Carboxymethyl chitosan azide, glucose responsive polysaccharide-based hydrogel as well as preparation method and application of glucose responsive polysaccharide-based hydrogel

Abstract

The invention belongs to the technical field of high polymer materials, and particularly relates to azidated carboxymethyl chitosan, glucose responsive polysaccharide-based hydrogel and a preparation method and application thereof. The azidation carboxymethyl chitosan is obtained by taking carboxymethyl chitosan and fluorosulfonyl azide as raw materials and carrying out azidation reaction. According to the method, the FSO2N3 directly reacts with the carboxymethyl chitosan to prepare the azidated carboxymethyl chitosan in one step for the first time, the reaction conditions are mild, the product purity is high, and a new technical route is provided for polysaccharide modification. Glucose-responsive polysaccharide-based hydrogel covalently linked with polyphenols is further prepared from the azidated carboxymethyl chitosan, has the performances of glucose-responsive release, oxidation resistance, bacteria resistance and cell migration promotion, and can effectively promote healing of diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology, specifically relating to an azide-carboxymethyl chitosan, a glucose-responsive polysaccharide hydrogel, its preparation method, and its application. Background Technology

[0002] Chitosan is a natural cationic polysaccharide, a product of chitin deacetylation, with its precursors mainly derived from marine arthropods, fungal cell wall structures, and insect cuticles. Due to its excellent biocompatibility, biodegradability, and antibacterial activity, chitosan has become an important direction in the research and development of biomedical materials. However, the intramolecular hydrogen bonds of chitosan cause it to dissolve only under acidic conditions, which severely limits its application in the biomedical field. Therefore, researchers often employ chemical modification strategies to modify the structure of chitosan. Among these, carboxymethyl chitosan (CMCS) is considered one of the most promising derivatization products due to its good solubility and functional tunability. CMCS retains the inherent bioactivity of chitosan while introducing a strongly hydrophilic carboxymethyl group, thus achieving stable dissolution over a wide pH range. CMCS azidation modification is a promising modification method. Azide-modified CMCS exhibits high reactivity and can undergo click chemistry reactions with alkyne groups under mild conditions. The reaction is highly efficient and specific, allowing for precise modification of CMCS, and the product exhibits good stability. Traditional methods for preparing azidated polysaccharides typically employ a two-step sulfonation-azidation approach. This involves first introducing a sulfonate group as a leaving group through sulfonation, followed by a nucleophilic substitution reaction with sodium azide. This method suffers from drawbacks such as demanding reaction conditions, numerous byproducts, and low product purity. Summary of the Invention

[0003] This invention provides a method for preparing azidated carboxymethyl chitosan, which involves the direct reaction of FSO2N3 with carboxymethyl chitosan, thus avoiding the defects of the two-step sulfonation-azidation method in traditional methods.

[0004] This invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing azidated carboxymethyl chitosan, which is obtained by azidation reaction of carboxymethyl chitosan and fluorosulfonyl azide as raw materials.

[0005] In a preferred embodiment of the present invention, the ratio of carboxymethyl chitosan to fluorosulfonyl azide solution is 220.0 mg : 0.01 mL to 0.5 mL, the concentration of fluorosulfonyl azide solution is 165 to 170 mmol / L, and the azidization reaction is carried out at 20 to 50 °C for 12 to 24 h.

[0006] In a second aspect, the present invention provides an azide-modified carboxymethyl chitosan prepared according to the above method.

[0007] In a third aspect, the present invention provides a method for preparing glucose-responsive polysaccharide-based hydrogels using the aforementioned azidomethyl chitosan, comprising the following steps: Add an alkyne-modified insulin-like growth factor 1 C-domain polypeptide, a copper source, and a reducing agent to the solution of the azidomethyl chitosan, and perform a click reaction to obtain modified carboxymethyl chitosan. Modified sodium alginate was obtained by condensation reaction of aldehyde-modified sodium alginate and 3-aminophenylboronic acid under the action of a coupling agent. Polyphenolic substances are added to the solution of modified carboxymethyl chitosan, and then mixed with the solution of modified sodium alginate. The amino groups of modified carboxymethyl chitosan and the carboxyl groups of modified sodium alginate are cross-linked through Schiff base reaction, and the polyphenolic substances are linked therein through boron ester bonds and hydrogen bonds, thus obtaining the glucose-responsive polysaccharide hydrogel.

[0008] In a preferred embodiment of the present invention, the aldehyde-modified sodium alginate is obtained by adding an oxidant to an aqueous solution of sodium alginate and carrying out an oxidation reaction.

[0009] In a preferred embodiment of the present invention, the click reaction is carried out at 30~50°C for 12~24 h; The condensation reaction was carried out at 30-50°C for 12-24 hours. The oxidant is sodium periodate, and the oxidation reaction is carried out at 20~50℃ for 2~8 h. After the reaction is completed, ethylene glycol is added to terminate the reaction, and the termination time is 1~3 h.

[0010] In a preferred embodiment of the present invention, the copper source is a water-soluble copper salt, the reducing agent is sodium ascorbate, the coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the polyphenolic substance is epigallocatechin gallate.

[0011] In a preferred embodiment of the present invention, the mass ratio of the azidomethyl chitosan to the C-domain polypeptide of alkynylated insulin-like growth factor 1 is 55:6.5~37.5. The mass ratio of the modified carboxymethyl chitosan, epigallocatechin gallate, and the modified sodium alginate is 1~10:0.1~1:1~10.

[0012] In a fourth aspect, the present invention provides a glucose-responsive polysaccharide hydrogel prepared according to the above method.

[0013] In a fifth aspect, the present invention provides the application of the glucose-responsive polysaccharide hydrogel described above in the preparation of wound-healing products.

[0014] In a preferred embodiment of the present invention, the product is a gel dressing.

[0015] In a sixth aspect, the present invention provides the application of the glucose-responsive polysaccharide hydrogel described above in the preparation of antioxidant products.

[0016] In a seventh aspect, the present invention provides the application of the glucose-responsive polysaccharide hydrogel described above in the preparation of antibacterial products.

[0017] In a preferred embodiment of the present invention, the glucose-responsive polysaccharide hydrogel inhibits Escherichia coli.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to prepare azidomethyl chitosan by directly reacting FSO2N3 with carboxymethyl chitosan in one step. The reaction conditions are mild and the product has high purity, providing a new technical route for polysaccharide modification.

[0019] This invention endows the hydrogel with anti-inflammatory and antioxidant activities by adding polyphenols such as epigallocatechin gallate, thereby improving the microenvironment of excessive inflammation and oxidative stress in diabetic wounds. Modification with the C-domain peptide of insulin-like growth factor 1 (IGF1C) promotes cell migration and angiogenesis. Using carboxymethyl chitosan and sodium alginate as a matrix imparts excellent mechanical properties to the hydrogel. The prepared polysaccharide-based hydrogel exhibits self-healing, glucose-responsive, antibacterial, anti-inflammatory, and antioxidant activities, promoting wound healing in diabetic patients. The hydrogel prepared by the method provided in this invention significantly accelerates wound healing in a mouse diabetic wound model.

[0020] The method for preparing IGF1C modified polysaccharides provided by this invention (i.e., the click reaction of azidomethyl chitosan with alkynylated insulin-like growth factor 1 C-domain polypeptide) has the advantages of controllable reaction region and degree of substitution, and good stability. Attached Figure Description

[0021] Figure 1 A schematic diagram illustrating the preparation of the glucose-responsive polysaccharide hydrogel provided by this invention; Figure 2 Infrared spectrum (A) of azidomethyl chitosan and infrared spectrum (B) of IGF1C modified carboxymethyl chitosan provided for this invention. Figure 3 A general view of the glucose-responsive polysaccharide hydrogel provided by the present invention; Figure 4SEM images of different hydrogels; Figure 5 Alternating step strain scanning image of glucose-responsive polysaccharide hydrogel provided by the present invention; Figure 6 Release curve of the glucose-responsive polysaccharide hydrogel provided by the present invention; Figure 7 The scavenging rates of different hydrogels for DPPH free radicals; Figure 8 Schematic diagram of the antibacterial effects of different hydrogels on Escherichia coli; Figure 9 The effects of different hydrogels on the migration of L929 cells; A. intercellular distance, B. migration rate statistics; Figure 10 Schematic diagram of different hydrogels used to treat a mouse model of full-thickness skin defects; A. Skin wound healing status; B. Wound healing rate. Figure 11 Schematic diagram of the treatment of diabetic mice with full-thickness skin defects using different hydrogels; A, skin wound healing status; B, wound healing rate. Detailed Implementation

[0022] To better understand the technical content of this invention, specific embodiments are provided below to clearly and completely describe the invention. The described specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] Azide-modified carboxymethyl chitosan exhibits high reactivity, undergoing click chemistry with alkynyl groups under mild conditions. This reaction is highly efficient and specific, allowing for precise modification of carboxymethyl chitosan, and the product demonstrates good stability. Traditional methods for preparing azide-modified polysaccharides typically employ a two-step sulfonation-azidation approach. This involves first introducing a sulfonation group as a leaving group through sulfonation, followed by a nucleophilic substitution reaction with sodium azide. This method suffers from drawbacks such as demanding reaction conditions, numerous byproducts, and low product purity.

[0024] Based on this, the present invention provides azidated carboxymethyl chitosan and its preparation method, which directly reacts carboxymethyl chitosan with FSO2N3, avoiding the defects of the two-step sulfonation-azidation method in the traditional method.

[0025] In addition, existing methods for preparing IGF1C-modified polysaccharides involve linking azide groups to IGF1C via a condensation reaction, followed by the synthesis of alkynylated chitosan through the condensation reaction of chitosan and 4-pentynic acid. Finally, a click reaction is used to synthesize the CS-IGF1C complex. This method has several drawbacks: First, the regio-reaction of propargyl acid with chitosan is uncontrollable, potentially leading to the formation of O-alkynylated products. Second, the degree of substitution in chitosan alkynylation is difficult to control, and the reaction reagent EDC is prone to hydrolysis. Furthermore, the alkynylated chitosan prepared by this method contains amide bonds, making it susceptible to degradation in a protease environment.

[0026] This invention utilizes a click reaction between azidomethyl chitosan and an alkyne-modified C-domain polypeptide of insulin-like growth factor 1 (IGF1C). This method for preparing IGF1C-modified polysaccharides has advantages such as controllable reaction region and degree of substitution, and good stability.

[0027] Diabetes is a complex metabolic disease often accompanied by a variety of complications, including poor wound healing, cardiovascular disease, eye disease, nerve damage, and lower limb amputation due to foot ulcers. Diabetic wounds are a serious complication of diabetes, accounting for approximately 20% of wound healing failures in diabetic patients worldwide. Skin regeneration in diabetic patients is often impaired, leading to poor wound healing and even amputation or death. It has been reported that approximately 50% to 70% of amputations are caused by diabetic wounds.

[0028] Normal wound healing typically involves four consecutive and overlapping phases: hemostasis, inflammation, proliferation, and remodeling. However, the healing process of diabetic wounds is disrupted by various factors, leading to delayed healing. The main factors affecting diabetic wound healing include: (1) Biochemical disorders: factors such as hyperglycemia, dyslipidemia, and insulin resistance can cause tissue damage and hinder the normal wound healing process. (2) Excessive inflammation: abnormal immune cells in diabetic wounds make it difficult to suppress inflammation, and the persistent inflammatory phase will maintain the inflammatory state of the wound, thereby hindering wound healing. (3) Oxidative stress: excessive oxidative stress and reduced tissue antioxidant capacity in diabetic wounds lead to an imbalance between redox and oxidation. (4) Reduced angiogenesis: the diabetic state leads to reduced angiogenesis at the wound site, thereby hindering normal healing and the recovery of a healthy vascular system.

[0029] Dressings for diabetic wounds can be divided into dry dressings and wet dressings. Dry dressings, including gauze, bandages, and plasters, are readily available, easy to make, and can absorb exudate from the wound. However, dry dressings are difficult to remove during dressing changes, which can damage granulation tissue, causing secondary damage to the wound and pain for the patient. Traditional wound healing theory holds that dry dressings maintain a dry environment for the wound, which is more conducive to wound healing. However, subsequent studies have shown that compared with traditional dry dressings, wet dressings can promote cell growth, proliferation, and migration, reduce bacterial infection, reduce patient pain during dressing changes, and are more conducive to wound healing. Hydrogel dressings are a type of wet dressing, formed by polymers through physical or chemical cross-linking, and have a three-dimensional network structure. Hydrogels have good water absorption and can swell in water but do not dissolve. In addition to strong water absorption and moisturizing properties, the hydrogels described in this invention also have many physical and chemical properties similar to the natural extracellular matrix, providing a more ideal treatment method for diabetic wounds. As a medical wound dressing, hydrogels can physically isolate wounds from the external environment while providing a moist environment. However, single-function and single-structure hydrogels cannot meet the needs of the complex environment of diabetic wounds. Therefore, designing multifunctional smart hydrogels for the treatment of diabetic wounds is a current research hotspot and trend.

[0030] Ideal diabetic wound dressings should have the following characteristics: (1) good biocompatibility; (2) good antibacterial properties to prevent wound infection; (3) the ability to remove excess glucose from the wound site and reduce oxidative stress; (4) anti-inflammatory effects to reduce inflammation at the wound site; (5) convenient to use and good compliance; (6) good stability and easy to store.

[0031] Based on this, the present invention utilizes azidomethyl chitosan to prepare a glucose-responsive polysaccharide hydrogel, which has self-healing, glucose-responsive, antibacterial, anti-inflammatory and antioxidant activities, and can promote the healing of diabetic wounds.

[0032] The following description is based on specific embodiments.

[0033] Example 1 A method for preparing azidomethyl chitosan comprises the following steps: 220.0 mg of carboxymethyl chitosan was dissolved in 4 mL of distilled water. 400.0 mg of potassium bicarbonate was added to the solution, and after dissolution, FSO₂N₃ solution (167.8 mmol / L, 0.06 mL) was added. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain azide-modified carboxymethyl chitosan.

[0034] Example 2 220.0 mg of carboxymethyl chitosan was dissolved in 4 mL of distilled water. 400.0 mg of potassium bicarbonate was added to the solution, and after dissolution, 0.01 mL of FSO₂N₃ solution (167.8 mmol / L) was added. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain azide-modified carboxymethyl chitosan.

[0035] Example 3 220.0 mg of carboxymethyl chitosan was dissolved in 4 mL of distilled water. 400.0 mg of potassium bicarbonate was added to the solution, and after dissolution, 0.5 mL of FSO₂N₃ solution (167.8 mmol / L) was added. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain azide-modified carboxymethyl chitosan.

[0036] Example 4 220.0 mg of carboxymethyl chitosan was dissolved in 4 mL of distilled water. 400.0 mg of potassium bicarbonate was added to the solution, and after dissolution, 0.01 mL of FSO₂N₃ solution (167.8 mmol / L) was added. The mixture was stirred at 50 °C for 12 h. After the reaction, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain azide-modified carboxymethyl chitosan.

[0037] Since the azidomethyl chitosan expected in this invention was obtained in all Examples 1-4, the following preparation of glucose-responsive polysaccharide hydrogels will only take the azidomethyl chitosan prepared in Example 1 as an example.

[0038] Example 5 A glucose-responsive polysaccharide-based hydrogel is prepared according to the following steps: (1) Preparation of IGF1C modified carboxymethyl chitosan 110.0 mg of azidomethyl chitosan was dissolved in 10 mL of distilled water. Then, 13.6 mg of IGF1C (purchased from Qiangyao Biotechnology Co., Ltd.), 0.3 mg of copper sulfate, and 0.6 mg of sodium ascorbate were added to the solution. After complete dissolution, the mixture was stirred at 37 °C for 24 h. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain IGF1C-modified carboxymethyl chitosan.

[0039] (2) Preparation of boric acid-modified aldehyde-modified sodium alginate 3.0 g of sodium alginate was dissolved in 300 mL of distilled water. 648.4 mg of sodium periodate was added to the solution, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was complete, 3 mL of sodium periodate was added to the solution and the mixture was stirred for 2 h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was freeze-dried for 24 h to obtain aldehyde-modified sodium alginate.

[0040] 0.3 g of aldehyde-modified sodium alginate was dissolved in 30 mL of distilled water. 0.41 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the solution and stirred until dissolved. Then, an ethanol solution of 3-aminophenylboronic acid (0.86 mg, 30 mL) was added dropwise to the solution, and the mixture was stirred at room temperature for 24 h. After the reaction, the pH of the mixture was adjusted to 7, transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain phenylboronic acid-modified aldehyde-modified sodium alginate.

[0041] (3) Preparation of glucose-responsive polysaccharide hydrogels 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution. 6 mg of EGCG was added to this solution and dissolved to obtain solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand for 5 min to obtain a glucose-responsive polysaccharide hydrogel, labeled CMCS-IGF1C / OSA /

[0042] EGCG. Adding them in this order will make gel formation easier.

[0043] Example 6 A glucose-responsive polysaccharide-based hydrogel is prepared according to the following steps: (1) Preparation of IGF1C modified carboxymethyl chitosan 110.0 mg of azide-modified carboxymethyl chitosan was dissolved in 10 mL of distilled water. Then, 0.8 mg of IGF1C, 0.9 mg of copper sulfate, and 1.8 mg of sodium ascorbate were added to the solution. After complete dissolution, the mixture was stirred at 37 °C for 24 h. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain IGF1C-modified carboxymethyl chitosan.

[0044] (2) Preparation of boric acid-modified aldehyde-modified sodium alginate 3.0 g of sodium alginate was dissolved in 300 mL of distilled water. 648.4 mg of sodium periodate was added to the solution, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was complete, 3 mL of sodium periodate was added to the solution and the mixture was stirred for 2 h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was freeze-dried for 24 h to obtain aldehyde-modified sodium alginate.

[0045] 0.3 g of aldehyde-modified sodium alginate was dissolved in 30 mL of distilled water. 0.41 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the solution and stirred until dissolved. Then, an ethanol solution of 3-aminophenylboronic acid (0.86 mg, 30 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 24 h. After the reaction, the pH of the mixture was adjusted to 7, transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain phenylboronic acid-modified aldehyde-modified sodium alginate.

[0046] (3) Preparation of glucose-responsive polysaccharide-based hydrogels: 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution. 6 mg of EGCG was added to this solution and dissolved to obtain solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand for 5 min to obtain a glucose-responsive polysaccharide hydrogel, labeled CMCS-IGF1C / OSA / EGCG.

[0047] Example 7 A glucose-responsive polysaccharide-based hydrogel is prepared according to the following steps: (1) Preparation of IGF1C modified carboxymethyl chitosan 110.0 mg of azidomethyl chitosan was dissolved in 10 mL of distilled water. Then, 74.8 mg of IGF1C, 1.8 mg of copper sulfate, and 3.6 mg of sodium ascorbate were added to the solution. After complete dissolution, the mixture was stirred at 37 °C for 24 h. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain IGF1C-modified carboxymethyl chitosan.

[0048] (2) Preparation of boric acid-modified aldehyde-modified sodium alginate 3.0 g of sodium alginate was dissolved in 300 mL of distilled water. 648.4 mg of sodium periodate was added to the solution, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was complete, 3 mL of sodium periodate was added to the solution and the mixture was stirred for 2 h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was freeze-dried for 24 h to obtain aldehyde-modified sodium alginate.

[0049] 0.3 g of aldehyde-modified sodium alginate was dissolved in 30 mL of distilled water. 0.41 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the solution and stirred until dissolved. Then, an ethanol solution of 3-aminophenylboronic acid (0.86 mg, 30 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 24 h. After the reaction, the pH of the mixture was adjusted to 7, transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain phenylboronic acid-modified aldehyde-modified sodium alginate.

[0050] (3) Preparation of glucose-responsive polysaccharide-based hydrogels: 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution. 6 mg of EGCG was added to this solution and dissolved to obtain solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand for 5 min to obtain a glucose-responsive polysaccharide hydrogel, labeled CMCS-IGF1C / OSA / EGCG.

[0051] Example 8 A glucose-responsive polysaccharide-based hydrogel is prepared according to the following steps: (1) Preparation of IGF1C modified carboxymethyl chitosan 110.0 mg of azidomethyl chitosan was dissolved in 10 mL of distilled water. Then, 3.6 mg of IGF1C, 0.3 mg of copper sulfate, and 0.6 mg of sodium ascorbate were added to the solution. After complete dissolution, the mixture was stirred at 50 °C for 12 h. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain IGF1C-modified carboxymethyl chitosan.

[0052] (2) Preparation of boric acid-modified aldehyde-modified sodium alginate 3.0 g of sodium alginate was dissolved in 300 mL of distilled water. 648.4 mg of sodium periodate was added to the solution, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was complete, 3 mL of sodium periodate was added to the solution and the mixture was stirred for 1 h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was freeze-dried for 24 h to obtain aldehyde-modified sodium alginate.

[0053] 0.3 g of aldehyde-modified sodium alginate was dissolved in 30 mL of distilled water. 0.41 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the solution and stirred until dissolved. Then, an ethanol solution of 3-aminophenylboronic acid (0.86 mg, 30 mL) was added dropwise to the solution. The reaction was carried out at 50 °C with stirring for 12 h. After the reaction, the mixed solution was adjusted to pH 7 and transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da). Dialysis was performed using deionized water as the dialysis medium for 72 h. The product was then freeze-dried for 24 h to obtain phenylboronic acid-modified aldehyde-modified sodium alginate.

[0054] (3) Preparation of glucose-responsive polysaccharide-based hydrogels: 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution. 4 mg of EGCG was added to this solution and dissolved to obtain solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand for 5 min to obtain a glucose-responsive polysaccharide hydrogel, labeled CMCS-IGF1C / OSA / EGCG.

[0055] Example 9 A glucose-responsive polysaccharide-based hydrogel is prepared according to the following steps: (1) Preparation of IGF1C modified carboxymethyl chitosan 110.0 mg of azidomethyl chitosan was dissolved in 10 mL of distilled water. Then, 3.6 mg of IGF1C, 0.3 mg of copper sulfate, and 0.6 mg of sodium ascorbate were added to the solution. After complete dissolution, the mixture was stirred at 37 °C for 24 h. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da) and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain IGF1C-modified carboxymethyl chitosan.

[0056] (2) Preparation of boric acid-modified aldehyde-modified sodium alginate 3.0 g of sodium alginate was dissolved in 300 mL of distilled water. 648.4 mg of sodium periodate was added to the solution, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction was complete, 3 mL of sodium periodate was added to the solution, and the mixture was stirred for 3 h to terminate the reaction. The mixture was then transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was freeze-dried for 24 h to obtain aldehyde-modified sodium alginate.

[0057] 0.15 g of aldehyde-modified sodium alginate was dissolved in 30 mL of distilled water. 0.41 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the solution and stirred until dissolved. Then, an ethanol solution of 3-aminophenylboronic acid (0.86 mg, 30 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 24 h. After the reaction, the pH of the mixture was adjusted to 7, transferred to a dialysis bag (molecular weight cutoff MW = 3500 Da), and dialyzed with deionized water for 72 h. The product was then freeze-dried for 24 h to obtain phenylboronic acid-modified aldehyde-modified sodium alginate.

[0058] (3) Preparation of glucose-responsive polysaccharide-based hydrogels: 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution. 2 mg of EGCG was added to this solution and dissolved to obtain solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand for 5 min to obtain a glucose-responsive polysaccharide hydrogel, labeled CMCS-IGF1C / OSA / EGCG.

[0059] Comparative Example 1 Preparation of carboxymethyl chitosan / modified sodium alginate hydrogel: 50 mg of carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% carboxymethyl chitosan solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand to obtain a carboxymethyl chitosan / modified sodium alginate hydrogel, labeled CMCS / OSA.

[0060] The preparation process of modified sodium alginate is the same as in Example 5.

[0061] Comparative Example 2 50 mg of modified carboxymethyl chitosan was dissolved in 1 mL of distilled water to obtain a 5% modified carboxymethyl chitosan solution A. 100 mg of modified sodium alginate was dissolved in 1 mL of distilled water to obtain solution B. Equal volumes of solutions A and B were mixed and allowed to stand to obtain a modified carboxymethyl chitosan / modified sodium alginate hydrogel, labeled CMCS-IGF1C / OSA.

[0062] The preparation process of modified carboxymethyl chitosan and modified sodium alginate is the same as in Example 5.

[0063] Since examples 5-9 all yielded the glucose-responsive polysaccharide hydrogels expected in this invention, the following description will only use the glucose-responsive polysaccharide hydrogel prepared in example 5 as an example to illustrate the effect.

[0064] Experimental Example 1: Infrared spectroscopy characterization Azide-modified carboxymethyl chitosan and IGF1C-modified carboxymethyl chitosan were mixed evenly with potassium bromide particles, ground into powder, and then compressed into tablets. The tablets were then scanned in the range of 400–4000 cm⁻¹. -1 Infrared spectroscopy characterization was performed.

[0065] The results showed that azidomethyl chitosan at 2114 cm⁻¹ -1 A stretching vibration peak of the azido group appeared at 1647 cm⁻¹, and the absorption peak intensity increased with the increase of the degree of substitution of the azido group. The characteristic peak of the azido group in IGF1C-modified carboxymethyl chitosan weakened, and the peak intensity decreased at 1647 cm⁻¹. -1 1516 cm -1 and 1234 cm -1 Characteristic absorption peaks of amide bands I, II, and III were observed. Therefore, azido-modified carboxymethyl chitosan and IGF1C-modified carboxymethyl chitosan were successfully prepared.

[0066] Experimental Example 2: Scanning electron microscopy of glucose-responsive polysaccharide hydrogels The morphology and structure of the hydrogels were observed using a MAIA3 LMH scanning electron microscope. Three hydrogel samples, namely CMCS-IGF1C / OSA / EGCG (Example 5), CMCS / OSA (Comparative Example 1), and CMCS-IGF1C / OSA (Comparative Example 2), were quenched with liquid nitrogen and then sputtered with gold for 2 min.

[0067] The results showed that the CMCS-IGF1C / OSA / EGCG hydrogels all exhibited a porous network structure with interconnected pores, and the pore diameter ranged from 30 to 100 μm.

[0068] Experimental Example 3: Alternating step strain scanning experiment of glucose-responsive polysaccharide hydrogel A CMCS-IGF1C / OSA / EGCG hydrogel sample with a thickness of 1.0 mm was placed between the plates of an MCR302 rotational rheometer. The hydrogel sample was subjected to a constant strain of 1% for 60 s at 25 °C, followed by a strain of 250% for 60 s. This cycle was repeated 5 times, and the changes in G' and G'' were measured to evaluate the self-healing ability of the hydrogel.

[0069] The results showed that when the strain was 300%, G' was less than G'', and the hydrogel network was disrupted, exhibiting a sol state. When the strain recovered to 1%, G' was greater than G'', and the hydrogel network reformed, exhibiting a gel state. After five cycles, the hydrogel could still recover to its gel state, and G' showed no significant change, indicating that the CMCS-IGF1C / OSA / EGCG hydrogel has good self-healing properties.

[0070] Experiment Example 4: EGCG glucose-responsive release assay using glucose-responsive polysaccharide hydrogels Two 1 mL aliquots of CMCS-IGF1C / OSA / EGCG hydrogel were added to 10 mL of PBS buffer (pH=7.4) and 10 mL of PBS buffer containing 50 mmol / L glucose, respectively, and incubated in a shaker at 100 rpm and 37 °C. 2 mL of supernatant was collected at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively, and 2 mL of the corresponding fresh buffer was added to the system. The absorbance of the supernatant was then measured at 260 nm using a UV-Vis spectrophotometer. Based on the standard curve, the cumulative EGCG release rate of the CMCS-IGF1C / OSA / EGCG hydrogel at different time points was calculated, and the EGCG release curve was plotted.

[0071] The results showed that the cumulative EGCG release rate in glucose-containing PBS buffer was approximately twice that in PBS buffer solution after 24 h. Therefore, the release of EGCG from the CMCS-IGF1C / OSA / EGCG hydrogel is glucose-responsive.

[0072] Experimental Example 5: Study on the scavenging activity of glucose-responsive polysaccharide-based hydrogels against DPPH free radicals Preparation of DPPH solution: Add 10 mg DPPH to 10 mL methanol, sonicate for 20 min to dissolve, then dilute with methanol 10 times to obtain a 0.1 mmol / L DPPH solution, and store in the dark.

[0073] Take 200 μL of each of the three hydrogels (CMCS / OSA, CMCS-IGF1C / OSA, and CMCS-IGF1C / OSA / EGCG), then add 3.0 mL of DPPH solution and mix with the hydrogel. Incubate at 37 ℃ in the dark for 30 min. Measure the absorbance of the samples at 517 nm using a microplate reader, with three replicates per group. The DPPH clearance rate is calculated using the following formula:

[0074] ; Among them, A0 and A t The light absorption intensities at 517 nm are for the blank group (DPPH+ethanol) and the hydrogel group (DPPH+ethanol+hydrogel), respectively.

[0075] The results showed that the CMCS-IGF1C / OSA / EGCG hydrogel could significantly scavenge DPPH free radicals, with a scavenging rate of up to 74.6%. Therefore, the CMCS-IGF1C / OSA / EGCG hydrogel has good antioxidant capacity.

[0076] Experimental Example 6: Study on the inhibitory effect of glucose-responsive polysaccharide hydrogels on Escherichia coli 0. Preparation of hydrogel extract Take 200 μL of each of the three hydrogels (CMCS / OSA, CMCS-IGF1C / OSA, CMCS-IGF1C / OSA / EGCG), place them in a centrifuge tube, add 2 mL of PBS, incubate at 37 °C for 24 h, and then collect the extract.

[0077] 1. Preparation of Escherichia coli suspension Escherichia coli was cultured overnight at 37 °C in LB medium, and then the E. coli suspension was diluted to 1 × 10⁻⁶ with LB medium. 6 CFU / mL.

[0078] 3. Inhibitory effect of glucose-responsive polysaccharide hydrogel on Escherichia coli Take 3 mL of E. coli suspension (1×10) 6 Add 1 mL of each of the three hydrogel extraction solutions (CFU / mL) to centrifuge tubes, and 1 mL of blank PBS to the control group. Incubate at 37 °C for 3 h. After incubation, resuspend the E. coli and dilute 1000 times with PBS. Take 100 μL of each E. coli suspension, spread it on solid agar plates, and incubate at 37 °C. After 24 h, photograph and record the colony count for each group.

[0079] The results showed that, compared with CMCS / OSA and CMCS-IGF1C / OSA hydrogels, CMCS-IGF1C / OSA / EGCG hydrogel had a significant antibacterial effect against Escherichia coli.

[0080] Experiment Example 7: Study on the promoting effect of glucose-responsive polysaccharide-based hydrogels on L929 cell migration Preparation of hydrogel extract: Take 100 μL of each of the three hydrogels (CMCS / OSA, CMCS-IGF1C / OSA, CMCS-IGF1C / OSA / EGCG), place them in centrifuge tubes, sterilize them by UV irradiation in a clean bench for 30 minutes, then add 1 mL of DMEM medium containing low serum (5%) to the centrifuge tubes, incubate them in a cell culture incubator at 37 ℃ for 24 h, and collect the extract.

[0081] Draw two parallel lines evenly on the bottom of the 24-well plate with a marker to facilitate microscopic observation. Resuspend the digested L929 cells, count them, and dilute with DMEM medium to adjust the cell concentration to 3 × 10⁻⁶. 5 Cells were cultured at a density of 500 μL / mL in each well and incubated overnight at 37 °C. The 24-well plate was then removed, and three parallel lines were drawn perpendicularly to the wells using a 100 μL pipette tip, ensuring the width of each scratch was as consistent as possible. The old culture medium was then aspirated, and the cells were washed three times with PBS to remove the drawn cells. The hydrogel extract was centrifuged (12000 rpm, 5 min), and 500 μL of the supernatant was added to each well. DMEM medium containing 5% serum was used as a blank control. Cells were cultured at 37 °C for 12 h, then removed and photographed under a microscope. The images were opened in ImageJ, and the mean intercellular distance was calculated to determine the migration rate.

[0082] The results showed that after 12 h of treatment, the CMCS-IGF1C / OSA / EGCG group had the smallest scratch width, indicating that CMCS-IGF1C / OSA / EGCG can significantly promote the migration of L929 cells.

[0083] Experimental Example 8: Study on the Healing-Promoting Effect of Glucose-Responsive Polysaccharide-Based Hydrogels on a Mouse Model of Full-Thickness Skin Defects Thirty Balb / C mice were randomly selected and divided into five groups: control group (saline), Tegaderm group, CMCS / OSA group, CMCS-IGF1C / OSA group, and CMCS-IGF1C / OSA / EGCG group. All surgeries were performed on anesthetized mice (0.5% sodium pentobarbital, 0.2 mL / 10 g intraperitoneally). The fur on the backs of the mice was shaved with an electric animal razor, and the surgical areas were disinfected with alcohol. A 1 cm diameter circular full-thickness skin wound was then created on the back of each mouse using a punch, photographed, and 0.3 mL of hydrogel was applied to the wound using a syringe. Dressings were changed and photographed on days 0, 3, 7, 10, and 14. The wound area was assessed using ImageJ software to quantify the wound healing rate.

[0084] The results showed that, compared with the control group, Tegaderm group, CMCS / OSA group and CMCS-IGF1C / OSA group, CMCS-IGF1C / OSA / EGCG hydrogel could better promote the healing of full-thickness skin wounds in mice.

[0085] Experiment Example 9: Study on the Healing Effect of Glucose-Responsive Polysaccharide-Based Hydrogels on Full-Thickness Skin Defect Model in Diabetic Mice Thirty male C57BL / 6 mice (20-25 g) were selected to establish a type 1 diabetes model. After fasting for 12 h, the mice were intraperitoneally injected with 1% STZ solution (dissolved in pH 4.5 citrate-sodium citrate buffer) at a dose of 40 mg / kg body weight for 4 consecutive days. Blood glucose levels were measured every 5 days. A successful model was defined as a blood glucose concentration greater than 11.1 mmol / L after 4 weeks. Twenty successfully modeled diabetic mice were then randomly divided into five groups: control group (saline), Tegaderm group, CMCS / OSA group, CMCS-IGF1C / OSA group, and CMCS-IGF1C / OSA / EGCG group. All surgeries were performed on anesthetized mice (0.5% sodium pentobarbital, 0.2 mL / 10 g intraperitoneally). The fur on the backs of the mice was shaved with an electric animal razor, and the surgical areas were disinfected with alcohol. Then, two circular, full-thickness skin wounds, each 8 mm in diameter, were created on the back of each mouse using a punch, photographed, and 0.3 mL of hydrogel was applied to the wounds using a syringe. Dressings were changed and photographed on days 0, 3, 7, 10, and 15. The wound area was assessed using ImageJ software to quantify the wound healing rate.

[0086] The results showed that, compared with the control group, Tegaderm group, CMCS / OSA group and CMCS-IGF1C / OSA group, CF1 / OSA / EGCG hydrogel could significantly promote the healing of full-thickness skin wounds in diabetic mice.

[0087] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing azidomethyl chitosan, characterized in that, The azidated carboxymethyl chitosan is obtained by azidation reaction using carboxymethyl chitosan and fluorosulfonyl azide as raw materials.

2. The preparation method according to claim 1, characterized in that, The ratio of carboxymethyl chitosan to fluorosulfonyl azide solution is 220.0 mg : 0.01 mL to 0.5 mL, the concentration of fluorosulfonyl azide solution is 165 to 170 mmol / L, and the azidization reaction is carried out at 20 to 50 °C for 12 to 24 h.

3. An azide-modified carboxymethyl chitosan prepared by the method according to any one of claims 1 to 2.

4. A method for preparing glucose-responsive polysaccharide-based hydrogels using the azide-modified carboxymethyl chitosan of claim 3, characterized in that, Includes the following steps: Add an alkyne-modified insulin-like growth factor 1 C-domain polypeptide, a copper source, and a reducing agent to the solution of the azidomethyl chitosan, and perform a click reaction to obtain modified carboxymethyl chitosan. Modified sodium alginate was obtained by condensation reaction of aldehyde-modified sodium alginate and 3-aminophenylboronic acid under the action of a coupling agent. Polyphenolic substances are added to the solution of modified carboxymethyl chitosan, and then mixed with the solution of modified sodium alginate. The amino groups of modified carboxymethyl chitosan and the carboxyl groups of modified sodium alginate are cross-linked through Schiff base reaction. The polyphenolic substances are linked to the hydrogel network through boron ester bonds and hydrogen bonds, thus obtaining the glucose-responsive polysaccharide hydrogel.

5. The method according to claim 4, characterized in that, The aldehyde-modified sodium alginate is obtained by adding an oxidizing agent to an aqueous solution of sodium alginate and carrying out an oxidation reaction.

6. The method according to claim 5, characterized in that, The click reaction was carried out at 30-50°C for 12-24 hours. The condensation reaction was carried out at 30-50°C for 12-24 hours. The oxidant is sodium periodate, and the oxidation reaction is carried out at 20~50℃ for 2~8 h. After the reaction is completed, ethylene glycol is added to terminate the reaction, and the termination time is 1~3 h.

7. The method according to claim 4, characterized in that, The copper source is a water-soluble copper salt, the reducing agent is sodium ascorbate, the coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the polyphenolic substance is epigallocatechin gallate.

8. The method according to claim 7, characterized in that, The mass ratio of the azidomethyl chitosan to the C-domain polypeptide of alkynylated insulin-like growth factor 1 is 55:6.5~37.

5. The mass ratio of the modified carboxymethyl chitosan, epigallocatechin gallate, and the modified sodium alginate is 1~10:0.1~1:1~10.

9. A glucose-responsive polysaccharide hydrogel prepared according to the method of claim 4.

10. The use of the glucose-responsive polysaccharide-based hydrogel according to claim 9, characterized in that, Including any of the following: (1) Prepare products that promote wound healing; (2) Preparation of antioxidant products; (3) Prepare antibacterial products.

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