Glucose response type hydrogel and preparation method thereof

Abstract

The invention provides glucose response type hydrogel and a preparation method thereof, and relates to the technical field of glucose response type hydrogel. The preparation method of the glucose response type hydrogel comprises the processes of preparation of the functional chitosan, preparation of the polyacrylate compound, preparation of the hydrogel and the like. According to the invention, a dual-response mechanism design is adopted, glucose oxidase is introduced to enhance response sensitivity on the basis of a 3-carboxyphenylboronic acid response mechanism, a dual-network structure combining physical crosslinking and chemical crosslinking is constructed, and nanocellulose is introduced to enhance the material, so that the mechanical property of the material is remarkably improved; a green crosslinking process of click chemistry is combined with chitosan modification, so that the response time of the glucose response type hydrogel is shortened, the compression modulus and the stability of the glucose response type hydrogel are improved, and the glucose response type hydrogel has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of glucose-responsive hydrogel technology, and more particularly to a glucose-responsive hydrogel and its preparation method. Background Technology

[0002] With the continued increase in the number of people with diabetes worldwide, traditional blood glucose management methods are no longer sufficient to meet the diverse needs of patients. Glucose-responsive hydrogels, as materials that can automatically sense changes in blood glucose concentration and respond accordingly, offer a new direction for precision treatment of diabetes. Glucose-responsive hydrogels can not only achieve intelligent insulin release but also have applications in continuous blood glucose monitoring, intelligent wound dressings, and many other fields, showing significant application potential.

[0003] The core of glucose-responsive hydrogels lies in their ability to specifically recognize glucose molecules and produce measurable physical or chemical changes. Based on different response mechanisms, they can be mainly divided into the following three categories:

[0004] (1) Enzyme catalysis mechanism

[0005] Enzymatic catalysis is currently the most widely used glucose response strategy, primarily relying on glucose oxidase (GOx) to catalyze the oxidation of glucose. The gluconic acid produced during the reaction lowers the local pH, while hydrogen peroxide can further participate in subsequent chemical reactions. The formation of these products causes changes in the hydrogel network structure, thereby achieving a response to glucose concentration. While enzyme catalysis offers advantages in terms of high specificity and sensitivity, maintaining enzyme stability and activity remains a major challenge.

[0006] (2) Boric acid-diol complexation mechanism

[0007] Phenylboronic acid and its derivatives can form reversible borate ester bonds with compounds having an ortho-dihydroxy structure. Glucose molecules contain multiple hydroxyl groups, which can specifically bind to borate groups. This binding alters the charge distribution and spatial configuration of the molecule, thereby affecting the crosslinking density and swelling properties of the hydrogel. The advantage of the borate mechanism lies in the reversibility of the reaction and the relatively simple molecular design, but its selectivity is relatively low and it is easily interfered with by other sugar molecules.

[0008] (3) lectin binding mechanism

[0009] Lectins are a class of proteins that can specifically recognize and bind to carbohydrate molecules. By integrating lectin molecules into hydrogel networks, selective recognition of specific carbohydrates can be achieved. When glucose binds to lectins, it causes a conformational change in the protein, thereby affecting the physical properties of the hydrogel. The lectin mechanism exhibits extremely high selectivity and biocompatibility, but protein stability and cost are important factors to consider for its industrial application.

[0010] The most important application of glucose-responsive hydrogels in diabetes treatment is the construction of insulin delivery systems. These systems can automatically adjust the amount of insulin released based on real-time changes in blood glucose concentration, mimicking the physiological function of a healthy pancreas. This avoids the need for patients to frequently monitor blood glucose and manually inject insulin, which not only increases the burden on patients but also easily leads to poor glycemic control. Glucose-responsive hydrogels can also be used to develop novel blood glucose monitoring devices, enabling real-time and continuous monitoring of blood glucose concentration.

[0011] Diabetic patients often experience slow wound healing due to poor blood sugar control. Glucose-responsive hydrogels can release effective wound-healing components based on changes in glucose concentration at the wound site, significantly improving wound healing outcomes in diabetic patients.

[0012] However, glucose-responsive hydrogels still have certain problems in terms of response speed, mechanical strength and long-term stability, which restricts their clinical translation and application. Summary of the Invention

[0013] In view of this, the present invention provides a glucose-responsive hydrogel and its preparation method. The present invention shortens the response time of the glucose-responsive hydrogel and improves its compressive modulus and stability through a dual crosslinking and dual response mechanism, thereby improving the response speed of the glucose-responsive hydrogel, maintaining the structural integrity of the material in complex working environments and promoting the transformation (stability) of the material in clinical applications.

[0014] The first aspect of this invention is to provide a method for preparing a glucose-responsive hydrogel, specifically comprising the following steps:

[0015] S1. Preparation of functionalized chitosan:

[0016] Glucose oxidase (GOx) was dissolved in HEPES buffer to obtain solution A; hydroxyethyl deacetylated chitosan (GC) was dissolved in HEPES buffer and stirred until completely dissolved, 3-carboxyphenylboronic acid (PBA) and DBCO-PEG-COOH were added, and stirred until homogeneous to obtain solution B; solution A was slowly added to solution B, and the mixture was stirred until homogeneous, then 4Arm-PEG10000-N3 was added, and the mixture was freeze-dried after reaction to obtain functionalized chitosan (GC-PBA-GOx).

[0017] S2, Preparation of polyacrylate complex:

[0018] Sodium polyacrylate (PAA) was dissolved in HEPES buffer, chitosan (CS) solution was added, the pH was adjusted to 7.0, dopamine hydrochloride (DA) and cellulose nanoparticles (CNC) were added, the mixture was ultrasonically dispersed and stirred to react, and then freeze-dried to obtain polyacrylate complex (PAA-DA-CS).

[0019] S3. Preparation of hydrogel:

[0020] GC-PBA-GOx aqueous solution, PAA-DA-CS aqueous solution, and sodium alginate solution were mixed and stirred evenly to obtain solution C. CaCl2 solution was added to solution C for ionic cross-linking to form a preliminary gel network. 4Arm-PEG10000-N3 was added for chemical cross-linking. The product was soaked in PBS buffer for 24 h to remove unreacted substances, resulting in a glucose-responsive hydrogel.

[0021] Preferably, in step S1, the pH of the HEPES buffer is 7.4; the mass ratio of GC to PBA is 1:(0.5-0.8); the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:(0.8-1.2):1.5; the amount of DBCO-PEG-COOH is 4.5wt.%-5.5wt.% of GC; the mass-volume ratio of GOx to HEPES buffer is 1 mg:(1.5-2.5)mL; the mass-volume ratio of GC to HEPES buffer is 1g:(80-120)mL; the volume ratio of solution A to solution B is 1:1; the mixing and stirring temperature of solution A and solution B is room temperature, and the stirring time is 3-5 h; the reaction temperature is room temperature, and the reaction time is 6-24 h.

[0022] Preferably, in step S2, the mass ratio of PAA, CS, DA, and CNC is (8-12):(2-3):(0.3-0.5):(0.8-1.2); the reaction temperature is room temperature, and the reaction time is 16-32 h; the mass-to-volume ratio of PAA to HEPES buffer is 1 g:(40-60) mL.

[0023] Preferably, in step S3, the concentration of the CaCl2 solution is 1 M, and the amount added is 2% (v / v) of solution C; the mass-to-volume ratio of 4Arm-PEG10000-N3 to solution C is 150 mg:250 mL; the chemical crosslinking temperature is 37°C, and the chemical crosslinking time is 4-8 h.

[0024] A second aspect of the present invention is to provide a glucose-responsive hydrogel prepared according to the above method.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0026] This invention employs a dual response mechanism design, introducing GOx to enhance response sensitivity based on the PBA response mechanism, and adding a HEPES buffer system to maintain the optimal pH.

[0027] This invention significantly improves the mechanical properties of materials by constructing a dual-network structure that combines physical and chemical cross-linking and introducing CNC-reinforced materials.

[0028] This invention employs a green crosslinking process based on click chemistry combined with CS modification to shorten the response time of glucose-responsive hydrogels, thereby improving their compressive modulus and stability, and demonstrating promising application prospects. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings.

[0030] Figure 1 This is a flowchart illustrating the preparation process of the glucose-responsive hydrogel of the present invention. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] The first aspect of this invention is to provide a method for preparing a glucose-responsive hydrogel, specifically comprising the following steps:

[0033] Preparation of S1 and GC-PBA-GOx:

[0034] GOx was dissolved in HEPES buffer to obtain solution A. GC was dissolved in HEPES buffer and stirred until completely dissolved. PBA and DBCO-PEG-COOH were added and stirred until homogeneous to obtain solution B. Solution A was slowly added to solution B and stirred until homogeneous. Then 4Arm-PEG10000-N3 was added. After reaction, the mixture was freeze-dried to obtain GC-PBA-GOx.

[0035] The pH of the HEPES buffer solution is 7.4;

[0036] The mass ratio of GC to PBA is 1:(0.5-0.8); the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:(0.8-1.2):1.5; the amount of DBCO-PEG-COOH is 4.5wt.%-5.5wt.% of GC; preferably, the mass ratio of GC to PBA is 1:0.6; the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:1:1.5; and the amount of DBCO-PEG-COOH is 5wt.% of GC.

[0037] The mass-to-volume ratio of GOx to HEPES buffer is 1 mg:(1.5-2.5) mL, preferably 1 mg:2 mL; the mass-to-volume ratio of GC to HEPES buffer is 1 g:(80-120) mL, preferably 1 g:100 mL; the stirring and dissolution temperature of GC is 60℃; the stirring and dissolution temperature of PBA and DBCO-PEG-COOH is room temperature, and the stirring time is 1-3 h, preferably 2 h; the volume ratio of solution A to solution B is 1:1; the mixing and stirring temperature of solution A and solution B is room temperature, and the stirring time is 3-5 h, preferably 4 h; the reaction temperature is room temperature, and the reaction time is 6-24 h, preferably 12 h.

[0038] Preparation of S2 and PAA-DA-CS:

[0039] PAA was dissolved in HEPES buffer, CS solution was added, the pH was adjusted to 7.0, DA and CNC were added, the mixture was ultrasonically dispersed and stirred to react, and then freeze-dried to obtain the PAA-DA-CS complex.

[0040] The CS solution is a 1% acetic acid solution of CS, and the CS concentration in the CS solution is 10 mg / mL; the mass ratio of PAA, CS, DA, and CNC is (8-12):(2-3):(0.3-0.5):(0.8-1.2), preferably 10:2.5:0.4:1; the ultrasonic dispersion time is 15-45 min, preferably 30 min; the reaction temperature is room temperature, and the reaction time is 16-32 h, preferably 24 h; the mass-to-volume ratio of PAA to HEPES buffer is 1 g:(40-60) mL, preferably 1 g:50 mL;

[0041] S3. Preparation of hydrogel:

[0042] GC-PBA-GOx aqueous solution, PAA-DA-CS aqueous solution, and sodium alginate solution were mixed and stirred evenly to obtain solution C. CaCl2 solution was added to solution C for ionic cross-linking to form a preliminary gel network. 4Arm-PEG10000-N3 was added for chemical cross-linking. The product was soaked in PBS buffer for 24 h to remove unreacted substances, resulting in a glucose-responsive hydrogel.

[0043] The concentration of the GC-PBA-GOx aqueous solution is 3%, the concentration of the PAA-DA-CS aqueous solution is 3%, and the concentration of the sodium alginate solution is 1%; the volume ratio of the GC-PBA-GOx aqueous solution, PAA-DA-CS aqueous solution, and sodium alginate solution is 2:2:1; the stirring time is 30 min; the concentration of the CaCl2 solution is 1 M, and the addition amount is 2% (v / v) of solution C; the mass-volume ratio of 4Arm-PEG10000-N3 to solution C is 150 mg:250 mL; the chemical crosslinking temperature is 37℃, and the chemical crosslinking time is 4-8 h, preferably 6 h.

[0044] In the hydrogel preparation process of this invention, PBA and GC form reversible borate ester bonds; the DBCO group in DBCO-PEG-COOH undergoes a strain-promoted azido-alkyne cycloaddition reaction (SPAAC) with the azide group in 4Arm-PEG10000-N3, and GOx is integrated into the polymer network through physical embedding or chemical coupling; the catechol group in DA is covalently bonded to the carboxyl group of PAA and / or the amino group of CS, and the phenolic hydroxyl group of dopamine forms a hydrogen bond network with the carboxyl group of PAA, the hydroxyl group of CS, and the amino group; nanocellulose, as a reinforcing phase, is bonded to the matrix material through hydrogen bonds to improve mechanical strength; Ca 2+Ions chelate with the carboxylic acid groups on the sodium alginate molecular chain to rapidly form a preliminary three-dimensional gel network. The azide groups of 4Arm-PEG10000-N3 continue to undergo click chemistry reactions with the residual DBCO groups in the system to form a stable covalent cross-linked network.

[0045] In the presence of glucose, glucose exhibits a stronger affinity for PBA, competitively binding to replace the original polysaccharide-PBA complex and forming a more stable borate ester complex. This leads to a looser gel network. GOx catalyzes the oxidation of glucose to gluconic acid, further altering the local pH and affecting the borate-diol complex equilibrium. Ultimately, the hydrogel of this invention achieves a specific response to glucose through a reversible complexation reaction of borate-polyol. Changes in glucose concentration cause reversible changes in the gel network structure, thereby realizing the intelligent response function. The multiple cross-linked network ensures the mechanical stability and reversibility of the gel's response.

[0046] A second aspect of the present invention is to provide a glucose-responsive hydrogel prepared according to the above method.

[0047] The hydrogel described in this invention is stored in a sterile environment.

[0048] To further illustrate the present invention, the following embodiments will be described in detail.

[0049] All raw materials used in the following embodiments of the present invention are commercially available.

[0050] Unless otherwise specified, all experiments were repeated three times. Analysis of variance (ANOVA) and Duncan's multiple comparison analysis were performed using SPSS 21.0. Results are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0051] Example 1 A glucose-responsive hydrogel, prepared as follows:

[0052] Preparation of S1 and GC-PBA-GOx

[0053] 100 mg of GOx (enzyme activity 280 U / mg) was dissolved in 200 mL of HEPES buffer to obtain solution A. 2 g of GC (degree of deacetylation 91%, viscosity 250 mPa·s, molecular weight 250,000 Da) was dissolved in 200 mL of HEPES buffer (pH 7.4) and stirred at 60 °C until completely dissolved. 1.2 g of PBA and 100 mg of DBCO-PEG-COOH (PEG molecular weight 2500 Da, DBCO reaction efficiency ≥98%, purity ≥95%) were added, and the mixture was stirred at room temperature for 2 h to obtain solution B. Solution A was slowly added to solution B, and the mixture was stirred for 4 h. Then, 150 mg of 4Arm-PEG10000-N3 (functionality ≥95%, PDI ≤1.05, moisture content ≤1%) was added, and the mixture was reacted at room temperature for 12 h. The mixture was then freeze-dried to obtain GC-PBA-GOx.

[0054] Preparation of S2 and PAA-DA-CS

[0055] 2 g of PAA (molecular weight 200,000 Da, residual monomer: ≤100 ppm) was dissolved in 100 mL of HEPES buffer, 50 mL of CS (degree of deacetylation 87%, molecular weight 120,000 Da, viscosity 120 mPa·s) solution was added, the pH was adjusted to 7.0, 80 mg of DA (purity ≥99%, oxidation degree ≤2%) was added, and 200 mg of CNC (aspect ratio 20, average diameter 10 nm) was added. The mixture was ultrasonically dispersed for 30 min, then stirred and reacted for 24 h. The PAA-DA-CS complex was obtained by freeze-drying.

[0056] S3, Preparation of hydrogels

[0057] Prepare 100 mL of 3 wt.% GC-PBA-GOx aqueous solution, 100 mL of 3 wt.% PAA-DA-CS aqueous solution, and 50 mL of 1 wt.% sodium alginate (molecular weight 1 million Da, M / G ratio 0.5, viscosity 400 mPa·s) solution. Mix the three solutions and add 5 mL of 0.1 M CaCl2 solution for ionic cross-linking to form a preliminary gel network. Add 150 mg of 4Arm-PEG10000-N3 and react at 37 °C for 6 h to complete chemical cross-linking. Soak in PBS buffer for 24 h to remove unreacted substances and obtain glucose-responsive hydrogel.

[0058] Comparative Example 1

[0059] 2 g of GC was dissolved in 200 mL of PBS buffer (pH 7.4) and stirred at room temperature for 2 h. Pre-activated PBA (1.2 g PBA + 500 mg EDC + 300 mg NHS) was added and reacted at room temperature for 12 h. The mixture was then freeze-dried to obtain GC-PBA. 100 mL of 2% GC-PBA solution and 100 mL of 2% PAA solution were mixed and stirred for 30 min. 50 mg of EDC was added for crosslinking. The mixture was reacted at 37 °C for 6 h and then soaked in PBS for 24 h to obtain the hydrogel.

[0060] Comparative Example 2

[0061] The difference from Implementation 1 is that GOx was not used and the preparation process was adjusted accordingly. The specific steps are as follows:

[0062] Preparation of S1 and GC-PBA

[0063] 2 g of GC (degree of deacetylation 91%, viscosity 250 mPa·s, molecular weight 250,000 Da) was dissolved in 200 mL of HEPES buffer (pH 7.4) and stirred at 60 °C until completely dissolved. 1.2 g of PBA and 100 mg of DBCO-PEG-COOH (PEG molecular weight 2500 Da, DBCO reaction efficiency ≥98%, purity ≥95%) were added and stirred at room temperature for 2 h to obtain solution B. Solution A was slowly added to solution B and mixed and stirred for 4 h. Then 150 mg of 4Arm-PEG10000-N3 (functionality ≥95%, PDI ≤1.05, moisture content ≤1%) was added and reacted at room temperature for 12 h. The mixture was then freeze-dried to obtain GC-PBA-GOx.

[0064] The remaining steps are the same as in Example 1.

[0065] Comparative Example 3

[0066] The difference from Example 1 is that it does not contain CNCCNC, and the CNC addition step is skipped during the preparation of PAA-DA-CS.

[0067] Comparative Example 4

[0068] The difference from Example 1 is that EDC / NHS is used for crosslinking. The crosslinking agents are: 800 mg EDC·HCl and 500 mg NHS. The pH of solution B is adjusted to 5.5-6.0.

[0069] Add pre-activated crosslinking agents: 800 mg EDC·HCl and 500 mg NHS to solution B, pre-activate at 4°C for 30 min, slowly add solution A, stir and react at 4°C for 12 h, wash with PBS to remove unreacted raw materials, freeze dry to obtain GC-PBA-GOx, the remaining steps are the same as in Example 1, skip the steps involving DBCO-PEG-COOH and Arm-PEG10000-N3.

[0070] The response time (time required to reach 90% equilibrium swelling ratio) of the hydrogels obtained in Example 1 and Comparative Examples 1-4. 90 The compressive modulus and stability (37℃, PBS, for a total of 60 days) were tested, and the results are shown in Table 1.

[0071] Table 1. Results of hydrogel performance testing

[0072]

[0073] In terms of response time:

[0074] In the hydrogel of Example 1, GOx enzyme catalyzes glucose oxidation, rapidly producing gluconic acid, lowering the local pH. The porous network structure facilitates glucose diffusion, and the enzyme-catalyzed reaction significantly accelerates the response rate. Comparative Example 1 lacks GOx enzyme and relies solely on the direct complexation of PBA with glucose, resulting in a single cross-linked network with limited diffusion channels. Only simple EDC / NHS cross-linking forms a dense structure. Comparative Example 2 exhibits no enzyme catalysis and a single response mechanism, but retains the porous structure of click chemical cross-linking, making its network structure superior to Comparative Example 1. Comparative Example 3 has GOx enzyme catalysis but lacks CNC, resulting in a relatively loose network structure that affects response efficiency. Comparative Example 4 contains GOx enzyme, but the network formed by EDC / NHS cross-linking is not uniform enough, and enzyme activity is somewhat affected.

[0075] In terms of compressive modulus:

[0076] Example 1: CNC provides excellent mechanical reinforcement. Click chemistry forms a regular cross-linked network, and the synergistic effect of multiple cross-linking (chemical + ionic) further improves the modulus of the hydrogel. Comparative Example 1 uses a single EDC / NHS cross-linking method, resulting in limited cross-linking density, lack of nano-reinforcing phase, and an irregular network structure. Comparative Example 2: Click chemistry cross-linking results in a regular network with synergistic effects of multiple cross-linking mechanisms, but lacks CNC reinforcement. Comparative Example 3: Lacks the reinforcing effect of CNC, leading to a decrease in modulus. Comparative Example 4: EDC / NHS cross-linking is less regular than click chemistry, and side reactions occur during the cross-linking process, affecting network integrity.

[0077] In terms of stability:

[0078] In Example 1, click chemistry forms stable covalent bonds, CNC improves structural stability, and GOx enzyme is effectively immobilized and protected, resulting in good hydrogel stability. In Comparative Example 1, the amide bonds of the EDC / NHS crosslinks are easily hydrolyzed, the single network structure is easily disintegrated, and the use of an enzyme-free system leads to poor functional stability. In Comparative Example 2, click chemistry crosslinking is stable, and multiple network structures coexist, but functional stability (without GOx) is not as good as in Example 1. Comparative Example 3 lacks the structural support of CNC, and the network integrity decreases rapidly over time. In Comparative Example 4, the long-term stability of the EDC / NHS crosslinking is insufficient, enzyme activity is poorly maintained in traditional crosslinking, and functional stability is poor.

[0079] In summary, in the hydrogel system of the present invention, GOx enzyme can significantly improve the response speed of the hydrogel, CNC can improve the compression modulus of the hydrogel and enhance its long-term stability, click chemistry crosslinking can form a more regular and stable three-dimensional network, improving the overall performance of the hydrogel, and the synergistic effect of multiple components and multiple mechanisms makes Example 1 perform well in all indicators.

[0080] Example 2

[0081] The difference from Example 1 is that the mass ratio of GC to PBA is 1:0.5.

[0082] Example 3

[0083] The difference from Example 1 is that the mass ratio of GC to PBA is 1:0.8.

[0084] Example 4

[0085] The difference from Example 1 is that the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:0.8:1.5.

[0086] Example 5

[0087] The difference from Example 1 is that the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:1.2:1.5.

[0088] Example 6

[0089] The difference from Example 1 is that the amount of DBCO-PEG-COOH used is 4.5 wt.% of GC.

[0090] Example 7

[0091] The difference from Example 1 is that the amount of DBCO-PEG-COOH used is 5.5 wt.% of GC.

[0092] Example 8

[0093] The difference from Example 1 is that the mass ratio of PAA, CS, DA, and CNC is 8:2:0.3:0.8.

[0094] Example 9

[0095] The difference from Example 1 is that the mass ratio of PAA, CS, DA, and CNC is 12:3:0.5:1.2.

[0096] Testing revealed no significant difference in the properties of the hydrogels in Examples 2-9 compared to Example 1 (p > 0.05).

[0097] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a glucose-responsive hydrogel, characterized in that, Includes the following steps: S1. Preparation of functionalized chitosan: Glucose oxidase GOx was dissolved in HEPES buffer to obtain solution A; hydroxyethyl deacetylated chitosan GC was dissolved in HEPES buffer and stirred until completely dissolved, 3-carboxyphenylboronic acid PBA and DBCO-PEG-COOH were added and stirred evenly to obtain solution B; solution A was slowly added to solution B and stirred evenly, then 4Arm-PEG10000-N3 was added, and after reaction, the mixture was freeze-dried to obtain functionalized chitosan. S2, Preparation of polyacrylate complex: Sodium polyacrylate (PAA) was dissolved in HEPES buffer, chitosan CS solution was added, the pH was adjusted to 7.0, dopamine hydrochloride (DA) and nanocellulose (CNC) were added, the mixture was ultrasonically dispersed and stirred to react, and then freeze-dried to obtain the polyacrylate complex. S3. Preparation of hydrogel: Functionalized chitosan polysaccharide aqueous solution, polyacrylate complex aqueous solution, and sodium alginate solution were mixed and stirred evenly to obtain solution C. CaCl2 solution was added to solution C for ionic cross-linking to form a preliminary gel network. 4Arm-PEG10000-N3 was added for chemical cross-linking. The product was soaked in PBS buffer to remove unreacted substances, resulting in a glucose-responsive hydrogel.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of GC to PBA is 1:(0.5-0.8); the mass ratio of DBCO-PEG-COOH, GOx, and 4Arm-PEG10000-N3 is 1:(0.8-1.2):1.5; and the amount of DBCO-PEG-COOH used is 4.5wt.%-5.5wt.% of GC.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of GOx to HEPES buffer is 1 mg:(1.5-2.5) mL.

4. The preparation method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of GC to HEPES buffer is 1 g:(80-120) mL.

5. The preparation method according to claim 1, characterized in that, In step S1, the volume ratio of solution A to solution B is 1:

1.

6. The preparation method according to claim 1, characterized in that, In step S1, the reaction temperature is room temperature and the reaction time is 6-24 h.

7. The preparation method according to claim 1, characterized in that, The mass ratio of PAA, CS, DA, and CNC in step S2 is (8-12):(2-3):(0.3-0.5):(0.8-1.2).

8. The preparation method according to claim 1, characterized in that, In step S2, the reaction temperature is room temperature and the reaction time is 16-32 h.

9. The preparation method according to claim 1, characterized in that, In step S3, the chemical crosslinking temperature is 37°C and the chemical crosslinking time is 4-8 h.

10. A glucose-responsive hydrogel, characterized in that, Prepared by the method described in any one of claims 1-9.

Images