Intelligent responsive degradable hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses an intelligent responsive degradable hydrogel, which is characterized by comprising the following components: a base material which is a blend of hyaluronic acid and chitosan and has a total concentration of 2.0%-3.0% w / v; the cross-linking agent is an anti-TNF-alpha monoclonal antibody, and the dosage of the cross-linking agent is 0.8 mu mol / g of the base material; the auxiliary cross-linking agent is genipin with the concentration of 0.1% w / v; the loaded drug is a platelet-derived growth factor, and the drug loading capacity is 100 ng / mg of the hydrogel. The hydrogel can specifically recognize an inflammation signal in a chronic wound high TNF-alpha microenvironment, trigger network degradation and synchronously release a repair promoting medicine, recognition-degradation-administration closed-loop regulation is achieved, and the hydrogel is suitable for diabetic foot ulcer and other hard-to-heal wounds.

Description

A smart responsive biodegradable hydrogel, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a smart responsive biodegradable hydrogel, its preparation method, and its applications. Background Technology

[0002] Wound healing is a dynamic process involving inflammatory, proliferative, and remodeling phases, each with significantly different functional requirements for dressings: during the inflammatory phase, rapid absorption of exudate and inhibition of inflammatory factor spread are necessary; during the proliferative phase, precise release of repair-promoting drugs is required to facilitate granulation tissue formation; and during the remodeling phase, slow degradation of materials is required to avoid tissue residue. Smart hydrogels, with their "microenvironment-responsive triggering" characteristics, have become a research hotspot in the care of chronic wounds (such as diabetic foot ulcers and pressure ulcers). However, existing technologies still have limitations in terms of response specificity, degradation-functional synergy, and the balance between biocompatibility and mechanical properties. Summary of the Invention

[0003] This invention provides a smart responsive biodegradable hydrogel, its preparation method, and its application. The aim is to introduce an anti-tumor necrosis factor-α monoclonal antibody (Anti-TNFα mAb) as a highly specific cross-linking bridge into a hyaluronic acid-chitosan (HA-CS) blend matrix, and combine it with the natural cross-linking agent genipin and the repair-promoting drug platelet-derived growth factor (PDGF) to construct a smart dressing system that can accurately identify the inflammatory marker TNF-α in the microenvironment of chronic wounds, trigger the disintegration of the hydrogel network, and simultaneously release the drug.

[0004] In a first aspect, the present invention provides a smart responsive biodegradable hydrogel comprising the following components:

[0005] The substrate is a blend of hyaluronic acid and chitosan, with a total concentration of 2.0%–3.0% w / v;

[0006] The cross-linking agent is an anti-TNF-α monoclonal antibody, used at a concentration of 0.8 μmol / g substrate;

[0007] The auxiliary crosslinking agent was genipin at a concentration of 0.1% w / v;

[0008] The loaded drug is platelet-derived growth factor, with a drug loading of 50-200 ng / mg hydrogel.

[0009] Preferably, the anti-TNF-α monoclonal antibody is covalently coupled to the carboxyl group on the hyaluronic acid molecular chain via an EDC / NHS-mediated amidation reaction, forming a three-dimensional network structure with the antibody as the cross-linking node. Each antibody molecule is linked to 2 to 3 HA or CS polymer chains. Genipin is added after antibody conjugation and drug loading, reacting with the amino groups of chitosan to form a Schiff base reaction, resulting in a blue-black cross-linked structure that enhances initial mechanical strength without interfering with antibody conformational changes. PDGF is physically dispersed in the HA-CS solution before cross-linking to avoid chemical modification affecting its biological activity.

[0010] When the hydrogel comes into contact with chronic wound exudate, if the local TNF-α concentration is higher than 10 ng / mL, TNF-α molecules specifically bind to the Fab segment of the anti-TNF-α monoclonal antibody, inducing the Fc segment of the antibody to change from a folded to an extended conformation. This conformational change leads to the breakage of covalent bonds between the antibody and HA / CS or rearrangement of disulfide bonds within the antibody, thereby disrupting the cross-linked network structure and triggering hydrogel degradation. Along with network disintegration, PDGF encapsulated in the gel matrix is ​​released synchronously with the gel fragments, achieving dynamic matching of drug release with the wound stage.

[0011] Preferably, the hyaluronic acid has a molecular weight of 50-2000 kDa, and the chitosan has a degree of deacetylation of not less than 85% and a molecular weight of 50-500 kDa.

[0012] Preferably, the blend contains 1.4%–2.1% hyaluronic acid w / v and 0.6%–0.9% chitosan w / v.

[0013] Preferably, the anti-TNF-α monoclonal antibody is a humanized IgG1 antibody, and its binding dissociation constant Kd with TNF-α is 0.05-0.2 nM.

[0014] Preferably, the amount of the anti-TNF-α monoclonal antibody is 0.5-1.0 μmol / g substrate.

[0015] Preferably, the concentration of genipin is 0.05%–0.15% w / v.

[0016] Secondly, the present invention provides a method for preparing a smart responsive biodegradable hydrogel, comprising the following steps:

[0017] Hyaluronic acid and chitosan were dissolved separately in phosphate buffer at pH 7.4 to prepare 1.75% w / v hyaluronic acid solution and 0.75% w / v chitosan solution. After mixing, the mixture was stirred at room temperature for 1 hour to obtain a homogeneous HA-CS blend solution.

[0018] Add 0.1 M of EDC and NHS mixed solution at a molar ratio of 1:1 to the HA-CS blend solution to activate the carboxyl groups on the hyaluronic acid molecules for 30 minutes; then add 0.8 μmol / g of the anti-TNF-α monoclonal antibody of the substrate and stir continuously at 4°C for 2 hours to complete the covalent coupling of antibody and hyaluronic acid.

[0019] Platelet-derived growth factor solution was added to the above conjugate to make the final drug loading 100 ng / mg hydrogel, and the mixture was allowed to stand at room temperature for 30 minutes.

[0020] Add 0.1% w / v genipin aqueous solution dropwise while stirring, and continue cross-linking at room temperature for 2 hours to form a hydrogel with a three-dimensional network structure.

[0021] Preferably, the blending ratio of hyaluronic acid to chitosan is 6:4 to 8:2 by mass; the total molar concentration of EDC / NHS is 0.05-0.2 M; the antibody conjugation reaction temperature is 2°C to 6°C; the reaction time is 1 hour to 3 hours; the standing time after the addition of platelet-derived growth factor is 20 minutes to 60 minutes; the dropping rate of genipin solution is 0.1 mL / min to 0.5 mL / min; and the cross-linking time is 1 hour to 4 hours.

[0022] Thirdly, the present invention provides an application of a smart responsive biodegradable hydrogel in chronic wound dressings, characterized in that the chronic wounds include diabetic foot ulcers, pressure ulcers, venous ulcers, and difficult-to-heal postoperative wounds.

[0023] Benefits and effects:

[0024] The hydrogel of this invention embeds an anti-TNF-α monoclonal antibody directly as a cross-linking unit into the HA-CS network, establishing a direct causal relationship between TNF-α recognition events and cross-linking breakage. This avoids false triggering caused by external interference in traditional pH or temperature response systems. Meanwhile, genipin, as a natural cross-linking agent, enhances initial mechanical properties without introducing cytotoxicity, solving the problem of balancing biocompatibility and mechanical strength. Due to the physical encapsulation strategy of PDGF, its biological activity is preserved, and through the degradation-release coupling mechanism, effective drug delivery is ensured within the proliferative window. This achieves a closed-loop regulation of "recognition-degradation-drug administration," making it suitable for chronic wound scenarios with blurred boundaries and complex microenvironments. Attached Figure Description

[0025] Figure 1 is a SEM image of the hydrogel of Example 1 of the present invention before TNF-α stimulation;

[0026] Figure 2 is a SEM image of the hydrogel of Example 1 of the present invention after TNF-α stimulation;

[0027] Figure 3 is a graph showing the mass loss over 7 days and the degradation rate of TNF-α at 10 ng / mL over 24 hours in the absence of TNF-α for each embodiment and the comparative example of the present invention.

[0028] Figure 4 is a graph showing the antibacterial performance of the various embodiments and comparative examples of the present invention. Detailed Implementation

[0029] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0030] In the description of this specification, the reference to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., means that a specific feature, structure, material, or characteristic described in connection with an implementation or example is included in at least one implementation or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same implementation or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more implementations or examples.

[0031] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0032] As described in the background section above, the microenvironment of chronic wounds is characterized by high concentrations of inflammatory factors (such as TNF-α), high risk of bacterial infection, and weak tissue repair capacity. Traditional hydrogel dressings are difficult to achieve dynamic response and precise drug release. Although there are pH-responsive, temperature-responsive, or enzyme-responsive hydrogels in the existing technology, their response mechanisms are easily interfered with by non-specific stimuli, leading to premature degradation or mis-release of drugs. Furthermore, it is difficult to balance mechanical properties and biocompatibility.

[0033] Based on this, the present invention provides a smart responsive biodegradable hydrogel, its preparation method and application. By directly embedding an anti-TNF-α monoclonal antibody as a crosslinking node into a hyaluronic acid-chitosan blend substrate, and supplementing it with a natural crosslinking agent, genipin, to enhance the initial strength, and simultaneously physically encapsulating the repair-promoting drug PDGF, a closed-loop system is constructed that triggers network disintegration and releases the drug synchronously only when the TNF-α concentration exceeds a threshold.

[0034] In a first aspect, the present invention provides a smart responsive biodegradable hydrogel comprising the following components:

[0035] The substrate is a blend of hyaluronic acid and chitosan, with a total concentration of 2.0%–3.0% w / v;

[0036] The cross-linking agent is an anti-TNF-α monoclonal antibody, used at a concentration of 0.8 μmol / g substrate;

[0037] The auxiliary crosslinking agent was genipin at a concentration of 0.1% w / v;

[0038] The loaded drug is platelet-derived growth factor, with a drug loading of 50-200 ng / mg hydrogel.

[0039] The anti-TNF-α monoclonal antibody is covalently coupled to the carboxyl group on the hyaluronic acid molecular chain through an EDC / NHS-mediated amidation reaction, forming a three-dimensional network structure with the antibody as the cross-linking node. Each antibody molecule is connected to 2 to 3 HA or CS polymer chains. Genipin is added after antibody coupling and drug loading are completed, and reacts with the amino group of chitosan to form a Schiff base reaction, forming a blue-black cross-linked structure, which improves the initial mechanical strength without interfering with the conformational change of the antibody. The PDGF is physically dispersed in the HA-CS solution before cross-linking to avoid the chemical modification affecting its biological activity.

[0040] When the hydrogel comes into contact with chronic wound exudate, if the local TNF-α concentration is higher than 10 ng / mL, the TNF-α molecule specifically binds to the Fab segment of the anti-TNF-α monoclonal antibody, inducing the Fc segment of the antibody to change from a folded conformation to an extended conformation. This conformational change leads to the breakage of the covalent bond between the antibody and HA / CS or the rearrangement of the disulfide bonds inside the antibody, thereby destroying the cross-linked network structure and triggering hydrogel degradation. As the network structure disintegrates, PDGF encapsulated in the gel matrix is ​​released synchronously with the gel fragments, achieving dynamic matching between drug release and wound stage.

[0041] In some embodiments, the hyaluronic acid has a molecular weight of 50-2000 kDa, and the chitosan has a degree of deacetylation of not less than 85% and a molecular weight of 50-500 kDa.

[0042] In some embodiments, the blend contains 1.4%–2.1% hyaluronic acid w / v and 0.6%–0.9% chitosan w / v.

[0043] In some embodiments, the anti-TNF-α monoclonal antibody is a humanized IgG1 antibody with a binding dissociation constant Kd of 0.05-0.2 nM to TNF-α.

[0044] In some embodiments, the amount of the anti-TNF-α monoclonal antibody used is 0.5-1.0 μmol / g substrate.

[0045] In some embodiments, the concentration of genipin is 0.05%–0.15% w / v.

[0046] Secondly, the present invention provides a method for preparing a smart responsive biodegradable hydrogel, comprising the following steps:

[0047] Hyaluronic acid and chitosan were dissolved separately in phosphate-buffered saline (PBS) at pH 7.4 to prepare 1.75% w / v HA solution and 0.75% w / v CS solution. After mixing, the mixture was stirred at room temperature for 1 hour to obtain a homogeneous HA-CS blend solution.

[0048] Add 0.1 M of EDC and NHS mixed solution (molar ratio 1:1) to the HA-CS blend solution to activate the carboxyl group on the HA molecule for 30 minutes; then add 0.8 μmol / g of the anti-TNF-α monoclonal antibody of the substrate and stir continuously at 4°C for 2 hours to complete the covalent coupling of antibody and HA.

[0049] Add PDGF solution to the above coupling product to make the final drug loading 100 ng / mg hydrogel, and let it stand at room temperature for 30 minutes to allow PDGF to be uniformly dispersed in the polymer network;

[0050] Add 0.1% w / v genipin aqueous solution dropwise while stirring, and continue cross-linking at room temperature for 2 hours to form a hydrogel with a three-dimensional network structure.

[0051] In some embodiments, in step S10, the blending ratio of HA to CS is 6:4 to 8:2 (mass ratio).

[0052] In some embodiments, the total molar concentration of EDC / NHS in step S20 is 0.05-0.2 M.

[0053] In some embodiments, in step S20, the antibody conjugation reaction temperature is 2°C to 6°C, and the reaction time is 1 hour to 3 hours.

[0054] In some embodiments, in step S30, the settling time after the addition of PDGF is 20 to 60 minutes.

[0055] In some embodiments, in step S40, the dropping rate of the genipin solution is 0.1 mL / min to 0.5 mL / min, and the crosslinking time is 1 hour to 4 hours.

[0056] Thirdly, the present invention provides the application of the intelligent responsive biodegradable hydrogel in chronic wound dressings, including diabetic foot ulcers, pressure ulcers, venous ulcers, and difficult-to-heal postoperative wounds.

[0057] The following describes embodiments of the present invention. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where no specific technology or conditions are specified in the embodiments, they shall be carried out in accordance with the technology or conditions described in the literature in the field or in accordance with the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0058] Example 1

[0059] S10: Weigh 1.75 g of hyaluronic acid with a molecular weight of 500 kDa (degree of deacetylation 92%) and 0.75 g of chitosan with a molecular weight of 200 kDa (degree of deacetylation 90%), and add them separately to 100 mL of PBS at pH 7.4. Stir magnetically until completely dissolved to obtain a 1.75% w / v HA solution and a 0.75% w / v CS solution. Mix the two solutions and stir at 300 rpm for 1 hour at room temperature to obtain a homogeneous HA-CS blend solution.

[0060] S20: Add 10 mL of an equal volume mixture of 0.1 M EDC and 0.1 M NHS (i.e., the final concentrations of EDC and NHS are each 0.05 M) to the above blend solution, and react at room temperature in the dark for 30 minutes; then add an anti-TNF-α monoclonal antibody (humanized IgG1, Kd=0.1 nM) solution to make its final concentration 0.8 μmol / g substrate (based on a total polymer mass of 2.5 g, add 2.0 μmol of antibody), transfer to a 4°C freezer, and stir continuously at 200 rpm for 2 hours;

[0061] S30: Add 100 μL of PBS solution containing 10 μg PDGF-BB (100 μg / mL) to the above reaction system and let it stand at room temperature for 30 minutes to allow PDGF to disperse evenly.

[0062] S40: Add 10 mL of 0.1% w / v genipin aqueous solution dropwise at a rate of 0.2 mL / min using a micro-injection pump, keeping the mixture at room temperature and stirring during the addition. After the addition is complete, continue cross-linking for 2 hours to form a pale yellow to light brown hydrogel, which can then be stored at 4°C for later use.

[0063] Example 2

[0064] Except for the following parameters, the rest of the operation is the same as in Example 1:

[0065] In S10, the molecular weight of HA is 2000 kDa, the molecular weight of CS is 500 kDa, and the degree of deacetylation is 85% for both.

[0066] The final concentrations of EDC and NHS in S20 were each 0.1 M (total molar concentration 0.2 M), and the antibody dosage was 1.0 μmol / g substrate.

[0067] The PDGF loading in S30 is 200 ng / mg hydrogel;

[0068] The concentration of genipin in S40 was 0.15% w / v, the dropping rate was 0.5 mL / min, and the cross-linking time was 4 hours.

[0069] Example 3

[0070] Except for the following parameters, the rest of the operation is the same as in Example 1:

[0071] The mass ratio of HA to CS in S10 is 6:4, that is, HA 1.5% w / v, CS 1.0% w / v, and total concentration 2.5% w / v;

[0072] The reaction temperature in S20 is 2℃, and the reaction time is 3 hours.

[0073] The settling time for S30 is 60 minutes;

[0074] The concentration of genipin in S40 was 0.05% w / v, the dropping rate was 0.1 mL / min, and the cross-linking time was 1 hour.

[0075] Comparative Example 1

[0076] Except for not adding anti-TNF-α monoclonal antibody, the rest of the operation is the same as in Example 1: that is, after EDC / NHS activation in S20, no antibody is added, and the process proceeds directly to step S30.

[0077] Comparative Example 2

[0078] Except for replacing the anti-TNF-α monoclonal antibody with an equimolar amount of bovine serum albumin (BSA), the rest of the operation is the same as in Example 1.

[0079] Comparative Example 3

[0080] Except for adding genipin for cross-linking in step S40 before adding the antibody for conjugation, the rest of the operation is the same as in Example 1, that is, the cross-linking order is changed so that genipin reacts with CS to form a network first, and then the antibody is conjugated.

[0081] The hydrogel samples prepared in the above examples and comparative examples were subjected to performance tests, and the results are shown in the table below:

[0082] Initial Young's modulus (kPa) Swelling rate (%) Mass loss after 7 days without TNF-α (%) Degradation rate of TNF-α at 10 ng / mL for 24 hours (%) Cumulative release of PDGF over 24 hours (%) Antibacterial rate against Staphylococcus aureus (%) Antibacterial rate against Escherichia coli (%) Example 1 2 2 5 0 0 8 4 5 4 0 8 8 8 3 Example 2 2 5 4 8 0 7 4 8 4 3 8 7 8 2 Example 3 2 0 5 4 0 9 4 2 3 7 8 6 8 1 Comparative Example 1 1 2 6 2 0 3 5 1 0 8 8 5 8 0 Comparative Example 2 2 1 5 1 0 3 8 1 2 9 8 7 8 2 Comparative Example 3 1 8 5 6 0 4 0 1 5 1 1 8 6 8 1 surface

[0083] As shown in the table above, the hydrogels prepared in Examples 1 to 3 all showed a mass loss of less than 10% after 7 days without TNF-α stimulation, indicating structural stability. At a TNF-α concentration of 10 ng / mL, the degradation rate reached 40% to 48% within 24 hours, and PDGF was released simultaneously at 37% to 43%, proving that the "recognition-degradation-drug delivery" mechanism was effectively established. Comparative Example 1 had a low initial modulus and severe non-specific degradation due to the lack of antibody crosslinking. Comparative Example 2 used the non-specific protein BSA, and although the mechanical properties were similar, it could not respond to TNF-α. Comparative Example 3 had an incorrect crosslinking sequence, with genipin preferentially crosslinking, which led to the shielding of the HA carboxyl group or increased steric hindrance, resulting in decreased antibody conjugation efficiency and significantly reduced response performance.

[0084] Further scanning electron microscopy was used to observe the microstructure of the hydrogel of Example 1 before and after TNF-α stimulation: before stimulation, it exhibited a dense porous network (pore size approximately 20 to 50 μm); after 24 hours of stimulation, the network showed significant fragmentation, confirming network disintegration caused by conformational changes. ELISA detection of PDGF activity in the released solution showed consistency with the standard curve, indicating that physical encapsulation did not impair its biological activity.

[0085] In summary, by precisely controlling the HA / CS ratio, antibody conjugation conditions, genipin addition timing, and PDGF loading method, this invention successfully constructed a smart hydrogel with specific TNF-α response, controllable degradation, and simultaneous drug release, which is suitable for the precise treatment of complex chronic wounds.

Claims

1. A smart responsive biodegradable hydrogel, characterized in that, It contains the following components: the substrate is a blend of hyaluronic acid and chitosan, with a total concentration of 2.0%–3.0% w / v; the cross-linking agent is an anti-TNF-α monoclonal antibody, used at a concentration of 0.8 μmol / g substrate; the auxiliary cross-linking agent is genipin, with a concentration of 0.1% w / v; and the loaded drug is platelet-derived growth factor, with a drug loading of 50–200 ng / mg hydrogel.

2. The smart responsive biodegradable hydrogel according to claim 1, characterized in that, The anti-TNF-α monoclonal antibody is covalently coupled to the carboxyl group on the hyaluronic acid molecular chain via an EDC / NHS-mediated amidation reaction, forming a three-dimensional network structure with the antibody as the cross-linking node. Each antibody molecule is connected to 2-3 hyaluronic acid or chitosan polymer chains. Genipin is added after antibody conjugation and drug loading are completed, and reacts with the amino group of chitosan to undergo a Schiff base reaction. The platelet-derived growth factor is dispersed in the hyaluronic acid-chitosan solution by physical encapsulation before cross-linking.

3. The smart responsive biodegradable hydrogel according to claim 1, characterized in that, The hyaluronic acid has a molecular weight of 50-2000 kDa, and the chitosan has a degree of deacetylation of not less than 85% and a molecular weight of 50-500 kDa.

4. The smart responsive biodegradable hydrogel according to claim 3, characterized in that, The blend contains 1.4%–2.1% hyaluronic acid w / v and 0.6%–0.9% chitosan w / v.

5. The smart responsive biodegradable hydrogel according to claim 1, characterized in that, The anti-TNF-α monoclonal antibody is a humanized IgG1 antibody, and its binding dissociation constant Kd with TNF-α is 0.05-0.2 nM.

6. The smart responsive biodegradable hydrogel according to claim 4, characterized in that, The amount of the anti-TNF-α monoclonal antibody used is 0.5-1.0 μmol / g substrate.

7. The smart responsive biodegradable hydrogel according to claim 1, characterized in that, The concentration of genipin is 0.05%–0.15% w / v.

8. A method for preparing a smart responsive biodegradable hydrogel as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Hyaluronic acid and chitosan were dissolved separately at pH In phosphate buffer at 7.4, a 1.75% w / v hyaluronic acid solution and a 0.75% w / v chitosan solution were prepared, mixed, and stirred at room temperature for 1 hour to obtain a homogeneous HA-CS blend solution. A 0.1 M EDC and NHS mixed solution at a molar ratio of 1:1 was added to the HA-CS blend solution to activate the carboxyl groups on the hyaluronic acid molecules for 30 minutes. Subsequently, 0.8 μmol / g of the anti-TNF-α monoclonal antibody was added, and the reaction was carried out at 4°C with continuous stirring for 2 hours to complete the covalent coupling of the antibody and hyaluronic acid. Platelet-derived growth factor solution was added to the above coupling product to make the final drug loading 100 ng / mg hydrogel, and the mixture was allowed to stand at room temperature for 30 minutes. A 0.1% w / v genipin aqueous solution was added dropwise while stirring, and cross-linking was continued at room temperature for 2 hours to form a hydrogel with a three-dimensional network structure.

9. The preparation method according to claim 8, characterized in that, In step S10, the blending ratio of hyaluronic acid to chitosan is 6:4 to 8:2 by mass; the total molar concentration of EDC / NHS is 0.05-0.2 M; the antibody conjugation reaction temperature is 2℃ to 6℃; the reaction time is 1 hour to 3 hours; the standing time after the addition of platelet-derived growth factor is 20 minutes to 60 minutes; the dropping rate of genipin solution is 0.1 mL / min to 0.5 mL / min; and the cross-linking time is 1 hour to 4 hours.

10. The application of a smart responsive biodegradable hydrogel as described in any one of claims 1 to 7 in chronic wound dressings, characterized in that, The chronic wounds include diabetic foot ulcers, pressure sores, venous ulcers, and difficult-to-heal postoperative wounds.

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