitosan solution, 0.75 mL of deionized water, 0.5 mL of Gox enzyme solution, and 0.25 mL of HRP enzyme solution, and stir magnetically for a certain period of time to gel. Name this hydrogel XG4 / CPA1 hydrogel.

[0061] Comparative Example 3: Preparation of xyloglucan / modified chitosan enzymatic polysaccharide hydrogels

[0062] Prepare a 3 wt% xyloglucan solution in deionized water; prepare a 4 wt% modified chitosan solution in deionized water; prepare a 16 U / ml Gox enzyme solution and a 48 U / ml HRP enzyme solution. Mix 2 mL of xyloglucan solution, 0.5 mL of modified chitosan solution, 0.75 mL of deionized water, 0.5 mL of Gox enzyme solution, and 0.25 mL of HRP enzyme solution, and stir magnetically for a certain period of time to gel. Name this hydrogel XG3 / CPA1 hydrogel.

[0063] Test Example 1

[0064] The chemical structures of Examples 2 and 3 were tested. The rheological properties, antioxidant properties, biocompatibility and hemostatic properties of the hydrogels obtained in Examples 3, Comparative Examples 1, 2 and 3 were tested. The therapeutic effect of Example 3 on chronic diabetic wounds was also tested.

[0065] Test Example 2: Scanning Electron Microscopy Observation

[0066] Example 3 was observed using a scanning electron microscope, and the results are as follows: Figure 2 As shown, the XG3 / CPA2 hydrogel has a stable and clear pore structure, which is conducive to cell migration and proliferation and promotes normal material exchange between cells and tissues.

[0067] Test Example 3 Rheological Test

[0068] Rheological tests were performed on the hydrogels obtained in Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 using a rheometer equipped with a 20 mm parallel plate. Time-scan testing (300 s, 1% strain, 10 rad / s frequency) and strain scan (1%-

[0069] The following tests were conducted: 1000% strain at 10 rad / s frequency; a "three-plateau" scan (10% strain at 10 rad / s and 50 rad / s frequencies for 1.5 cycles, lasting 80 s); and an alternating time scan (1% and 1000% strain for 3.5 cycles, lasting 60 s and 30 s respectively, at a frequency of 10 rad / s). Each experiment used 300 μL of hydrogel, was conducted at 25 °C, and had a gap of 500 μm. Results are as follows: Figure 3 As shown in the figure. Time-scan results show that the storage modulus (G') of all hydrogels consistently exceeded the loss modulus (G"), indicating that a stable hydrogel network was formed in each group. Among them, the XG3 / CPA1 hydrogel exhibited the lowest G', at 198.3±10.2 Pa, while the XG4 / CPA2 hydrogel exhibited the highest G', at 595.5±30.5 Pa. This indicates that as the polysaccharide concentration increases, the modulus of the hydrogel increases accordingly, improving its mechanical strength and making it less susceptible to damage from external forces, thus adapting to more practical applications. When the applied stress exceeds the fracture stress (1000%), the hydrogel transitions from a gel state to a sol state. When the stress recovers to 1%, the modulus recovers to [the required value]. The hydrogel returned to its original state and then transformed from a sol state to a gel state again. This indicates that the XG3 / CPA2 hydrogel possesses self-healing properties. Through a "three-plateau" scanning test with varying shear frequencies, within the range of 0–80 s, the hydrogel's shear frequency was 10 rad / s, and its viscosity was 89.9 ± 4.6 Pa·s. A sudden increase in the shear frequency to 500 rad / s caused the hydrogel's viscosity to instantly decrease to 14.4 ± 1.3 Pa·s; subsequently, a sudden decrease in the shear frequency to 10 rad / s restored the hydrogel's viscosity to its initial value of 91.3 ± 3.3 Pa·s, simulating the extrusion process of the hydrogel from a syringe needle and further verifying the hydrogel's injectability.

[0070] Test Example 4 Antioxidant Test

[0071] The hydrogels prepared in Examples 2, 1, 2, and 3 were each immersed in anhydrous ethanol (300 μL each), followed by the addition of 100 μL of 0.5 mM 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH·) solution, and allowed to stand for 1 h and 24 h, respectively. The absorbance at approximately 520 nm was then measured using a UV-Vis spectrophotometer. The free radical scavenging rate was calculated using the following formula:

[0072]

[0073] Among them, A B A HThe absorbance at 520 nm is for the blank group (DPPH·free radical + ethanol) and the hydrogel group (DPPH·free radical + ethanol + hydrogel), respectively.

[0074] XG / CPA hydrogels both exhibited good free radical scavenging capabilities, such as Figure 4 Within 24 hours, the scavenging rate of DPPH free radicals in each group of hydrogels was over 75%.

[0075] Test Example 5: Cell Compatibility Test

[0076] Hydrogels from Examples 2, 1, 2, and 3 were prepared under aseptic conditions and sterilized under UV light for 2 hours. 1 mL of the hydrogel was placed in 10 mL of DMEM complete medium and incubated at 37°C for 24 hours. L929 cells were collected and divided into 3 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in a 96-well plate. After 24 hours, the medium in the wells was replaced with 0.1 mL of filtered extract. On days 1, 2, and 3 of co-culture, the medium in the wells was aspirated, and 10 μL of CCK-8 reagent and 90 μL of cell culture medium were added. After incubation in a cell culture incubator for 1 hour, the absorbance was measured at 450 nm. Cell viability was calculated as follows:

[0077]

[0078] At, A0, and Ac represent the absorbance of the hydrogel group, the cck-8 blank group, and the control group, respectively.

[0079] The cell viability of all hydrogel cells was greater than 100%. Figure 5 This indicates that all hydrogels exhibited good cell compatibility.

[0080] Test Example 6: Liver Hemostasis Performance Test

[0081] First, via intraperitoneal injection Fifty male ICR mice (20-25g) were anesthetized and fixed on a vertical operating table. The abdominal cavity of the mice was opened, the liver lobe was located, and the surrounding tissue fluid was drained with gauze. A pre-weighed sheet of filter paper was placed under the liver, and a 3mm incision was made in the liver with a scalpel. Immediately afterward, 0.2mL of the hydrogel obtained in Example 3 was injected into the incision. Untreated wounds were used as a blank control, and blood loss was calculated using filter paper.

[0082] XG3 / CPA2 hydrogel can rapidly stop liver bleeding in mice. Figure 6 Furthermore, the liver blood loss in the control group was approximately one-third of the liver blood loss in the untreated control group.

[0083] Test Example 7: Chronic Wound Test in Diabetic Mice

[0084] A full-thickness skin wound was created on the back of diabetic mice, and the hydrogel prepared in Example 3 was applied to the wound. The wound was compared with that treated mice using commercial excipients; the healing process was photographed and the wound contraction rate was calculated. Figure 7 As shown, by day 14, the wound healing rate in the hydrogel treatment group had exceeded 80%, and by day 21 it was almost completely healed, while the control group still had obvious wounds by day 21.

Claims

1. An injectable enzymatically catalyzed polysaccharide hydrogel, characterized in that: The hydrogel is formed by cross-linking a mixture of polysaccharide, modified polysaccharide, and enzyme. The polysaccharide is a polysaccharide containing galactose side chains, and the modified polysaccharide is a polysaccharide with amino groups. The specific weight ratio is as follows: polysaccharide 0.5-20%, modified polysaccharide 0.5-20%, enzyme 0.001-0.1%, and the remainder is water. The modified polysaccharide is chitosan (CPA) grafted with arginine and phenol groups. The enzymes are galactose oxidase (Gox) and horseradish peroxidase (HRP). A method for preparing injectable enzymatically catalyzed polysaccharide hydrogels includes the following steps: (1) Extracting polysaccharides from raw materials; (2) Modified polysaccharides were synthesized in one pot using polysaccharides and grafts as raw materials; Step (2) includes the following steps: Step 1: Dissolve 1 g of polysaccharide in acetic acid solution at room temperature to obtain solution A; Step 2: At room temperature, dissolve 1g of 3-(4-hydroxyphenyl)propionic acid in an ethanol solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, stir to mix evenly, adjust the pH, and activate the reaction for 0.5h to obtain solution B; Step 3: At room temperature, dissolve 2.11 g of L-arginine in acetic acid solution, add EDC and NHS, stir to mix evenly, adjust pH, activate reaction for 2 hours to obtain solution C; Step 4: Add solution B and solution C dropwise to solution A in sequence, stir to mix thoroughly, adjust pH and couple the reaction for 24 hours; after the reaction is complete, dialyze to remove reactants and byproducts, and freeze-dry to obtain modified polysaccharide; (3) Mixed polysaccharides, modified polysaccharides and enzyme solutions.

2. The injectable enzymatic polysaccharide hydrogel according to claim 1, characterized in that, In step (1), 10 g of raw material is dissolved in 1000 mL of deionized water, the temperature is gradually raised to 40°C, and the mixture is stirred continuously. Then, impurities are removed by centrifugation. The reaction time in the process is 1-48 h.

3. The injectable enzymatic polysaccharide hydrogel according to claim 1, characterized in that, Step (3) includes the following steps: (1) Dissolve the polysaccharide, modified polysaccharide, and enzyme; (2) Mix the polysaccharide, modified polysaccharide and enzyme in the proportions specified in claim 1 and stir until homogeneous.

4. The injectable enzymatic polysaccharide hydrogel according to claim 1, characterized in that, The solvent used in the process is one or more of water, phosphate buffer, Green's solution, and glycerol.

5. The application of the injectable enzymatic polysaccharide hydrogel according to claim 1 in the preparation of medical wound dressings; the application includes dressings for mechanically damaged wounds and ulcerative wounds.

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