A dual-network crosslinked hydrogel and a preparation method and application thereof

Abstract

The application discloses a kind of double-network crosslinked hydrogel and its preparation method and application, belong to the field of biomedical materials.The double-network crosslinked hydrogel is composed of two aqueous solutions, one of which is an aqueous solution containing 1-15% carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosan by mass volume ratio, the other aqueous solution is an aqueous solution containing 1-15% dialdehyde-based oxidized polysaccharide by mass volume ratio, and the two aqueous solutions are mixed to form a double-network crosslinked hydrogel.The hydrogel of the present application undergoes temperature-sensitive physical crosslinking and Schiff base chemical crosslinking at body temperature to form a double-network hydrogel, which has better gel strength than single temperature-sensitive physical crosslinking hydrogel or single Schiff base chemical crosslinking hydrogel, prolongs the in vivo degradation time of the hydrogel, improves the cell compatibility of the hydrogel, is easy to use and operate, and can be used to prepare in vivo or in vitro absorbable medical devices or medical materials or drugs.

Description

Technical Field

[0001] This invention relates to a medical and bioengineering product, and in particular to a method for preparing a dual-network cross-linked hydrogel and its application, belonging to the field of biomedical materials. Background Technology

[0002] Natural biopolymer hydrogels are a type of three-dimensional network hydrogel formed by the cross-linking of biopolymers. They can absorb large amounts of water and possess extracellular matrix-like properties, providing a favorable microenvironment for cell adhesion and tissue remodeling. Biopolymer hydrogels have great application potential in the field of biomedical materials, such as wound healing dressings, hemostatic materials, tissue engineering cell scaffolds, and small molecule drug and growth factor delivery carriers. From the perspective of medical materials, higher requirements are also placed on the performance and safety of biopolymer hydrogels.

[0003] Thermosensitive hydrogels are typical physically cross-linked hydrogels, exhibiting temperature-responsiveness. They exist in a liquid state at lower temperatures below their temperature-sensitive point and in a gel state above it. Hydroxybutyl chitosan hydrogel is a thermosensitive hydrogel, with a temperature-sensitive point typically between 15 and 18°C. Lower temperature-sensitive points can occur depending on the preparation conditions. The doctoral dissertation, "Preparation of Hydroxybutyl Chitosan and its Hydrogel Sensitivity (Temperature, pH) and Biocompatibility Study" (Li Jingjing, 2011), detailed the rheological properties of hydroxybutyl chitosan hydrogels. The abstract stated that the gelation temperature of 5% hydroxybutyl chitosan with distilled water as a solvent was 16.5°C, and the gelation temperature with PBS buffer at pH 7.4 as a solvent was 15.5°C. This indicates that the gelation temperature of hydroxybutyl chitosan hydrogel under near-neutral normal pH conditions is less than 17°C, which is relatively low. The abstract of the thesis also recorded that hydroxybutyl chitosan exhibited a strong inhibitory effect on L929 cell migration, with an inhibition rate of 48% at a concentration of 0.25 mg / ml. The master's thesis, "Study on the Biosafety of Thermosensitive Hydroxybutyl Chitosan" (Chen Yinsheng, 2016), studied the biosafety of hydroxybutyl chitosan in detail. The abstract of the thesis recorded the toxic effects of hydroxybutyl chitosan extract on L929 cells. In the 25% extract group, the cell proliferation rates on days 2 and 4 were 44.6% and 14.5%, respectively, and apoptosis occurred in both groups. Therefore, the application of hydroxybutyl chitosan thermosensitive hydrogels in the field of biomedical materials still needs to solve two key technical problems: First, improving the temperature sensitivity of the hydroxybutyl chitosan thermosensitive hydrogel. Existing hydroxybutyl chitosan has a low temperature sensitivity, typically below 17°C. It is liquid at low temperatures and a non-flowing gel above 17°C. This low temperature sensitivity makes injection, introduction, and application of the thermosensitive hydrogel at room temperature difficult, resulting in a short operable time at room temperature. Second, improving the cell compatibility of hydroxybutyl chitosan. Existing hydroxybutyl chitosan exhibits inhibitory effects on the growth and migration of L929 fibroblasts, indicating poor cell compatibility, which is detrimental to its application in in vivo tissues. In chemically cross-linked hydrogels, biopolymers form Schiff bases through their own aldehyde and amino groups, resulting in chemically cross-linked hydrogels without the need for external cross-linking agents and without residual harmful substances. For example, when sodium alginate aqueous solution is mixed with hydroxypropyl chitosan aqueous solution, the aldehyde group of the former cross-links with the amino group of the latter through Schiff base to form a hydrogel. Therefore, biopolymers form hydrogels by cross-linking aldehyde and amino groups to form Schiff bases, which neither introduces nor produces harmful residues, making them a promising candidate for medical applications. However, the cross-linking strength of Schiff bases is relatively weak, and they are prone to hydrolysis, exhibiting inherent instability. Summary of the Invention

[0004] To address the aforementioned problems, the first objective of this invention is to provide a dual-network cross-linked hydrogel formed from two aqueous solutions, which has better gel strength and cell compatibility, thereby overcoming the shortcomings of single-cross-linked hydrogels.

[0005] One aqueous solution of the present invention is an aqueous solution containing carboxylated hydroxybutyl chitosan, and another aqueous solution is an aqueous solution containing a dialdehyde-containing oxidized polysaccharide. The two aqueous solutions are mixed to form a double-network cross-linked hydrogel. The dialdehyde-containing oxidized polysaccharide is, for example, oxidized hydroxyethyl cellulose, oxidized hydroxypropyl cellulose, sodium or potassium salt of oxidized carboxymethyl cellulose, oxidized chitosan, oxidized hydroxyethyl chitosan, oxidized hydroxypropyl chitosan, sodium or potassium salt of oxidized carboxymethyl chitosan, oxidized hydroxyethyl chitin, oxidized hydroxypropyl chitin, or sodium salt of oxidized carboxymethyl chitin. The product is a potassium salt, a sodium or potassium salt of oxidized hyaluronic acid containing a dialdehyde group, or a sodium or potassium salt of oxidized alginate containing a dialdehyde group; the carboxylated hydroxybutyl chitosan is a chitosan backbone with side chains having hydroxybutyl and carboxyl groups, wherein the carboxyl side chain refers to a side chain with a carboxyl group structure, such as carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, or succinyl, attached to the -NH2 position of the chitosan; the aqueous solution refers to a solution in which the solvent is water, physiological saline, phosphate buffer, Green's solution, glucose solution, or other commonly used aqueous solvents; the dual-network hydrogel is a dual-network crosslinked hydrogel formed by a thermosensitive physical crosslinking network and a Schiff base formed by the reaction of aldehyde and amino groups.

[0006] Furthermore, the aqueous solution containing carboxylated hydroxybutyl chitosan of the present invention may also contain other water-soluble chitosans, such as one or more of sodium or potassium salts of carboxymethyl chitosan, sodium or potassium salts of carboxyethyl chitosan, hydroxyethyl chitosan, and hydroxypropyl chitosan.

[0007] The above-mentioned dual-network crosslinked hydrogel consists of two aqueous solutions, one of which is an aqueous solution containing carboxylated hydroxybutyl chitosan or containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans (hereinafter referred to as Solution A). This aqueous solution is thermosensitive, with a temperature sensitivity range of 20–37°C. The thermosensitive characteristic of this aqueous solution originates from carboxylated hydroxybutyl chitosan, which is a key component of the physical crosslinking network in the dual-network hydrogel. The carboxylated hydroxybutyl chitosan has side chains with both hydroxybutyl and carboxyl groups in its molecular structure. The presence of the carboxyl side chains... The original hydrogen bond balance of hydroxybutyl chitosan hydrogel is disrupted, thereby increasing the temperature sensitivity of the carboxylated hydroxybutyl chitosan aqueous solution to a range of 20–37°C, overcoming the low temperature sensitivity of existing hydroxybutyl chitosan thermosensitive hydrogels. Simultaneously, the carboxyl group structure in carboxylated hydroxybutyl chitosan has a negative charge, weakening the original positive charge of -NH2 and reducing the negative effects of positive charge on cells. This significantly improves the cell compatibility of the aforementioned carboxylated hydroxybutyl chitosan, eliminating cytotoxicity and compensating for the unsatisfactory cell compatibility of hydroxybutyl chitosan thermosensitive hydrogels.

[0008] Another aqueous solution in the aforementioned dual-network cross-linked hydrogel is an aqueous solution of oxidized polysaccharide containing dialdehyde groups (hereinafter referred to as solution B). When solution A and solution B are mixed at a temperature below the temperature-sensitive point of solution A, and then the temperature is increased, the mixture forms a dual-network cross-linked hydrogel. When solution A and solution B are mixed, Schiff base cross-linking occurs between the amino groups of carboxylated hydroxybutyl chitosan in solution A or the amino groups of carboxylated hydroxybutyl chitosan and water-soluble chitosan and the aldehyde groups of oxidized polysaccharide containing dialdehyde groups in solution B, forming a chemically cross-linked hydrogel. At the same time, when the temperature is higher than the temperature-sensitive point of solution A, the carboxylated hydroxybutyl chitosan in the mixture undergoes a temperature response, forming a physically cross-linked hydrogel. Thus, when solution A and solution B are mixed, under body temperature conditions, both temperature-sensitive physical cross-linking and Schiff base chemical cross-linking occur simultaneously, forming a dual-network hydrogel. This dual-network hydrogel exhibits better gel strength than either a single thermosensitive physically cross-linked hydrogel or a single Schiff base chemically cross-linked hydrogel, prolongs the in vivo degradation time of the hydrogel, improves the cell compatibility of the hydrogel, and is easy to use.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned dual-network cross-linked hydrogel composed of two aqueous solutions, comprising the following steps: 1. Preparing carboxylated hydroxybutyl chitosan to obtain a carboxylated hydroxybutyl chitosan hydrogel with a higher temperature sensitivity point, thereby overcoming the shortcomings of the prior art where the temperature sensitivity point of the hydroxybutyl chitosan thermosensitive hydrogel is low; 2. Preparing oxidized dialdehyde polysaccharide; 3. Preparing an aqueous solution (solution A) containing carboxylated hydroxybutyl chitosan or containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans to obtain a thermosensitive physically cross-linked hydrogel network, and obtaining an amino donor for the chemical cross-linking of Schiff bases; 4. Preparing an aqueous solution (solution B) containing dialdehyde oxidized polysaccharide to obtain an aldehyde donor for the chemical cross-linking of Schiff bases; 5. Mixing solution A and solution B to form a dual-network cross-linked hydrogel.

[0010] A third objective of this invention is to provide applications of the aforementioned dual-network crosslinked hydrogel composed of two aqueous solutions.

[0011] The specific technical solution of the present invention is as follows:

[0012] A dual-network crosslinked hydrogel, characterized in that the dual-network crosslinked hydrogel is composed of two aqueous solutions, wherein one aqueous solution is an aqueous solution containing 1-15% (m / v) carboxylated hydroxybutyl chitosan, and the other aqueous solution is an aqueous solution containing 1-15% (m / v) dialdehyde-containing oxidized polysaccharide, the two aqueous solutions being mixed to form the dual-network crosslinked hydrogel; wherein the dialdehyde-containing oxidized polysaccharide is dialdehyde-containing oxidized hydroxyethyl cellulose, dialdehyde-containing oxidized hydroxypropyl cellulose, dialdehyde-containing sodium or potassium salt of oxidized carboxymethyl cellulose, dialdehyde-containing oxidized chitosan, dialdehyde-containing oxidized hydroxyethyl chitosan, dialdehyde-containing oxidized hydroxypropyl chitosan, or dialdehyde-containing... The polysaccharide comprises one of the following: sodium or potassium salt of oxidized carboxymethyl chitosan, oxidized hydroxyethyl chitosan containing a dialdehyde group, oxidized hydroxypropyl chitosan containing a dialdehyde group, sodium or potassium salt of oxidized carboxymethyl chitosan containing a dialdehyde group, sodium or potassium salt of oxidized hyaluronic acid containing a dialdehyde group, or sodium or potassium salt of oxidized alginate containing a dialdehyde group. The percentage of dialdehyde-containing sugar units in the oxidized polysaccharide is 5%–100% of the total sugar units of the polysaccharide. The carboxylated hydroxybutyl chitosan is a chitosan backbone with side chains containing hydroxybutyl and carboxyl groups. The carboxyl-containing side chains refer to carboxymethyl side chains, carboxyethyl side chains, carboxypropyl side chains, carboxybutyl side chains, succinyl side chains, or other aliphatic or aromatic side chains with carboxyl groups.

[0013] Furthermore, the aqueous solution containing carboxylated hydroxybutyl chitosan of the present invention may also contain other water-soluble chitosans, such as one or more of sodium or potassium salts of carboxymethyl chitosan, sodium or potassium salts of carboxyethyl chitosan, hydroxyethyl chitosan, and hydroxypropyl chitosan.

[0014] The temperature-sensitive point of the carboxylated hydroxybutyl chitosan or an aqueous solution containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans can be adjusted between 20 and 37°C by controlling the different lengths and degrees of substitution of the side chains containing carboxyl groups in the carboxylated hydroxybutyl chitosan.

[0015] The aqueous solution refers to a solution in which the solvent is water, physiological saline, phosphate buffer, Green's solution, glucose solution or other commonly used aqueous solvents; the dual-network hydrogel is a dual-network hydrogel formed by a thermosensitive physical cross-linking network and a chemical cross-linking network of aldehyde and amino Schiff bases.

[0016] The method for preparing the above-mentioned dual-network crosslinked hydrogel is characterized by comprising the following steps:

[0017] (1) Prepare carboxylated hydroxybutyl chitosan by any of the following methods:

[0018] 1) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of 30-90% (v / v) organic solvent, stir and disperse to prepare a 0.5-20% (m / v) hydroxybutyl chitosan suspension; add carboxylating agent at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to carboxylating agent glyoxylic acid or levulinic acid, stir and react for 1-24 h at room temperature or under heating, and adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution after the reaction; slowly add 1-10% NaBH4 aqueous solution to the reaction system at room temperature for reduction reaction for 1-5 h; wash the reaction product with ethanol aqueous solution to desalt, dehydrate with ethanol, and dry, or dialyze the reaction product with deionized water at 4-10℃ or room temperature, and freeze-dry the dialysate to obtain carboxymethyl hydroxybutyl chitosan or carboxybutyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan.

[0019] 2) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of 30-90% (v / v) organic solvent, stir and disperse to prepare a 0.5-20% (m / v) hydroxybutyl chitosan suspension; adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution, add an aqueous solution of carboxylating agent dropwise at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to carboxylating agent chloroacetic acid, chloropropionic acid, chlorolactic acid, or acrylic acid, and stir the reaction at room temperature or under heating for 1-24 h; after the reaction, adjust the pH of the reaction system to neutral with dilute acid or dilute alkali solution, wash the reaction product with aqueous ethanol solution to desalt, dehydrate with ethanol, and dry, or dialyze the reaction product with deionized water at 4-10℃ or room temperature, freeze-dry the dialysate under vacuum to obtain carboxymethyl hydroxybutyl chitosan or carboxyethyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan.

[0020] 3) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of a common organic solvent at a concentration of 80-100% (v / v), stir and disperse to prepare a 0.5-20% (m / v) hydroxybutyl chitosan suspension; add an organic solvent solution of the carboxylating agent at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to the carboxylating agent succinic anhydride (succinic anhydride), and react at room temperature or with heating and stirring for 1-24 h; after the reaction, adjust the pH of the reaction system to neutral with a dilute alkaline solution, wash the reaction product with an aqueous ethanol solution to desalt, dehydrate with ethanol, and dry; or dialyze the reaction product with deionized water at 4-10℃ or room temperature, and freeze-dry the dialysate under vacuum to obtain succinyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan.

[0021] 4) Add chitosan to an aqueous solution of a common organic solvent at a concentration of 50-90% (v / v), stir and disperse to prepare a chitosan suspension at a concentration of 1-15% (m / v). Add an aqueous solution of the carboxylating agent (concentration range may be required) dropwise according to a molar ratio of chitosan to carboxylating agent glyoxylic acid or levulinic acid of 1:(0.1-5). Stir the reaction at room temperature or under heating for 1-24 h. After the reaction, adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution. Slowly add 1-10% NaBH4 aqueous solution to the reaction system at room temperature for a reduction reaction of 1-5 h. Wash the reactants with an aqueous ethanol solution to desalt, dehydrate with ethanol, and dry to obtain carboxymethyl chitosan or carboxybutyl chitosan, i.e., carboxylated chitosan. Carboxylated chitosan is added to an aqueous solution of a common organic solvent at a concentration of 50–90% (v / v), stirred and dispersed to prepare a 1–15% (m / v) carboxylated chitosan suspension. The pH of the suspension is adjusted to alkaline with an alkaline solution. While heating and stirring, 1,2-epoxybutane is added dropwise at a molar ratio of 1:(5–30) of carboxylated chitosan N. The reaction is carried out under heating and stirring for 12–48 h. After the reaction, the pH of the reaction system is adjusted to neutral with a dilute acid solution. The reactants are washed with an aqueous ethanol solution to desalt, dehydrated with ethanol, and dried. Alternatively, the reactants are dialyzed with deionized water at 4–10 °C or room temperature. The dialysate is then freeze-dried under vacuum to obtain carboxylated hydroxybutylated chitosan.

[0022] 5) Add carboxylated chitosan to an aqueous solution of 50-90% (v / v) of a common organic solvent, stir and disperse to prepare a 1-15% (m / v) carboxylated chitosan suspension. Adjust the pH of the suspension to alkaline with alkali solution. Under heating and stirring, add 1,2-epoxybutane dropwise at a molar ratio of 1:(5-30) of carboxylated chitosan moles to 1,2-epoxybutane. Stir and react for 12-48 hours under heating. After the reaction, adjust the pH of the reaction system to neutral with dilute acid solution. Wash the reactants with an aqueous ethanol solution to desalt, dehydrate with ethanol, and dry, or dialyze the reactants with deionized water at 4-10°C or room temperature. Freeze-dry the dialysate under vacuum to obtain carboxylated hydroxybutylated chitosan. The carboxylated chitosan refers to one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, carboxybutyl chitosan, or succinoyl chitosan.

[0023] (2) Preparation of oxidized polysaccharides containing dialdehyde groups:

[0024] Prepare a 1-15% (v / v) polysaccharide aqueous solution. Add solid NaIO4 or KIO4 oxidant under stirring at a polysaccharide to oxidant molar ratio of 1:(0.01-1). React at 4℃ to room temperature in the dark for 2-36 hours. The reactants are precipitated with ethanol, washed with ethanol aqueous solution for desalting, dehydrated with anhydrous ethanol, and dried. Alternatively, the reactants are dialyzed with deionized water at 4-10℃ or room temperature. The dialysate is freeze-dried under vacuum to obtain oxidized polysaccharides containing dialdehyde groups.

[0025] (3) Prepare an aqueous solution containing carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans:

[0026] Take carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans, and prepare an aqueous solution of 1-15% (m / v), wherein the proportion of carboxylated hydroxybutyl chitosan in the carboxylated hydroxybutyl chitosan and other water-soluble chitosans is not less than 50% (m / m), to obtain a temperature-sensitive aqueous solution containing carboxylated hydroxybutyl chitosan and / or other water-soluble chitosans.

[0027] (4) Preparation of aqueous solutions of oxidized polysaccharides containing dialdehyde groups:

[0028] Take the oxidized polysaccharide containing dialdehyde group and prepare an aqueous solution of 1-15% (m / v) to obtain the aqueous solution of the oxidized polysaccharide containing dialdehyde group.

[0029] (5) Preparation of a dual-network cross-linked hydrogel composed of two aqueous solutions:

[0030] Take equal volumes of an aqueous solution containing carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans, and an aqueous solution of oxidized polysaccharide containing dialdehyde groups. Mix the two equal volumes of solutions evenly, or put them into the two syringes of a double syringe and push them out simultaneously and mix them evenly to obtain a double-network cross-linked hydrogel composed of the two aqueous solutions.

[0031] The carboxylating agent is not limited to the specific carboxylating agents mentioned above. Those skilled in the art can achieve the purpose of this invention by using other carboxylating agents to graft other side chains with carboxyl groups onto the -NH2 position of chitosan, such as carboxymethyl side chains, carboxyethyl side chains, carboxypropyl side chains, carboxybutyl side chains, succinyl side chains, or other aliphatic or aromatic side chains with carboxyl groups. The organic solvents mentioned above refer to ethanol, isopropanol, acetone, methanol, or other commonly used organic solvents. The aqueous solutions mentioned above refer to solutions with water, physiological saline, phosphate buffer, Green's solution, glucose solution, or other commonly used aqueous solvents.

[0032] The application of the dual-network cross-linked hydrogel is characterized by its use in the preparation of medical devices, medical materials, or drugs for hemostasis, wound exudate sealing, and cerebrospinal fluid sealing.

[0033] The application of the dual-network cross-linked hydrogel is characterized by its use in the preparation of medical devices, medical materials, or drugs for preventing postoperative tissue adhesions, abdominal adhesions, and wound healing.

[0034] The application of the dual-network crosslinked hydrogel is characterized by its use in the preparation of medical devices, medical materials, or drugs for digestive tract mucosal protection, lacrimal duct plugs, and tissue filling.

[0035] The application of the dual-network cross-linked hydrogel is characterized by its use in the preparation of medical devices, medical materials, and tissue engineering scaffolds for cell carriers, 3D cell culture, drug carriers, and active factor carriers.

[0036] The dual-network cross-linked hydrogel of this invention, composed of two aqueous solutions, is a dual-network hydrogel formed by thermosensitive physical cross-linking and Schiff base chemical cross-linking under body temperature conditions. This dual-network hydrogel exhibits better gel strength than either a single thermosensitive physical cross-linked hydrogel or a single Schiff base chemical cross-linked hydrogel, prolongs the in vivo degradation time, improves cell compatibility, and is convenient to use. The dual-network cross-linked hydrogel of this invention, composed of two aqueous solutions, can be degraded and absorbed in vivo, laying an important foundation for its application in the preparation of absorbable medical devices, medical materials, or drugs for in vivo and in vitro use. Attached Figure Description

[0037] Figure 1This is a diagram showing the subcutaneous tissue filling and degradation of a dual-network cross-linked hydrogel composed of two aqueous solutions. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and tables, and through specific embodiments.

[0039] Example 1: Preparation of carboxylated hydroxybutyl chitosan-1

[0040] 7 g (approximately 0.03 mol) of thermosensitive hydroxybutyl chitosan was added to 100 ml of 70% (v / v) ethanol aqueous solution and stirred to disperse, thus preparing a 7% (m / v) hydroxybutyl chitosan suspension. At a molar ratio of 1:1 between hydroxybutyl chitosan and the carboxylating agent glyoxylic acid, 10 ml (approximately 0.03 mol) of an aqueous solution containing 2.22 g of glyoxylic acid was added dropwise. The mixture was stirred at 40°C for 3 h. After the reaction, the pH of the reaction system was adjusted to 7.5 with dilute alkali solution. 6 ml of 20% NaBH4 aqueous solution was slowly added dropwise to the reaction system at room temperature for a reduction reaction of 2 h. The reaction product was dialyzed against deionized water at 6–10°C, and the dialysate was freeze-dried under vacuum to obtain carboxymethyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-1.

[0041] In Example 1, the method for preparing carboxylated hydroxybutyl chitosan-1 can be summarized as follows: Hydroxybutyl chitosan with temperature sensitivity is added to an aqueous solution of 30–90% (v / v) ethanol, isopropanol, acetone, methanol, or other commonly used organic solvents, and the mixture is stirred and dispersed to prepare a 0.5–20% (m / v) hydroxybutyl chitosan suspension; a carboxylating agent is added at a molar ratio of 1:(0.1–5) of hydroxybutyl chitosan to the carboxylating agent glyoxylic acid or levulinic acid, and the mixture is stirred and reacted at room temperature or under heating for 1–24 h; after the reaction, the pH of the reaction system is adjusted to neutral or alkaline with dilute alkali solution; a 1–10% NaBH4 aqueous solution is slowly added dropwise to the reaction system at room temperature for a reduction reaction of 1–5 h. h; The reaction product is washed with an aqueous ethanol solution to desalt, dehydrated with ethanol, and dried, or the reaction product is dialyzed with deionized water at 4-10°C or room temperature, and the dialysate is freeze-dried under vacuum to obtain carboxymethyl hydroxybutyl chitosan or carboxybutyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-1.

[0042] Example 2: Preparation of carboxylated hydroxybutyl chitosan-2

[0043] 4.67 g (approximately 0.02 mol) of thermosensitive hydroxybutyl chitosan was added to 100 ml of 50% (v / v) ethanol aqueous solution and stirred to disperse, thus preparing a 4.67% (m / v) hydroxybutyl chitosan suspension. The pH of the reaction system was adjusted to 11 with dilute alkali solution. 7.5 ml of 50% ethanol aqueous solution containing 1.5 g of chloroacetic acid was added dropwise at a molar ratio of 1:0.8 between hydroxybutyl chitosan and the carboxylating agent chloroacetic acid. The reaction was heated at 55 °C and stirred for 12 h. After the reaction, the pH of the reaction system was adjusted to 7.5 with dilute acid or dilute alkali solution. The reaction product was washed with 75% ethanol aqueous solution to desalt, dehydrated with ethanol, and dried to obtain carboxymethyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-2.

[0044] In Example 2, the method for preparing carboxylated hydroxybutyl chitosan-2 can be summarized as follows: Hydroxybutyl chitosan with temperature sensitivity is added to an aqueous solution of 30–90% (v / v) ethanol, isopropanol, acetone, methanol, or other commonly used organic solvents, and the mixture is stirred and dispersed to prepare a 0.5–20% (m / v) hydroxybutyl chitosan suspension; the pH of the reaction system is adjusted to neutral or alkaline with dilute alkali solution; an aqueous solution of the carboxylating agent is added dropwise at a molar ratio of hydroxybutyl chitosan to the carboxylating agent chloroacetic acid, chloropropionic acid, chlorolactic acid, or acrylic acid of 1:(0.1–5); the reaction is stirred at room temperature or under heating for 1–24 hours. h; After the reaction, the pH of the reaction system is adjusted to neutral with dilute acid or dilute alkali solution. The reaction product is washed with ethanol aqueous solution to desalt, dehydrated with ethanol, and dried. Alternatively, the reactants are dialyzed with deionized water at 4-10℃ or room temperature. The dialysate is freeze-dried under vacuum to obtain carboxymethyl hydroxybutyl chitosan or carboxyethyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-2.

[0045] Example 3: Preparation of carboxylated hydroxybutyl chitosan-3

[0046] 9.32 g (approximately 0.04 mol) of thermosensitive hydroxybutyl chitosan was added to 100 ml of 90% (v / v) aqueous ethanol solution and stirred to disperse, thus preparing a 9.3% (m / v) hydroxybutyl chitosan suspension. 15 ml of an ethanol solution containing 1.2 g of succinic anhydride was added at a molar ratio of 1:0.3 between hydroxybutyl chitosan and the carboxylating agent succinic anhydride. The mixture was stirred at room temperature for 6 h. After the reaction, the pH of the reaction system was adjusted to 7.0 with a dilute alkaline solution. The reactants were washed with 75% aqueous ethanol solution to desalt, dehydrated with anhydrous ethanol, and dried to obtain succinyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-3.

[0047] In Example 3, the method for preparing carboxylated hydroxybutyl chitosan-3 can be summarized as follows: Hydroxybutyl chitosan with temperature sensitivity is added to an aqueous solution of 80–100% (v / v) ethanol, isopropanol, acetone, methanol, or other commonly used organic solvents, and the mixture is stirred and dispersed to prepare a 0.5–20% (m / v) hydroxybutyl chitosan suspension; an organic solvent solution of the carboxylating agent is added according to a molar ratio of hydroxybutyl chitosan to the carboxylating agent succinic anhydride (succinic anhydride) of 1:(0.1–5), and the reaction is carried out at room temperature or with heating and stirring for 1–24 h; after the reaction, the pH of the reaction system is adjusted to neutral with a dilute alkaline solution, and the reaction product is washed with an aqueous ethanol solution for desalting, dehydrated with ethanol, and dried; or the reaction product is dialyzed with deionized water at 4–10°C or room temperature, and the dialysate is freeze-dried under vacuum to obtain succinyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan-3.

[0048] In Examples 1-3 above, hydroxybutyl chitosan was provided by Ocean University of China, and the temperature-sensitive point of its aqueous solution was 17.5°C. The carboxylating agent is not limited to the specific carboxylating agent mentioned above. Those skilled in the art can use other carboxylating agents to graft side chains with carboxyl structures at the C2-NH2 position of hydroxybutyl chitosan, such as carboxymethyl side chains, carboxyethyl side chains, carboxypropyl side chains, carboxybutyl side chains, succinoyl side chains, or other aliphatic or aromatic side chains with carboxyl structures.

[0049] Example 4: Preparation of carboxylated hydroxybutyl chitosan-4

[0050] Add 5 g of carboxylated chitosan (approximately 0.025 mol) to 100 ml of 90% (v / v) aqueous ethanol solution, stir and disperse to prepare a 5% (m / v) carboxylated chitosan suspension. Adjust the pH of the suspension to 11 with alkali solution, heat and stir at 40 °C, and add 32.5 ml of 1,2-epoxybutane dropwise at a molar ratio of 1:15 for carboxylated chitosan 2. Stir and react for 24 h under heating. After the reaction, adjust the pH of the reaction system to 7.5 with dilute acid solution. Dialyze the reactants to deionized water at 4–10 °C or room temperature, and freeze-dry the dialysate under vacuum to obtain carboxylated hydroxybutylated chitosan-4.

[0051] In Example 4, the method for preparing carboxylated hydroxybutyl chitosan-4 can be summarized as follows: Carboxylated chitosan is added to an aqueous solution of 50-90% (v / v) ethanol, isopropanol, acetone, methanol, or other commonly used organic solvents, and stirred to disperse, forming a 1-15% (m / v) carboxylated chitosan suspension. The pH of the suspension is adjusted to alkaline with an alkaline solution. Under heating and stirring, 1,2-epoxybutane is added dropwise at a molar ratio of 1:(5-30) of carboxylated chitosan to 1,2-epoxybutane. The reaction is stirred under heating for 12-48 h. After the reaction, the pH of the reaction system is adjusted to neutral with a dilute acid solution. The reactants are washed with an aqueous ethanol solution to desalt, dehydrated with ethanol, and dried, or dialyzed with deionized water at 4-10 °C or room temperature. The dialysate is then freeze-dried under vacuum to obtain carboxylated hydroxybutyl chitosan-4. The carboxylated chitosan refers to one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, carboxybutyl chitosan, or succinoyl chitosan.

[0052] Example 5: Preparation of oxidized polysaccharide-1 containing dialdehyde groups

[0053] Prepare 200 ml of 2% (m / v) sodium alginate aqueous solution, add 0.5 g of NaIO4 solid oxidant, and stir at room temperature in the dark for 12 h. After the reaction is complete, stir and add 4 times the volume of 95% ethanol aqueous solution, precipitate, filter, wash with 95% ethanol aqueous solution to desalt, dehydrate with anhydrous ethanol, and vacuum dry to obtain sodium alginate oxidized polysaccharide containing dialdehyde group, namely dialdehyde oxidized polysaccharide-1, with a dialdehyde group percentage of 11.2% (the dialdehyde group percentage refers to the percentage of sugar units containing dialdehyde group in the polysaccharide molecule to the total sugar units of the polysaccharide molecule).

[0054] Example 6: Preparation of oxidized polysaccharide-2 containing dialdehyde groups

[0055] Prepare 200 ml of 2.5% (m / v) sodium carboxymethyl cellulose aqueous solution, add 1.2 g of solid KIO4 oxidant, and stir at room temperature in the dark for 24 h. After the reaction is complete, dialyze the reactants with deionized water and freeze-dry to obtain sodium carboxymethyl cellulose oxidized polysaccharide containing dialdehyde group, namely dialdehyde-containing oxidized polysaccharide-2, with a dialdehyde group percentage of 34.2%.

[0056] In Examples 5 and 6, the method for preparing oxidized polysaccharides containing dialdehyde groups can be summarized as follows: Prepare a 1-15% (v / v) polysaccharide aqueous solution, add solid NaIO4 or KIO4 oxidant under stirring at a polysaccharide to oxidant molar ratio of 1:(0.01-1), and react at 4°C to room temperature in the dark for 2-36 hours; the reactants are precipitated with ethanol, washed with ethanol aqueous solution for desalting, dehydrated with anhydrous ethanol, and dried, or the reactants are dialyzed with deionized water at 4-10°C or room temperature, and the dialysate is freeze-dried under vacuum to obtain oxidized polysaccharides containing dialdehyde groups.

[0057] The dialdehyde-containing oxidized polysaccharides described in Examples 5 and 6 can be prepared by oxidizing hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose (sodium or potassium salt), chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, carboxymethyl chitosan (sodium or potassium salt), hydroxyethyl chitin, hydroxypropyl chitin, carboxymethyl chitin (sodium or potassium salt), hyaluronic acid (sodium or potassium salt), or alginate (sodium or potassium salt) with an oxidizing agent to form dialdehyde groups in the molecules. The resulting dialdehyde-containing oxidized polysaccharides are oxidized hydroxyethyl cellulose containing dialdehyde groups. The oxidized polysaccharide comprises: hydroxypropyl cellulose containing dialdehyde groups, hydroxymethyl cellulose containing dialdehyde groups (sodium or potassium salts), oxidized chitosan containing dialdehyde groups, hydroxyethyl chitosan containing dialdehyde groups, hydroxypropyl chitosan containing dialdehyde groups, hydroxymethyl chitosan containing dialdehyde groups (sodium or potassium salts), hydroxyethyl chitin containing dialdehyde groups, hydroxypropyl chitin containing dialdehyde groups, hydroxymethyl chitin containing dialdehyde groups (sodium or potassium salts), oxidized hyaluronic acid containing dialdehyde groups (sodium or potassium salts), or oxidized alginate containing dialdehyde groups (sodium or potassium salts). The molar ratio of polysaccharide to oxidant is 1:(0.01–1), the reaction temperature can be 4℃ to room temperature, and the reaction time is 2–36 h. The percentage of dialdehyde-containing sugar units in the oxidized polysaccharide is 5%–100% of the total sugar units of the polysaccharide.

[0058] Example 7: Preparation of an aqueous solution containing carboxylated hydroxybutyl chitosan-1:

[0059] Take the carboxylated hydroxybutyl chitosan-1 prepared in Example 1 and prepare 100 ml of a 3% (m / v) aqueous solution at 20°C. The temperature-sensitive point of the aqueous solution is tested to be about 23°C, thus obtaining a temperature-sensitive aqueous solution containing carboxylated hydroxybutyl chitosan, namely, a temperature-sensitive aqueous solution containing carboxylated hydroxybutyl chitosan-1.

[0060] Example 8: Preparation of an aqueous solution containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans - 2:

[0061] All solvents, solutes, and equipment used were sterilized, and the operation was carried out under aseptic conditions. 3 g of carboxylated hydroxybutyl chitosan-1 prepared in Example 1 was dissolved in 100 ml of water at 20°C to obtain an aqueous solution of carboxylated hydroxybutyl chitosan. 2 g of water-soluble chitosan hydroxyethyl chitosan was added to the carboxylated hydroxybutyl chitosan aqueous solution and stirred to dissolve, yielding a 5% (m / v) aqueous solution containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans. The temperature-sensitive point of this aqueous solution was tested to be approximately 31°C, i.e., a temperature-sensitive aqueous solution containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans -2 (sterile).

[0062] The aqueous solutions of carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans described in Examples 7 and 8 are thermosensitive. The thermosensitivity is derived from carboxylated hydroxybutyl chitosan. The temperature sensitivity point can be adjusted between 20 and 37°C by controlling the length of the side chains containing carboxyl groups and the degree of substitution of the carboxylated hydroxybutyl chitosan.

[0063] In Examples 6 and 7, the method for preparing an aqueous solution containing carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans can be summarized as follows: Under aseptic conditions, carboxylated hydroxybutyl chitosan or carboxylated hydroxybutyl chitosan and other water-soluble chitosans are taken and prepared into an aqueous solution of 1-15% (m / v), wherein the proportion of carboxylated hydroxybutyl chitosan in the carboxylated hydroxybutyl chitosan and / or other water-soluble chitosans is not less than 50% (m / m), and the temperature-sensitive point of the aqueous solution is tested to obtain a temperature-sensitive aqueous solution (sterile) containing carboxylated hydroxybutyl chitosan and / or other water-soluble chitosans.

[0064] Example 9: Preparation of an aqueous solution of an oxidized polysaccharide containing a dialdehyde group:

[0065] All solvents, solutes, and equipment used were sterilized, and the operation was performed under aseptic conditions. 3 g of the dialdehyde-containing oxidized polysaccharide sodium alginate prepared in Example 5 was dissolved in 50 ml of water with stirring to obtain a 6% (m / v) aqueous solution of the dialdehyde-containing oxidized polysaccharide-1 (sterile). 1.5 g of the dialdehyde-containing oxidized polysaccharide sodium carboxymethyl cellulose prepared in Example 6 was dissolved in 50 ml of water with stirring to obtain a 3% (m / v) aqueous solution of the dialdehyde-containing oxidized polysaccharide-2 (sterile).

[0066] In Example 9, the method for preparing an aqueous solution of oxidized polysaccharide containing dialdehyde group can be summarized as follows: Under sterile conditions, oxidized polysaccharide containing dialdehyde group is taken and prepared into an aqueous solution of 1-15% (m / v) to obtain an aqueous solution of oxidized polysaccharide containing dialdehyde group (sterile).

[0067] In Examples 7-9, the aqueous solution refers to a solvent such as water, physiological saline, phosphate buffer, Green's solution, glucose solution, or other commonly used aqueous solvents.

[0068] Example 10: Preparation of a dual-network crosslinked hydrogel-1 composed of two aqueous solutions:

[0069] Take 3 ml of aqueous solution-2 containing carboxylated hydroxybutyl chitosan and / or other water-soluble chitosan prepared in Example 8, and 3 ml of aqueous solution-1 containing dialdehyde oxidized polysaccharide prepared in Example 9, respectively, and put them into the two syringes of a double syringe. They are then pushed out simultaneously and mixed evenly to form a hydrogel, thus obtaining a double-network crosslinked hydrogel-1 composed of the two aqueous solutions.

[0070] Example 11: Preparation of a dual-network crosslinked hydrogel-2 composed of two aqueous solutions:

[0071] Take 3 ml of aqueous solution-1 containing carboxylated hydroxybutyl chitosan prepared in Example 7 and 3 ml of aqueous solution-2 containing dialdehyde oxidized polysaccharide prepared in Example 9, respectively, and put them into the two syringes of a double syringe. They are then pushed out simultaneously and mixed evenly to form a hydrogel, thus obtaining a double-network crosslinked hydrogel-2 composed of the two aqueous solutions.

[0072] The methods for preparing dual-network cross-linked hydrogels composed of two aqueous solutions in Examples 10 and 11 can be summarized as follows: Take equal volumes of aqueous solutions containing carboxylated hydroxybutyl chitosan and other water-soluble chitosans, and aqueous solutions containing dialdehyde-containing oxidized polysaccharides. Mix the two equal volumes of solutions evenly, or load them into the two syringes of a dual syringe, push them out simultaneously, mix them evenly, and form a gel to obtain a dual-network cross-linked hydrogel composed of two aqueous solutions.

[0073] Example 12: Evaluation of the hemostatic effect of dual-network cross-linked hydrogels

[0074] Twelve male SD rats, weighing 200–240 g, were randomly divided into two groups: a double-network cross-linked hydrogel group (Group A) and a gauze control group (Group B), with six rats in each group. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital saline solution (30 mg / kg). The rats were fixed to a surgical frame with their abdomens facing upwards. The abdomen was shaved with a razor, disinfected with iodine, and the liver was surgically exposed. Pre-weighed gauze was placed under the liver to absorb bleeding. A 1.5 cm × 0.5 cm section of liver apex tissue was removed using surgical scissors. The wounds were covered with the double-network cross-linked hydrogel-1 prepared in Example 11 and gauze, respectively, according to the group. Hemostasis time was recorded for each group, the amount of blood-absorbing gauze was weighed, and the amount of bleeding was calculated. The experimental results are shown in Table 1. Regarding hemostasis time, the average hemostasis time for Group A and Group B was 70.43 ± 7.66 s and 125.33 ± 16.80 s, respectively. The hemostasis time of the double-network hydrogel group was significantly shorter than that of the gauze group, showing a significant difference. P <0.01). Regarding bleeding volume, the double-network hydrogel group showed significantly less bleeding than the gauze group, and also exhibited a significant reduction in bleeding time (…). P <0.01). This indicates that the dual-network cross-linked hydrogel composed of two aqueous solutions of the present invention has excellent hemostatic properties, and can form a gel in situ on the wound to seal the bleeding site, thereby playing a role in hemostasis.

[0075] Table 1 Hemostasis Time and Bleeding Volume

[0076] Group Group A Group B Hemostasis time (s) 70.426±7.663** 125.328±16.801 Blood loss (g) 0.326±0.137** 0.619±0.235

[0077] Compared with the gauze group (Group B), **P <0.01.

[0078] Example 13: Tissue filling and degradation absorption of dual-network cross-linked hydrogels

[0079] Rats were used as experimental animals and divided into two groups, A and B, with 20 rats in each group. Rats in group A were subcutaneously injected with 0.5 ml of the dual-network cross-linked hydrogel-1 prepared in Example 11, and rats in group B were subcutaneously injected with 0.5 ml of the dual-network cross-linked hydrogel-2 prepared in Example 11. The rats were fed normally after surgery. The tissue filling and bulging of the hydrogel were observed post-surgery, and the degradation of the hydrogel was observed by dissection at 1, 2, 4, and 6 weeks. The experimental results are as follows: Figure 1 As shown, the dual-network cross-linked hydrogel of the present invention, composed of two aqueous solutions, forms a visible bulge after subcutaneous injection in rats. Undegraded gel blocks are still visible 6 weeks after the operation, but the residual gel blocks are significantly smaller than the gel blocks at 1 week. As the gel remains in the body for a longer period of time, it gradually degrades and is absorbed. The local tissue of the gel does not have the irritation of foreign bodies such as redness and swelling, and it has good biocompatibility.

[0080] The dual-network cross-linked hydrogel composed of two aqueous solutions forms a gel block in the local tissue after injection, thus filling the tissue. Therefore, it can be injected submucosally during gastrointestinal mucosal surgery to form a protrusion and protect the mucosa; it can also be injected into the lacrimal duct to form a lacrimal plug. Therefore, the dual-network cross-linked hydrogel of the present invention, composed of two aqueous solutions, can be used in the preparation of medical devices, medical materials, or drugs for tissue filling, such as in the preparation of gastrointestinal mucosal protectants, lacrimal plugs, and tissue filling materials.

[0081] The dual-network cross-linked hydrogel of the present invention, composed of two aqueous solutions, forms a gel in situ on the wound surface to cover and seal the wound, thus enabling it to stop bleeding, seal tissue exudate, and seal cerebrospinal fluid and other exudates. At the same time, covering the wound surface also promotes wound healing. Therefore, it can be used in the preparation of medical devices, medical materials or drugs for hemostasis, wound healing, wound exudate sealing and cerebrospinal fluid sealing.

[0082] Example 14: Evaluation of the effect of dual-network cross-linked hydrogels in preventing postoperative tissue adhesions

[0083] Thirty female SD rats, weighing 200–230 g, were randomly divided into three groups: Group A (double-network cross-linked hydrogel-1), Group B (double-network cross-linked hydrogel-2), and Group C (blank control group), with 10 rats in each group. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital saline solution (30 mg / kg). The abdomen was shaved and disinfected with iodine. A 3 cm incision was made in the midline of the abdomen to open the abdominal cavity. Under aseptic conditions, the cecum was located, and its contents were squeezed out from the distal end. The cecum surface was continuously rubbed with sterile gauze until the outer serosa was damaged, resulting in beaded bleeding on the cecum surface. According to the groups, double-network cross-linked hydrogel-1, double-network cross-linked hydrogel-2, and saline were applied evenly to the injured cecum areas, respectively. The cecum was then returned to the abdominal cavity, and the incision was sutured. Postoperatively, the animals were housed separately. All animals were sacrificed 14 days later, and intestinal adhesions were observed through dissection. The degree of intestinal adhesion was classified into five levels, and the following grading standards were determined: (1) Grade 0: No adhesion at all, and the intestinal serosa is well repaired; (2) Grade I: The cecum is loosely and easily separated from the surrounding tissues, with no bleeding; (3) Grade II: The cecum is slightly to moderately adhered to the surrounding tissues, and there is local bleeding during separation; (4) Grade III: The intestinal loop is extensively adhered to the surrounding tissues, which is difficult to separate, and there is no intestinal obstruction; Grade IV: The intestinal loop is tightly adhered to the surrounding tissues, which is difficult to separate and causes intestinal obstruction.

[0084] The experimental results are shown in Table 2. The results show that, compared with the blank control group, the thermosensitive hydrogel A group and the thermosensitive hydrogel B group showed better repair of the abraded cecum in rats and less intestinal adhesion, with significant differences (P < 0.01). This indicates that the dual-network cross-linked hydrogel composed of two aqueous solutions of the present invention has a good effect in preventing postoperative tissue adhesion. At the same time, it was observed that the healing state of the damaged intestinal tissue in the hydrogel group was significantly better than that in the blank control group, which also shows that the dual-network cross-linked hydrogel composed of two aqueous solutions of the present invention has the effect of promoting the healing of damaged tissue. Therefore, it can be used in the preparation of medical devices, medical materials or drugs for preventing postoperative tissue adhesion, such as the preparation of medical devices or medical materials for preventing postoperative abdominal adhesion, pelvic adhesion, tendon adhesion, isolating soft tissue and hard tissue to prevent adhesion, and wound healing.

[0085] Table 2 Results of thermosensitive hydrogel in preventing postoperative tissue adhesions

[0086]

[0087] The dual-network cross-linked hydrogel of the present invention, composed of two aqueous solutions, can also be used in the preparation of medical devices, medical materials, and tissue engineering scaffolds for cell carriers, 3D cell culture, drug carriers, and active factor carriers.

Claims

1. A dual-network cross-linked hydrogel composed of two aqueous solutions, characterized in that, One of them The aqueous solution contains 1-15% by mass / volume of carboxylated hydroxybutyl chitosan, and the other aqueous solution contains 1-15% by mass / volume of an oxidized polysaccharide with dialdehyde groups. The two aqueous solutions are mixed to form a double-network cross-linked hydrogel. The carboxylated hydroxybutyl chitosan has side chains with hydroxybutyl and carboxyl groups on the chitosan backbone. These carboxyl side chains can be carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, or succinyl side chains. The carboxylated hydroxybutyl chitosan has side chains with both hydroxybutyl and carboxyl groups in its molecular structure, and its temperature sensitivity is 20-37°C. The oxidized polysaccharide containing dialdehyde groups is... The polysaccharide contains one of the following: oxidized hydroxyethyl cellulose, oxidized hydroxypropyl cellulose containing a dialdehyde group, sodium or potassium salt of oxidized carboxymethyl cellulose containing a dialdehyde group, oxidized chitosan containing a dialdehyde group, oxidized hydroxyethyl chitosan containing a dialdehyde group, oxidized hydroxypropyl chitosan containing a dialdehyde group, sodium or potassium salt of oxidized carboxymethyl chitosan containing a dialdehyde group, oxidized hydroxyethyl chitin containing a dialdehyde group, oxidized hydroxypropyl chitin containing a dialdehyde group, sodium or potassium salt of oxidized carboxymethyl chitin containing a dialdehyde group, oxidized hyaluronic acid containing a dialdehyde group, or oxidized alginate containing a dialdehyde group. In the oxidized polysaccharide containing a dialdehyde group, the percentage of sugar units containing a dialdehyde group in the total sugar units of the polysaccharide is 5% to 100%.

2. The dual-network crosslinked hydrogel as described in claim 1, characterized in that... The aqueous solution containing carboxylated hydroxybutyl chitosan also contains other water-soluble chitosans; the other water-soluble chitosans are one or more of sodium or potassium salts of carboxymethyl chitosan, sodium or potassium salts of carboxyethyl chitosan, hydroxyethyl chitosan, and hydroxypropyl chitosan.

3. The dual-network crosslinked hydrogel as described in claim 1, characterized in that... The aqueous solution refers to a solution in which the solvent is water, physiological saline, phosphate buffer, Green's solution, or glucose solution.

4. The method for preparing the dual-network crosslinked hydrogel as described in claim 1, characterized in that, Includes the following steps: (1) Prepare carboxylated hydroxybutyl chitosan by any of the following methods: 1) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of 30-90% v / v organic solvent, stir and disperse to prepare a 0.5-20% m / v hydroxybutyl chitosan suspension; add carboxylating agent at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to carboxylating agent glyoxylic acid or levulinic acid, stir and react for 1-24 h at room temperature or under heating, and adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution after the reaction; slowly add 1-10% NaBH4 aqueous solution to the reaction system at room temperature for reduction reaction for 1-5 h; wash the reaction product with ethanol aqueous solution to desalt, dehydrate with ethanol, and dry, or dialyze the reaction product with deionized water at 4-10℃ or room temperature, and freeze-dry the dialysate to obtain carboxymethyl hydroxybutyl chitosan or carboxybutyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan; 2) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of an organic solvent at 30-90% v / v, stir and disperse to prepare a 0.5-20% m / v hydroxybutyl chitosan suspension; adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution, add an aqueous solution of carboxylating reagent dropwise at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to carboxylating reagent chloroacetic acid, chloropropionic acid, chlorolactic acid, or acrylic acid, and stir the reaction at room temperature or under heating for 1-24 h; after the reaction, adjust the pH of the reaction system to neutral with dilute acid or dilute alkali solution, wash the reaction product with an aqueous ethanol solution to desalt, dehydrate with ethanol, and dry, or dialyze the reaction product with deionized water at 4-10℃ or room temperature, freeze-dry the dialysate under vacuum to obtain carboxymethyl hydroxybutyl chitosan or carboxyethyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan; 3) Add thermosensitive hydroxybutyl chitosan to an aqueous solution of a common organic solvent at 80-100% v / v, stir and disperse to prepare a 0.5-20% m / v hydroxybutyl chitosan suspension; add an organic solvent solution of the carboxylating agent at a molar ratio of 1:(0.1-5) of hydroxybutyl chitosan to the carboxylating agent succinic anhydride, and react at room temperature or with heating and stirring for 1-24 h; after the reaction, adjust the pH of the reaction system to neutral with a dilute alkaline solution, wash the reaction product with an aqueous ethanol solution to desalt, dehydrate with ethanol, and dry; or dialyze the reaction product with deionized water at 4-10℃ or room temperature, and freeze-dry the dialysate under vacuum to obtain succinyl hydroxybutyl chitosan, i.e., carboxylated hydroxybutyl chitosan; 4) Add chitosan to an aqueous solution of a common organic solvent (50-90% v / v), stir and disperse to prepare a chitosan suspension (1-15% m / v). Add an aqueous solution of the carboxylating agent dropwise at a molar ratio of chitosan to the carboxylating agent glyoxylic acid or levulinic acid of 1:(0.1-5). Stir the reaction at room temperature or under heating for 1-24 hours. After the reaction, adjust the pH of the reaction system to neutral or alkaline with dilute alkali solution. Slowly add a 1-10% NaBH4 aqueous solution to the reaction system at room temperature to carry out a reduction reaction for 1-5 hours. h; The reactants are washed with aqueous ethanol solution to desalt, dehydrated with ethanol, and dried to obtain carboxymethyl chitosan or carboxybutyl chitosan, i.e., carboxylated chitosan; Carboxylated chitosan is added to an aqueous solution of a common organic solvent at 50-90% v / v, stirred and dispersed to prepare a carboxylated chitosan suspension at 1-15% m / v, the pH of the suspension is adjusted to alkaline with alkali solution, and epoxide is added dropwise under heating and stirring at a molar ratio of carboxylated chitosan N to 1,2-epoxybutane of 1:(5-30), and the reaction is stirred under heating for 12-48 h. After the reaction, the pH of the reaction system is adjusted to neutral with dilute acid solution; The reactants are washed with aqueous ethanol solution to desalt, dehydrated with ethanol, and dried, or the reactants are dialyzed with deionized water at 4-10℃ or room temperature, and the dialysate is freeze-dried under vacuum to obtain carboxylated hydroxybutyl chitosan; (2) Preparation of aqueous solutions of oxidized polysaccharides containing dialdehyde groups: Prepare a 1-15% v / v polysaccharide aqueous solution. Add solid NaIO4 or KIO4 oxidant under stirring at a polysaccharide to oxidant molar ratio of 1:(0.01-1). React at 4℃ to room temperature in the dark for 2-36 hours. The reactants are precipitated with ethanol, washed with ethanol aqueous solution for desalting, dehydrated with anhydrous ethanol, and dried. Alternatively, the reactants are dialyzed with deionized water at 4-10℃ or room temperature. The dialysate is freeze-dried under vacuum to obtain oxidized polysaccharides containing dialdehyde groups. Take the oxidized polysaccharides containing dialdehyde groups and prepare an aqueous solution of 1-15% m / v to obtain an aqueous solution of oxidized polysaccharides containing dialdehyde groups. (3) Preparation of an aqueous solution containing carboxylated hydroxybutyl chitosan: Take carboxylated hydroxybutyl chitosan and prepare an aqueous solution of 1-15% m / v to obtain an aqueous solution containing carboxylated hydroxybutyl chitosan; (4) Preparation of a dual-network cross-linked hydrogel composed of two aqueous solutions: Take equal volumes of an aqueous solution containing carboxylated hydroxybutyl chitosan and an aqueous solution of oxidized polysaccharide containing dialdehyde groups, mix the two solutions evenly, or put them into the two syringes of a double syringe, push them out simultaneously, and mix them evenly to obtain a double-network cross-linked hydrogel composed of the two aqueous solutions.

5. The application of the dual-network cross-linked hydrogel as described in claim 1 in the preparation of medical devices, medical materials, or drugs for hemostasis, wound exudate sealing, and cerebrospinal fluid sealing.

6. The application of the dual-network cross-linked hydrogel as described in claim 1 in the preparation of medical devices, medical materials, or drugs for preventing postoperative tissue adhesions, tendon adhesions, abdominal adhesions, pelvic adhesions, isolating soft tissues from hard tissues to prevent adhesions, and wound healing.

7. The use of the dual-network crosslinked hydrogel as described in claim 1 in the preparation of medical devices, medical materials, or drugs for digestive tract mucosal protection agents, lacrimal duct plugs, and tissue filling agents.

8. The application of the dual-network cross-linked hydrogel as described in claim 1 in the preparation of medical devices, medical materials, and tissue engineering scaffolds for cell carriers, 3D cell culture, drug carriers, and active factor carriers.

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