Polysaccharide-based polymeric crosslinking agents, polysaccharide-based biomaterials, manufacturing methods and uses

Abstract

The present invention relates to polysaccharide-based polymeric crosslinkers, polysaccharide-based biomaterials, preparation methods, and uses. In the present invention, the polysaccharide-based polymeric crosslinker is a polysaccharide-based polymeric crosslinker modified with ortho-phthalaldehyde groups, which does not rely on a synthetic hydrophilic polymer backbone or complex chemical synthesis processes. The polysaccharide-based polymeric crosslinker provided by the present invention, together with water-soluble small molecules or polymers containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups, forms a two-component polysaccharide-based hydrogel material in an aqueous medium. This two-component polysaccharide-based hydrogel material has the advantages of being injectable, having low swelling, and having high tissue adhesion. Furthermore, the two-component polysaccharide-based hydrogel material provided by the present invention can be further fabricated into polysaccharide-based biomaterials in the form of membranes, powders, sponges, and other forms, and can be used as tissue adhesives, sealants, hemostats, tissue engineering materials, and other applications, demonstrating its potential for a wide range of biomedical applications.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomaterials, and in particular to polysaccharide-based polymeric crosslinkers, polysaccharide-based biomaterials, preparation methods and uses. [Background technology]

[0002] Hydrogels are polymeric materials with high water content and a three-dimensional crosslinked network structure. They have tunable physical and chemical properties, and are widely used in many biomedical fields due to their high water content, biocompatibility, and versatility. Two-component hydrogels can be clinically used as hemostats, tissue adhesives, and sealants due to their in situ molding and hardening properties. For example, DuraSeal rubber manufactured by Confluent Surgical, Inc. of the United States and CoSeal rubber manufactured by Confluent Surgical, Inc. of the United States, use polyethylene glycol as a backbone and crosslink through the reaction of the amino or mercapto groups at the end of the polyethylene glycol backbone with a carboxylic acid active ester. The carboxylic acid active ester reacts with active groups such as amino groups on the tissue surface to adhere to the tissue and seal the wound. Furthermore, Chinese Patents CN111440310A, CN111440334A, CN111574756A, CN111621038A, CN113509591A, CN113694249A, and CN114767920A disclose a series of ortho-phthalaldehyde-modified polyethylene glycol crosslinkers. Two-component hydrogels prepared by crosslinking ortho-phthalaldehyde with amino-containing polymers can be used as tissue sealants. However, due to the highly hydrophilic nature of the polyethylene glycol backbone, these hydrogels generally have a high swelling ratio (>100%). This high swelling not only significantly reduces the hydrogel's mechanical properties, but also increases the risk of compression of surrounding tissues and gel detachment, posing safety risks in clinical applications. For example, polyethylene glycol-based sealants, such as DuraSeal and CoSeal rubbers, are often used as auxiliary sealants after dural closure in neurosurgery to prevent cerebrospinal fluid leakage. However, in actual clinical applications, hydrogels can absorb body fluids, swell, and compress nerves, resulting in adverse events leading to limb paralysis (D Thavarajah, P De Lacy, R Hussain, RM Redfern, Spine. 2010, 35, 25-26).

[0003] To address the swelling issue, Chinese Patent CN114907558A discloses a method for producing a polyethylene glycol-based low-swelling hydrogel by modifying the polyethylene glycol polymer backbone to make it hydrophobic, thereby reducing its hydrophilicity. However, this method involves complex modifications to the polyethylene glycol backbone and involves multi-step chemical synthesis. Summary of the Invention

[0004] To solve the problem of high swelling ratio common to polyethylene glycol-based hydrogels in the prior art, the present invention provides a polysaccharide-based macromolecular crosslinker, a polysaccharide-based biomaterial, a method for preparing it and its use.

[0005] Specifically, the present invention provides polysaccharide-based polymeric crosslinkers, methods for producing polysaccharide-based polymeric crosslinkers, polysaccharide-based biomaterials, methods for producing polysaccharide-based biomaterials, and uses of polysaccharide-based biomaterials.

[0006] The object of the present invention can be achieved by the following technical means.

[0007] According to a first aspect of the present invention, there is provided a polysaccharide-based polymeric crosslinking agent.

[0008] The polysaccharide-based polymer crosslinking agent is a polysaccharide-based polymer crosslinking agent modified with an orthophthalaldehyde group, and has a structure as shown in Formula 1: [ka] In formula 1, P is a natural polysaccharide polymer or a modified or degraded product thereof, and P is one or more selected from hyaluronic acid, cellulose, cellulose derivatives, alginic acid, dextran, agarose, heparin, chondroitin sulfate, carrageenan, tragacanth gum, xanthan gum, gellan gum, guar gum, gum arabic, locust bean gum, starch, starch hydrolysates, starch derivatives, etc.; In Formula 1, n≧2 means that the average number of ortho-phthalaldehyde functional groups in a single polysaccharide polymer chain is 2 or more; In Formula 1, the ortho-phthalaldehyde group and P are linked by a covalent bond.

[0009] In some embodiments of the invention, P is selected from hyaluronic acid, cellulose derivatives, alginic acid, heparin, chondroitin sulfate, xanthan gum, gellan gum, starch, starch hydrolysates, and starch derivatives.

[0010] The cellulose derivative may be selected from cellulose ethers, including, but not limited to, methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose; Preferably, the cellulose derivative is selected from carboxymethylcellulose.

[0011] The starch derivative is selected from oxidized starch, esterified starch, etherified starch and alkylated starch.

[0012] Preferably, the starch derivative is selected from carboxymethyl starch.

[0013] In some specific embodiments of the present invention, the ortho-phthalaldehyde group-modified polysaccharide polymer crosslinker is [ka] This is one of the structures selected from However, 1≦r≦20, 1≦s≦20, 1≦t≦50, and n≧2. Preferably, 1≦r≦6, 1≦s≦10, 1≦t≦30, and n≧2.

[0014] According to a second aspect of the present invention, there is provided a method for producing the polysaccharide-based polymer crosslinking agent.

[0015] The polysaccharide-based polymer crosslinking agent can be produced as follows.

[0016] An ortho-phthalaldehyde precursor derivative whose aldehyde group has been protected in advance is covalently linked to the active group of a polysaccharide polymer to obtain a polysaccharide polymer modified with the ortho-phthalaldehyde precursor derivative, and then the aldehyde group of the polysaccharide polymer modified with the ortho-phthalaldehyde precursor derivative is deprotected to obtain a polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group.

[0017] The ortho-phthalaldehyde precursor derivative in which the aldehyde group is protected in advance comprises an ortho-phthalaldehyde group in which the aldehyde group is protected in advance and a substituent R capable of linking to an active group of a polysaccharide polymer, and has a structure as shown in Formula 2: [ka] In Formula 2, R includes, but is not limited to, carboxyl substituents, vinyl sulfone substituents, epoxy substituents, halogenated alkane substituents, isocyanate substituents, and amino substituents.

[0018] In formula 2, R may be directly linked to the benzene ring, or may be linked to the benzene ring via a single or multiple alkylene chains or alkoxy chains, and multiple alkylene chains or alkoxy chains are linked via ether bonds, amide bonds, ester bonds, urethane bonds, or urea bonds.

[0019] In some specific embodiments of the present invention, the ortho-phthalaldehyde precursor derivative in which the aldehyde group is pre-protected is [ka] [ka] is one selected from the compounds However, 1≦r≦20, 1≦s≦20, and 1≦t≦50. Preferably, 1≦r≦6, 1≦s≦10, and 1≦t≦30.

[0020] The active group of the polysaccharide polymer may be an active group of the polysaccharide polymer itself, or may be a reactive group of the polysaccharide polymer after modification or decomposition.

[0021] In some embodiments of the present invention, the active groups of the polysaccharide polymer are selected from hydroxyl groups and carboxyl groups.

[0022] The covalent bond between the ortho-phthalaldehyde precursor derivative in which the aldehyde group has been protected in advance and the active group of the polysaccharide polymer is selected from the group consisting of an ether bond, an amide bond, an ester bond, a urethane bond, and a urea bond, and preferably an ether bond, an amide bond, an ester bond, or a urethane bond.

[0023] More specifically, when the active group of the polysaccharide polymer is selected from hydroxyl groups, the covalent bond is based on an ether bond between a vinyl sulfone substituent, an epoxy substituent, or a halogenated alkane substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer, an ester bond between a carboxyl substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer, or a urethane bond between an isocyanate substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer; When the polysaccharide polymer active group is selected from carboxyl groups, the covalent linkage is based on an amide bond between the amino substituent of the orthophthalaldehyde precursor derivative and the carboxyl group of the polysaccharide polymer.

[0024] In some specific embodiments of the present invention, the covalent linkage is preferably based on an ether bond between the vinyl sulfone substituent of the ortho-phthalaldehyde precursor derivative and the hydroxyl group of the polysaccharide polymer, an ether bond between the epoxy substituent of the ortho-phthalaldehyde precursor derivative and the hydroxyl group of the polysaccharide polymer, an ester bond between the carboxyl substituent of the ortho-phthalaldehyde precursor derivative and the hydroxyl group of the polysaccharide polymer, and an amide bond between the amino substituent of the ortho-phthalaldehyde precursor derivative and the carboxyl group of the polysaccharide polymer.

[0025] Deprotection of the aldehyde group of a polysaccharide polymer modified with an ortho-phthalaldehyde precursor derivative involves deprotecting the ortho-phthalaldehyde functional group, which has been previously protected and is linked to the polysaccharide polymer, under acidic conditions to obtain a polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group.

[0026] The structures of the ortho-phthalaldehyde group-modified polysaccharide polymer crosslinkers represented by Formulae 1-1 to 1-12 in the first aspect of the present invention are merely illustrative examples of ortho-phthalaldehyde group-modified polysaccharide polymer crosslinkers obtained by a specific production method using the above-mentioned ortho-phthalaldehyde precursor derivative in which the aldehyde group has been previously protected and a polysaccharide polymer. Based on the contents of the present application, a person skilled in the art can imagine that the ortho-phthalaldehyde group-modified polysaccharide polymer crosslinkers can be realized using the structure of another ortho-phthalaldehyde precursor derivative in which the aldehyde group has been previously protected.

[0027] According to a third aspect of the present invention, there is provided a method for producing a polysaccharide-based two-component hydrogel as follows: Component A and component B are dissolved in a solvent, respectively, to obtain a solution of component A and a solution of component B, which are the dual components of the polysaccharide-based two-component hydrogel, and the solution of component A and the solution of component B are mixed to obtain the polysaccharide-based two-component hydrogel.

[0028] The component A is a polysaccharide-based polymer crosslinking agent modified with an ortho-phthalaldehyde group, and is represented by formula 1, where n is 2 or more, Component B is a water-soluble small molecule, a water-soluble synthetic polymer, or a water-soluble natural polymer (e.g., a protein, a nucleic acid, or a polysaccharide) containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, or mercapto groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or mercapto functional groups contained in a single molecule is two or more.

[0029] In some embodiments of the present invention, preferably, component B is one or more selected from the group consisting of polyethylene glycol derivatives, polyethyleneimines, polyamino acids, proteins, protein modifications, protein denaturations, protein degradation products, polysaccharides, polysaccharide modifications, and polysaccharide degradation products, and the molecular structure of the substance selected as component B contains one or more groups selected from primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto functional groups contained in a single molecule is 2 or more; Preferably, the polyethylene glycol derivative is a polyethylene glycol derivative modified with a primary amine, a hydrazine, a hydrazide, a hydroxylamine, or a mercapto group; Preferably, the proteins include collagen, serum proteins, fibrinogen and fibrin, and the protein degradation products include gelatin or polypeptides; Preferably, the polysaccharide, modified polysaccharide, or degraded polysaccharide is selected from chitosan, modified chitosan, and degraded chitosan; hyaluronic acid, alginic acid, chondroitin sulfate, heparin, cellulose, chitin, and their respective modified and degraded products, which are modified with primary amines, hydrazines, hydrazides, hydroxylamine, or mercapto groups.

[0030] More preferably, component B is selected from amino-modified polyethylene glycol derivatives, hydrazide-modified hyaluronic acid, collagen, serum proteins, gelatin, polypeptides, polyamino acids, and chitosan.

[0031] In some embodiments of the invention, the solvent is selected from water, saline, a buffer solution, a decellularized matrix, and a cell culture medium solution.

[0032] In some embodiments of the present invention, the solid content of component A in the component A solution is preferably 0.1 to 40 wt%, preferably 0.5 to 20 wt%, and more preferably 0.5 to 10 wt%, and the solid content of component B in the component B solution is 0.1 to 40 wt%, preferably 0.5 to 20 wt%, and more preferably 0.5 to 10 wt%. Increasing the proportion of component A in the polysaccharide-based two-component hydrogel can effectively improve the tissue adhesiveness and hemostatic performance of the hydrogel.

[0033] In some embodiments of the present invention, the production temperature for producing a hydrogel by mixing a solution of component A and a solution of component B is 0 to 80°C, and the production pH is 1 to 12.

[0034] According to a fourth aspect of the present invention, there is provided a polysaccharide-based two-component hydrogel produced according to the method of the third aspect of the present invention.

[0035] According to a fifth aspect of the present invention, there are provided a plurality of polysaccharide-based biomaterials.

[0036] In some embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based membrane material obtained by drying a polysaccharide-based two-component hydrogel provided by the fourth aspect of the present invention.

[0037] In some embodiments of the present invention, the drying method includes air drying and oven drying.

[0038] Preferably, the oven drying conditions are 30° C. for 12 hours.

[0039] In some embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based powder material obtained by mechanical ball milling of the polysaccharide-based membrane material. Mechanical ball milling involves placing the polysaccharide-based membrane material in a ball mill and placing it between vigorously agitated grinding balls, subjecting it to repeated impact, crushing, shear, and pressure forces, thereby continuously deforming and crushing the material to obtain a fine powder. Liquid surfactants and lubricants, such as ethanol, can be added to reduce interparticle adhesion and prevent particle aggregation. Liquid additives must not corrode or chemically react with the particles.

[0040] In some other embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based powder material obtained by mixing a solution of component A and a solution of component B provided by the third aspect of the present invention, followed by preparing microgel particles by an emulsification method, a microfluidic method, a mechanical grinding method, or the like, and then drying and sieving the microgel particles.

[0041] Preferably, the method for producing the microgel particles is a mechanical pulverization method in which a polysaccharide hydrogel obtained by mixing a solution of component A and a solution of component B is cut into particles having a uniform particle size distribution using a high-shear homogenizer. Preferably, the particle size distribution of the microgel particles is 1 to 1000 μm.

[0042] In some specific embodiments of the present invention, the drying method is vacuum freeze-drying or oven drying. Preferably, the freeze-drying conditions are freezing at -20°C for 6 hours, followed by freeze-drying at -60°C for 48 hours, and the oven-drying conditions are oven-drying at 30°C for 12 hours. In some specific embodiments of the present invention, sieving is an operation process in which a pulverized powder with a large difference between coarseness and fineness is separated into coarse powder and fine powder using a mesh-shaped tool (e.g., a vibrating sieve). Preferably, the particle size distribution of the powder after sieving is 20 mesh to 500 mesh.

[0043] In some embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based sponge material obtained by mixing a solution of component A, a solution of component B, and a pore-forming agent provided by the third aspect of the present invention to obtain a polysaccharide-based hydrogel material having a pore structure, and then drying the resulting material.

[0044] Component A is a polysaccharide-based polymer crosslinker modified with an ortho-phthalaldehyde group, and component B is a water-soluble small molecule, water-soluble synthetic polymer, or water-soluble natural polymer (e.g., protein, nucleic acid, or polysaccharide) containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, or mercapto groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or mercapto functional groups contained in a single molecule is two or more.

[0045] In some embodiments of the present invention, the pore-forming agent is an additive that causes a material to form a pore structure, and includes substances that easily decompose into gas, polymer microspheres, polyethylene glycol, polyvinylpyrrolidone, surfactants, water, etc.

[0046] When a substance that easily decomposes into gas, such as ammonium bicarbonate, is added to the material and heated, carbon dioxide and ammonia gas are released, which overflow from the material and create a pore structure. Examples of polymer microspheres include polymethyl methacrylate microspheres, silica microspheres, and polystyrene microspheres.

[0047] Preferably, the pore-forming agent is water, and the hydrogel becomes a porous honeycomb sponge structure upon freeze-drying.

[0048] Preferably, the freeze-drying conditions are freezing at -20°C for 6 hours, followed by freeze-drying at -60°C for 48 hours.

[0049] According to a sixth aspect of the present invention, Use of said polysaccharide-based two-component hydrogel in the manufacture of a tissue repair product. Use of the polysaccharide-based two-component hydrogel in the manufacture of a tissue fluid leakage closure product. Use of the polysaccharide-based two-component hydrogel in the manufacture of a tissue air leak closure product; and There is provided a use of a polysaccharide-based two-component hydrogel according to a fourth aspect of the present invention, which is one selected from the uses of the polysaccharide-based two-component hydrogels described above in the manufacture of a hemostatic product.

[0050] Tissue repair includes skin repair, abdominal wall repair, etc. Tissue fluid leak closure includes, for example, pancreatic fluid leak closure, cerebrospinal fluid leak closure, intestinal leak closure, gastric leak closure, etc. Tissue air leak closure includes pulmonary parenchymal air leak closure, etc. Hemostasis includes liver hemostasis, kidney hemostasis, spleen hemostasis, pancreatic hemostasis, bone cross-section hemostasis, arterial hemostasis, and cardiac hemostasis, etc.

[0051] According to a seventh aspect of the present invention there is provided the use of a polysaccharide-based biomaterial according to the fifth aspect of the present invention.

[0052] When the polysaccharide-based biomaterial is a polysaccharide-based membrane material, the use of the polysaccharide-based membrane material includes: Use of said polysaccharide-based membrane material in the manufacture of a sutureless surgical wound closure product; and and the use of said polysaccharide-based film material in the manufacture of a patch product, Patch products made using polysaccharide-based film materials can be used for abdominal hernia repair, etc.

[0053] When the polysaccharide-based biomaterial is a polysaccharide-based powder material, the use of the polysaccharide-based powder material includes: Use of said polysaccharide-based powder material in the manufacture of a hemostatic product; and and the use of said polysaccharide-based powder material in the manufacture of cell, factor, and drug carrier products; Hemostatic products made using polysaccharide powder materials can be used for hemostasis during minimally invasive surgery, hemostasis of the liver, kidney, spleen, pancreas, bone cross-section, artery, and heart, etc., and cell, factor, and drug carriers include stem cell carriers, growth-promoting factor carriers, antibacterial drug carriers, and anti-inflammatory drug carriers, etc.

[0054] When the polysaccharide-based biomaterial is a polysaccharide-based sponge material, the use of the polysaccharide-based sponge material may include: Use of said polysaccharide-based sponge material in the manufacture of a wound dressing product; and The use of said polysaccharide-based sponge material in the manufacture of hemostatic products is one of the preferred embodiments.

[0055] The hemostatic products manufactured using the polysaccharide sponge material can be used for hemostasis of the liver, kidney, spleen, pancreas, bone cross section, artery, heart, and non-compression penetrating wound.

[0056] Polysaccharides are natural water-soluble polymers that are widely available in various sources and have good biocompatibility.

[0057] The present invention provides a polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group. The polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group of the present invention does not rely on a synthesized hydrophilic polymer backbone or a complicated chemical synthesis process, and has the advantages of being available from a wide range of raw material sources and being easily prepared.

[0058] The present invention further provides a two-component polysaccharide-based hydrogel material in an aqueous medium, which is composed of a polysaccharide-based polymer crosslinker and a small molecule or polymer derivative containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups. The hydrogel material is composed of two crosslinked components: the first is a polysaccharide-based polymer crosslinker modified with ortho-phthalaldehyde groups; and the second is a water-soluble small molecule, water-soluble synthetic polymer, or water-soluble natural polymer (e.g., protein, nucleic acid, or polysaccharide) containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups, each of which has two or more corresponding functional groups. The two-component polysaccharide-based hydrogel material provided by the present invention has the advantages of being injectable, having low swelling, and having high tissue adhesiveness, making it suitable for use as a tissue adhesive, sealant, hemostat, tissue engineering material, and the like.

[0059] The present invention further provides a number of different forms of polysaccharide-based biomaterials (e.g., membranes, powders, and sponges) based on polysaccharide-based polymer crosslinkers or hydrogel materials. The two-component hydrogels, membranes, powders, and sponges provided by the present invention can be used as tissue adhesives, sealants, hemostats, tissue engineering materials, etc., and have the potential for a wide range of biomedical applications and are of great significance in clinical hemostasis, tissue adhesion, and sealing. [Brief explanation of the drawings]

[0060] [Figure 1] 1 is a nuclear magnetic resonance hydrogen spectrum of a hyaluronic acid-based crosslinking agent (i.e., component A-1.2) in Example 2. [Figure 2] 1 is a nuclear magnetic resonance hydrogen spectrum of a carboxymethyl cellulose-based crosslinking agent (i.e., component A-2.2) in Example 3. [Figure 3] 1 shows photographs of a precursor solution of a polysaccharide-based two-component hydrogel (Composition 7) in Example 11 and after gelation. [Figure 4]7d shows photographs of Example 16 in which a polysaccharide-based two-component hydrogel (group a) and sutures (group b) were used for skin tissue repair. [Figure 5] 10 is a photograph taken 7 days after surgery in Example 18, in which a polysaccharide-based two-component hydrogel (group a) was used on the pancreatic stump to close pancreatic juice leakage. [Figure 6] 10 shows photographs of Example 19, in which a polysaccharide-based two-component hydrogel (group a) and bone wax (group b) were used to seal cerebrospinal fluid leaks. [Figure 7] 10 shows photographs comparing the results of H&E staining 10 days after surgery in Example 24 between a group a in which a polysaccharide-based membrane material was used for non-suture closure of a surgical wound and a group b in which a suture was used. [Figure 8] 2 shows the microscopic morphology of polysaccharide-based powder materials of different particle sizes in Example 26. [Figure 9] FIG. 2 is a statistical graph showing the colon lengths of normal mice and mice whose acute enteritis was treated with a polysaccharide-based powder material with budesonide supported on its surface (group a), a budesonide enema solution (group b), and a blank group (group c) in Example 29. [Figure 10] 10 is a comparative photograph showing the use of a sponge material (group a) and a commercially available absorbent hemostatic gauze (group b) for splenic hemostasis in Example 32. [Figure 11] 13 is a photograph showing the use of a sponge material (group a) for cardiac hemostasis in Example 33. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention will now be described in detail with reference to the drawings and specific examples.

[0062] Example 1 Synthesis of orthophthalaldehyde precursor derivatives with pre-protected aldehyde groups (i.e., compounds 1-13)

[0063] (1) Synthesis of Compounds 1-3 (s=2, t=7) [ka]

[0064] (1) Synthesis of Compound 1: The synthesis process was carried out in accordance with the method disclosed in Zhen Zhang, Chaoliang He, Yan Rong, Hui Ren, Tianran Wang, Zheng Zou, Xuesi Chen, National Science Review, Volume 8, Issue 4, April 2021, nwaa128. 1 H NMR (400 MHz, DMSO-d6): δ = 12.74 (brs, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.12 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 6.30 (s, 1H), 6.11 (s, 1H), 3.40-3.30 (m, 6H).

[0065] (2) Synthesis of Compound 2: Compound 1 (10 g, 45 mmol) was dissolved in 10 mL of anhydrous dichloromethane (DCM), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 10.38 g, 67 mmol) and N-hydroxysuccinimide (NHS, 7.69 g, 67 mmol) were added. The mixture was stirred at room temperature for 30 min. Ethylenediamine (27.05 g, 0.45 mmol) was dissolved in anhydrous DCM and stirred. The reaction mixture was rapidly added dropwise to the ethylenediamine solution and stirred at room temperature for 12 h. After the reaction was completed, most of the solvent was removed, and the remaining compound was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting crude product was purified by silica gel column chromatography to obtain compound 2 (9.0 g, 75% yield). 1H NMR (400 MHz, DMSO-d6): δ = 8.18 (t, 1H), 8.07 (d, 1H), 7.80 (dd, 1H), 7.56 (dd, 1H), 6.05 (h, 2H), 4.59 (dt, 1H), 4.25 (dt, 1H), 3.38 (tt, J = 5.1 Hz, 2H), 3.34 (s, 3H), 3.03 (tt, J = 6.3, 2H).

[0066] (3) Synthesis of Compound 3: α,ω-Dicarboxypolyethylene glycol (COOH-PEG-COOH, t = 7, 8.26 g) was dissolved in 100 mL of anhydrous DCM, and EDC (3.52 g, 22.7 mmol) and NHS (2.61 g, 22.7 mmol) were added and stirred at room temperature for 30 min. Compound 2 (5 g, 18.8 mmol) was dissolved in anhydrous DCM (50 mL) and slowly added dropwise to the above system. The mixture was stirred at room temperature for 12 h. After the reaction was completed, most of the solvent was removed, and the remaining compound was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting crude product was purified by silica gel column chromatography to obtain Compound 3 (10.5 g, 79% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 8.86 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.93 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 6.38 (s, 1H), 6.30 (s, 1H), 3.80-3.48 (m, 28H), 3.40-3.30 (m, 6H).

[0067] (iv) Synthesis of compounds 4 to 8 (r = 2, s = 6, t = 7) [ka]

[0068] (1) Synthesis of Compound 4: The synthesis process was carried out in accordance with the method disclosed in Chun Ling Tung, Clarence TT Wong, Eva Yi Man Fung and Xuechen Li. Org. Lett. 2016, 18, 11, 2600-2603. 1 H NMR (400 MHz, DMSO-d6): δ = 11.82 (s, 1H), 7.56 (dq, 1H), 7.43 (dd, 1H), 7.16 (ddt, 1H), 6.05 (m, 1H), 5.97 (q, 1H), 3.53 (dt, 2H), 3.40-3.30 (m, 6H).

[0069] (2) Synthesis of Compound 5: The synthesis process was carried out in accordance with the synthesis of Compound 2. 1 H NMR (400 MHz, DMSO-d6): δ = 7.56 (t, 1H), 7.48 (m, 2H), 7.14 (ddt, 1H), 6.06 (s, 1H), 5.97 (q, 1H), 3.40-3.30 (m, 6H), 3.07 (q, 2H), 2.90-2.72 (m, 4H), 2.38 (t, J = 8.2 Hz, 2H), 1.87 (t, J = 6.5 Hz, 2H), 1.59-1.43 (m, 4H), 1.46-1.30 (m, 4H).

[0070] (3) Synthesis of Compound 6: Compound 5 (5 g, 14.3 mmol) was dissolved in 100 mL of toluene, and succinic anhydride (1.5 g, 15 mmol) and triethylamine (TEA, 2.1 mL, 15 mmol) were added. The mixture was stirred at room temperature for 4 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the resulting crude product was purified by silica gel column chromatography to obtain Compound 6 (5.2 g, 80% yield). 1H NMR (400 MHz, DMSO-d6): δ = 11.87 (s, 1H), 7.62 (t, 1H), 7.56 (t, 1H), 7.44-7.36 (m, 2H), 7.14 (ddt, 1H), 6.08-6.02 (m, 1H), 5.97 (q, 1H), 3.40-3.30 (m, 6H), 3.19-3.03 (m, 4H), 2.77 (tt, J = 8.7 Hz, 2H), 2.55 (dd, 2H), 2.48-2.34 (m, 4H), 1.56-1.43 (m, 4H), 1.40-1.27 (m, 4H).

[0071] (4) Synthesis of Compound 7: α,ω-diaminopolyethylene glycol (NH2-PEG-NH2, t = 7, 8.8 g) was dissolved in 50 mL of anhydrous DCM. Compound 4 (5 g, 19.8 mmol) was dissolved in anhydrous DCM (50 mL), and EDC (4.65 g, 30 mmol) and NHS (3.45 g, 30 mmol) were added and stirred at room temperature for 30 min. The reaction mixture was gradually added dropwise to the NH2-PEG-NH2 solution and stirred at room temperature for 12 h. After the reaction was completed, most of the solvent was removed, and the remaining compound was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting crude product was purified by silica gel column chromatography to obtain Compound 7 (10.4 g, 81% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.43-7.36 (m, 2H), 7.14 (ddt, 1H), 3.61-3.43 (m, 28H), 3.36-3.26 (m, 6H), 2.88-2.71 (m, 2H), 2.54-2.36 (m, 2H).

[0072] (5) Synthesis of Compound 8: Hexamethylene diisocyanate (HDI, 1.28 g, 7.6 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 50 mL). Compound 7 (5 g, 7.6 mmol) was dissolved in anhydrous THF (50 mL) and slowly added dropwise to the rapidly stirring HDI solution. The reaction was allowed to proceed with stirring at room temperature for 2 h. After removing the solvent by rotary evaporation, the mixture was redissolved in a small amount of DCM and poured into n-pentane to precipitate a solid. This was repeated twice, and the mixture was dried by suction to obtain Compound 8 (5.5 g, 92% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.53 (t, 1H), 7.44-7.36 (m, 2H), 7.14 (ddt, 1H), 6.047 (s, 1H), 5.97 (s, 1H), 5.87 (t, 1H), 5.65 (t, 1H), 3.63-3.51 (m, 32H), 3.36-3.28 (m, 4H), 3.39-3.32 (m, 6H), 3.31-3.18 (m, 4H), 3.10 (tdd, J = 5.7, 4.7, 1.0 Hz, 2H), 2.90-2.71 (m, 4H), 2.41 (t, J = 8.3 Hz, 2H), 1.87 (t, J = 6.5 Hz, 2H), 1.59-1.27 (m, 8H).

[0073] (3) Synthesis of Compounds 9-12 [ka]

[0074] (1) Synthesis of Compound 9: Intermediate 1 (5 g, 25.5 mmol) was dissolved in anhydrous DCM (40 mL), 0.2 M NaOH solution (85 mL) was added, followed by tetrabutylammonium bromide (TBAB, 0.82 g, 2.55 mmol), and the mixture was stirred at room temperature for 30 min. 1-Bromo-2-chloroethane (8.41 mL, 0.102 mol) was added dropwise, and the mixture was stirred at room temperature for 48 h. The phases were then separated, and the organic phase was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain Compound 9 (5.6 g, 85% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.45 (dd, 1H), 7.15 (dd, 1H), 6.88 (dd, 1H), 6.07-5.97 (m, 2H), 4.24 (q, J = 2.2 Hz, 2H), 3.81 (t, J = 2.2 Hz, 2H), 3.37-3.34 (s, 6H).

[0075] (2) Synthesis of Compound 10: HDI (6.43 g, 38.3 mmol) was dissolved in anhydrous DCM. Intermediate 1 (5 g, 25.5 mmol) was dissolved in anhydrous DCM, and TEA (3.53 mL, 25.5 mmol) was added. The mixture was slowly added dropwise to the rapidly stirring HDI solution and stirred at room temperature for 30 min. After removing the solvent by rotary evaporation, the mixture was redissolved in a small amount of DCM and poured into n-pentane to precipitate a solid. This process was repeated twice to obtain Compound 10 (9.76 g, 70% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.56 (dd, J = 8.4, 1H), 7.40 (s, 1H), 7.07 (dd, J = 8.3, 1H), 6.34 (t, J = 5.3 Hz, 1H), 6.03 - 5.93 (m, 2H), 3.38-3.31 (s, 6H), 3.20-3.13 (m, 2H), 3.06 (q, J = 5.5 Hz, 2H), 1.67-1.27 (m, 8H).

[0076] (3) Synthesis of Compound 11: Intermediate 1 (5 g, 25.5 mmol) was dissolved in anhydrous DCM under anhydrous and oxygen-free conditions. 2-Chloroethanesulfonyl chloride (5 g, 30.6 mmol) was slowly added dropwise to the solution in an ice bath at 0 °C. The reaction was allowed to proceed for 10 min. Anhydrous TEA (4.23 mL, 30.6 mmol) was then added dropwise and the reaction was allowed to proceed for 12 h at room temperature. After the reaction was completed, the solvent was removed by rotary evaporation. The resulting crude product was purified by silica gel column chromatography to obtain Compound 11 (6.9 g, 95% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.48-7.41 (m, 1H), 7.35 (s, 1H), 7.08 (dd, J = 8.2, 1H), 6.26 (dd, J = 18.5, 1H), 6.15 (dd, J = 18.4, 1H), 6.03 (dq, J = 2.9, 1.7 Hz, 2H), 3.41-3.33 (s, 6H).

[0077] (4) Synthesis of Compound 12: Intermediate 1 (5 g, 25.5 mmol) was suspended in 30 mL of water, and 1.5 M aqueous NaOH (10 mL) was added. The mixture gradually became clear. After stirring for 40 minutes, epichlorohydrin (7.1 g, 76.5 mmol) was added and stirred at room temperature for 24 hours. After the reaction was completed, most of the solvent was removed, and the remaining compound was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting crude product was purified by silica gel column chromatography to obtain Compound 12 (5.8 g, 90% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 7.45 (s, 1H), 7.14 (s, 1H), 6.91 (dd, J = 8.3, 1H), 6.07-5.99 (m, 2H), 4.11 (d, J = 3.3 Hz, 2H), 3.42 (s, 1H), 3.38-3.32 (m, 6H), 2.91-2.78 (m, 2H).

[0078] Example 2 Synthesis of Representative Components A-1.1 to A-1.4 of Ortho-Phthalaldehyde-Modified Hyaluronic Acid Crosslinkers

[0079] [ka]

[0080] (1) Synthesis of component A-1.1: Hyaluronic acid (HA, 5 g, 890 kDa) was dissolved in 500 mL of 0.01 mol / L 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution (pH = 5.2) and stirred until completely dissolved. Compound 2 (0.54 g, 2 mmol, s = 2) was weighed and dissolved in 10 mL of dimethyl sulfoxide (DMSO), and then added to the reaction solution. 4-(4,6-dimethoxytriazin-2-yl)4-methylmorpholine hydrochloride (DMTMM, 1.4 g, 5 mmol) was weighed and added to the reaction system in three portions at 1-hour intervals. Each portion was dissolved in 3 mL of MES buffer solution, and the reaction was carried out at 37 °C for 24 hours. After the reaction was complete, the pH was adjusted to 8-9, stirred for 1 h, and dialyzed against deionized water for 2-3 d (MWCO 14000). The product was collected, and then 10% trifluoroacetic acid was added. The reaction mixture was allowed to react for 1 h, and the pH was adjusted to neutral. The reaction mixture was then dialyzed against deionized water for 1 d (MWCO 14000). The product was collected and lyophilized to obtain component A-1.2. The labeling of compound 2 on HA was identified by nuclear magnetic resonance spectroscopy. The amount of remaining compound 2 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 2 was calculated to be approximately 6.78% (w / w).

[0081] [ka]

[0082] (2) Synthesis of Component A-1.2: HA (5 g, 340 kDa) was dissolved in 250 mL of MES buffer solution (0.01 M, pH 7.0), and compound 5 (420 mg, 1.2 mmol, r = 2, s = 6) was dissolved in 10 mL of DMSO and added to the reaction system. A dialysis bag with a MWCO of 7000 was used for dialysis. The other synthesis steps were performed using the synthesis of component A-1.1. The nuclear magnetic resonance (NMR) spectrum of a representative hyaluronic acid-based crosslinker (i.e., component A-1.2) in Example 2 is shown in Figure 1. The NMR spectrum identified the labeling of compound 5 on HA. The supernatant was collected, and the amount of remaining compound 5 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 5 was calculated to be approximately 4.21% (w / w).

[0083] [ka]

[0084] (3) Synthesis of component A-1.3: HA (5 g, 1.2 MDa) was dissolved in 50 mL of 0.2 M NaOH solution and stirred until completely dissolved. Compound 11 (343 mg, 1.2 mmol) was added to the above system and reacted at 50 °C for 6 h. After the reaction was completed, the system was neutralized to pH 7 with dilute hydrochloric acid and dialyzed against deionized water (MWCO 14000) for 2–3 d. The product was collected, and then 10% trifluoroacetic acid was added and reacted for 1 h. The pH was further adjusted to neutral. The reaction system was then dialyzed against deionized water (MWCO 14000) for 1 d. The product was collected and lyophilized to obtain component A-1.3. The labeling of compound 11 on HA was identified by nuclear magnetic resonance spectroscopy. The supernatant was collected, and the amount of remaining compound 11 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 11 was calculated to be approximately 4.12% (w / w).

[0085] [ka]

[0086] (4) Synthesis of component A-1.4: HA (5 g, 1.2 MDa) was dissolved in 50 mL of 0.25 M NaOH solution and stirred until completely dissolved. Compound 12 (302 mg, 1.2 mmol) was added to the above system and reacted at 40 °C for 12 h. After the reaction was complete, the system was neutralized to pH 7 with dilute hydrochloric acid and dialyzed against deionized water (MWCO 14000) for 2–3 d. The product was collected, and then 10% trifluoroacetic acid was added. The reaction system was then reacted for 1 h and further adjusted to neutral. The reaction system was then dialyzed against deionized water (MWCO 14000) for 1 d. The product was collected and lyophilized to obtain component A-1.4. The labeling of compound 12 on HA was identified by nuclear magnetic resonance spectroscopy. The amount of remaining compound 12 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 12 was calculated to be approximately 4.01% (w / w).

[0087] The above structural formula and examples provide exemplary structures of hyaluronic acid-based polymer crosslinkers modified with ortho-phthalaldehyde groups and methods for synthesizing the same. Based on the disclosure of this application, those skilled in the art can imagine that other ortho-phthalaldehyde protected derivatives can be used to synthesize the same ortho-phthalaldehyde-based polymer crosslinkers.

[0088] Example 3 Synthesis of Representative Components A-2.1 to A-2.4 of Carboxymethylcellulose-Based Crosslinkers Modified with Orthophthalaldehyde Groups

[0089] [ka]

[0090] (1) Synthesis of component A-2.1: Carboxymethyl cellulose (CMC, 5 g, viscosity (2%, 25 °C) 1200 mPa·s, degree of carboxymethyl substitution 0.7) was dissolved in 500 mL of deionized water and stirred until completely dissolved. Compound 1 (269 mg, 1.2 mmol), ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 1.86 g, 12 mmol), and 4-methylbenzenesulfonic acid N,N-dimethylpyridin-4-amine (DPTS, 3 g, 12 mmol) were added sequentially to the above solution. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was complete, the mixture was dialyzed for 2-3 d against a dilute hydrochloric acid-containing sodium chloride solution (pH = 4) (MWCO 14000). The product was collected, and then 10% trifluoroacetic acid was added and reacted for 1 h. The pH was further adjusted to neutral. The reaction mixture was then dialyzed for 1 d against deionized water (MWCO 14000). The product was collected and lyophilized to obtain component A-2.1. The labeling of compound 1 on the CMC was identified by nuclear magnetic resonance spectroscopy. The supernatant was collected and the amount of remaining compound 1 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 1 was calculated to be approximately 2.48% (w / w).

[0091] [ka]

[0092] (2) Synthesis of Component A-2.2: CMC (5 g, viscosity (2%, 25°C) 675 mPa·s, degree of carboxymethyl substitution 0.7) was dissolved in 250 mL of MES buffer solution (0.01 M, pH = 5.2) and stirred until completely dissolved. Compound 5 (280 mg, 0.8 mmol, r = 2, s = 6) was weighed and dissolved in DMSO and added to the reaction system. For other synthesis steps, refer to the synthesis of Component A-1.2. The nuclear magnetic resonance (NMR) spectrum of a representative carboxymethyl cellulose-based crosslinker (i.e., component A-2.2) in Example 3 is shown in Figure 2. The labeling of Compound 5 on CMC was identified by NMR spectroscopy. The supernatant was collected, and the amount of remaining Compound 5 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of Compound 5 was calculated to be approximately 3.31% (w / w).

[0093] [ka]

[0094] (3) Synthesis of component A-2.3: CMC (5 g, viscosity (2%, 25 °C) 675 mPa·s, degree of carboxymethyl substitution 0.7) was dissolved in 250 mL of MES buffer solution (0.01 M, pH = 5.2) and stirred until completely dissolved. Compound 7 (0.78 g, 1.2 mmol, r = 2, t = 7) was added directly to the reaction mixture. A 7000 MWCO dialysis bag was selected for dialysis. For other synthesis procedures, refer to the synthesis of component A-2.1. The labeling of compound 7 on CMC was identified by nuclear magnetic resonance spectroscopy. The supernatant was collected and the amount of remaining compound 7 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 7 was calculated to be approximately 3.67% (w / w).

[0095] [ka]

[0096] (4) Synthesis of Component A-2.4: CMC (5 g, viscosity (2%, 25 °C) 2000 mPa·s, degree of carboxymethyl substitution 0.85) was dissolved in 50 mL of 0.25 M NaOH solution and stirred until completely dissolved. Compound 9 (309 mg, 1.2 mmol) was added to the above system and reacted at 40 °C for 12 h. After the reaction was completed, the system was neutralized to pH 7 with dilute hydrochloric acid and dialyzed against deionized water (MWCO 14000) for 2–3 d. The product was collected, and then 10% trifluoroacetic acid was added and reacted for 1 h. The pH was further adjusted to neutral. The reaction system was then dialyzed against deionized water (MWCO 14000) for 1 d. The product was collected and lyophilized to obtain Component A-2.4. The labeling of compound 9 on CMC was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of compound 9 remaining in the supernatant was measured by high performance liquid chromatography. The labeling rate of compound 9 was calculated to be about 3.01% (w / w).

[0097] The above structural formula and examples provide exemplary structures of ortho-phthalaldehyde-modified carboxymethyl cellulose-based polymeric crosslinkers and methods for synthesizing them. Based on the disclosure of this application, those skilled in the art can imagine that other ortho-phthalaldehyde-modified carboxymethyl cellulose-based polymeric crosslinkers can be synthesized using ortho-phthalaldehyde precursor derivatives in which the aldehyde groups are previously protected.

[0098] Example 4 Synthesis of Representative Components A-3.1 to A-3.3 of Alginate-Based Crosslinkers Modified with Ortho-Phthalaldehyde Groups

[0099] [ka]

[0100] (1) Synthesis of component A-3.1: Alginic acid (sodium alginate, Alg, 5 g, viscosity (2%, 25 °C) 50 mPa·s, t = 7) was dissolved in 250 mL of deionized water and stirred until completely dissolved. Compound 3 (0.65 g, 1 mmol, s = 2, t = 7), EDC (1.6 g, 10 mmol), and DPTS (2.5 g, 10 mmol) were weighed and added sequentially to the solution. A dialysis bag with a MWCO of 3500 was selected for dialysis. For other synthesis steps, refer to the synthesis of component A-2.1. The labeling of compound 3 on Alg was identified by nuclear magnetic resonance spectroscopy. The supernatant was collected and the amount of remaining compound 3 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 3 was calculated to be approximately 2.63% (w / w).

[0101] [ka]

[0102] (2) Synthesis of Component A-3.2: Alg (5 g, viscosity (2%, 25 °C) 980 mPa·s) was dissolved in 250 mL of MES buffer solution (0.01 M, pH = 5.2) and stirred until completely dissolved. EDC (1.54 g, 10 mmol) and NHS (1.14 g, 10 mmol) were added and stirred at room temperature for 30 min. Compound 5 (350 mg, 1 mmol, r = 2, s = 6) was weighed and added to the reaction mixture, followed by stirring at room temperature for 24 h. After the reaction was complete, the mixture was neutralized to pH 7 with dilute hydrochloric acid and dialyzed against deionized water for 2–3 d (MWCO 7000). The product was collected, followed by the addition of 10% trifluoroacetic acid, reacted for 1 h, and further adjusted to neutrality. The reaction mixture was then dialyzed against deionized water for 1 d (MWCO 7000). The product was collected and lyophilized to obtain Component A-3.2. The labeling of compound 5 on Alg was identified by nuclear magnetic resonance hydrogen spectroscopy. The supernatant of the reaction system was collected, and the amount of compound 5 remaining in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 5 was calculated to be approximately 2.57% (w / w).

[0103] [ka]

[0104] (3) Synthesis of component A-3.3: Alg (5 g, viscosity (2%, 25°C) 980 mPa·s) was dissolved in 40 mL of 0.2 M NaOH solution and stirred until completely dissolved. Compound 11 (286 mg, 1 mmol) was added to the above system. For other synthesis steps, refer to the synthesis of component A-1.3. The labeling of compound 11 on Alg was identified by nuclear magnetic resonance hydrogen spectroscopy. The supernatant of the reaction system was collected, and the amount of remaining compound 11 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 11 was calculated to be approximately 2.20% (w / w).

[0105] The above structural formulas and examples provide exemplary structures of ortho-phthalaldehyde-modified alginic acid-based polymeric crosslinkers and methods for synthesizing the same. Based on the disclosure of this application, those skilled in the art will understand that the ortho-phthalaldehyde-modified alginic acid-based polymeric crosslinkers can be synthesized using other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0106] Example 5 Synthesis of Representative Components A-4.1 and A-4.2 of Carboxymethyl Starch-Based Crosslinkers Modified with Orthophthalaldehyde Groups

[0107] [ka]

[0108] (1) Synthesis of Component A-4.1: Carboxymethyl starch sodium (CMS, type A, 5 g, pH 5.0-7.5) was dispersed in 250 mL of deionized water. Compound 4 (0.3 g, 1.2 mmol, r = 2), EDC (1.86 g, 12 mmol), and DPTS (3 g, 12 mmol) were added sequentially to the above system. The reaction was allowed to proceed at room temperature for 24 h. After completion of the reaction, the mixture was reprecipitated with 10 times the volume of absolute ethanol to precipitate a white powder. The solid was collected and redispersed in 250 mL of deionized water. 10% trifluoroacetic acid was added, the mixture was reacted for 1 h, and the pH was adjusted to neutral. The mixture was reprecipitated with 10 times the volume of absolute ethanol to precipitate a white powder, i.e., component A-4.1. The labeling of compound 4 on CMS was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of compound 4 remaining in the supernatant was measured by high performance liquid chromatography. The labeling rate of compound 4 was calculated to be about 2.15% (w / w).

[0109] [ka]

[0110] (2) Synthesis of component A-4.2: CMS (form A, 5 g, pH 5.0-7.5) was dissolved in 40 mL of 0.25 M NaOH solution, and compound 12 (0.3 g, 1.2 mmol) was added to the above system. Finally, the system was reprecipitated with 10 volumes of absolute ethanol to obtain a white powder. For other synthesis steps, refer to the synthesis of component A-1.4. The labeling of compound 12 on CMS was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of remaining compound 12 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 12 was calculated to be approximately 1.78% (w / w).

[0111] The above structural formula and examples provide exemplary structures of ortho-phthalaldehyde-modified carboxymethyl starch-based polymeric crosslinkers and methods for their synthesis. Based on the contents of this application, those skilled in the art can similarly imagine that the ortho-phthalaldehyde-modified carboxymethyl starch-based polymeric crosslinkers can be synthesized using carboxymethyl starches of different sources or structures, and that the ortho-phthalaldehyde-modified carboxymethyl starch-based polymeric crosslinkers can be synthesized using other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0112] Example 6 Synthesis of Representative Components A-5.1 and A-5.2 of Hydroxyethylcellulose-Based Crosslinkers Modified with Orthophthalaldehyde Groups (m≧1)

[0113] [ka]

[0114] (1) Synthesis of Component A-5.1: Hydroxyethyl cellulose (HEC, Mw = 1 MDa, 5 g) was dissolved in 250 mL of anhydrous DMSO and stirred until completely dissolved. Compound 8 (1.63 g, 2 mmol, r = 2, t = 7) was dissolved in 5 mL of DMSO and added to the above system. Dibutyltin dilaurate (13 μL, 20 μmol) was added as a catalyst. The reaction was stirred at 70 °C for 24 h. After the reaction was complete, an equal volume of ice water was slowly added. The mixture was dialyzed against deionized water (MWCO 14000) for 2-3 d. The product was collected, followed by the addition of 10% trifluoroacetic acid, and the mixture was allowed to react for 1 h. The pH was further adjusted to neutral. The reaction mixture was then dialyzed against deionized water (MWCO 14000) for 1 d. The product was collected and lyophilized to obtain Component A-5.1. The labeling of compound 8 on HEC was identified by nuclear magnetic resonance hydrogen spectroscopy. The supernatant of the reaction system was collected, and the amount of remaining compound 8 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 8 was calculated to be approximately 4.12% (w / w).

[0115] [ka]

[0116] (2) Synthesis of component A-5.2: HEC (MW = 300 kDa, 5 g) was dissolved in 250 mL of anhydrous DMSO and stirred until completely dissolved. Compound 10 (0.73 g, 2 mmol) was dissolved in 5 mL of DMSO and added to the above system. Zinc octoate (6 μL, 20 μmol) was added as a catalyst. The reaction was stirred at 70 °C for 24 h. After the reaction was complete, an equal volume of ice water was slowly added, and the mixture was dialyzed against deionized water (MWCO 14000) for 2-3 d. The product was collected, followed by the addition of 10% trifluoroacetic acid, reacted for 1 h, and further adjusted to neutral. The reaction mixture was then dialyzed against deionized water (MWCO 7000) for 1 d. The product was collected and lyophilized to obtain component A-5.2. The labeling of compound 10 on HEC was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of Compound 10 remaining in the supernatant was measured by high performance liquid chromatography. The labeling rate of Compound 10 was calculated to be about 3.14% (w / w).

[0117] The above structural formula and examples provide exemplary structures of ortho-phthalaldehyde-modified hydroxyethyl cellulose-based polymeric crosslinkers and their synthesis methods. Based on the disclosure of this application, those skilled in the art can imagine that other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups are previously protected can be used to synthesize the ortho-phthalaldehyde-modified hydroxyethyl cellulose-based polymeric crosslinkers.

[0118] Example 7 Synthesis of A-6.1, a representative component of ortho-phthalaldehyde-modified heparin-based crosslinkers

[0119] [ka]

[0120] Heparin sodium (Hep, 5 g, 15 kDa) was dissolved in 150 mL of deionized water and stirred until completely dissolved. Compound 6 (0.97 g, 1.2 mmol, r = 2, t = 6), EDC (1.86 g, 12 mmol), and DPTS (3 g, 12 mmol) were added sequentially to the above solution. The reaction was allowed to proceed at room temperature for 24 h. After completion of the reaction, the mixture was dialyzed against a dilute aqueous solution of sodium chloride containing hydrochloric acid (pH = 4) for 2–3 d (MWCO 14000). The product was collected, and then 10% trifluoroacetic acid was added and the mixture was allowed to react for 1 h. The pH was further adjusted to neutral. The reaction mixture was then dialyzed against deionized water for 1 d (MWCO 14000). The product was collected and lyophilized to obtain component A-6.1. The labeling of compound 6 on Hep was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of Compound 6 remaining in the supernatant was measured by high performance liquid chromatography. The labeling rate of Compound 6 was calculated to be about 2.56% (w / w).

[0121] The above structural formulas and examples provide exemplary structures of ortho-phthalaldehyde-modified heparin-based polymeric crosslinkers and methods for synthesizing the same. Based on the disclosure of this application, those skilled in the art can understand that the ortho-phthalaldehyde-modified heparin-based polymeric crosslinkers can be synthesized using other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0122] Example 8 Synthesis of A-7.1, a representative component of ortho-phthalaldehyde-modified chondroitin sulfate-based crosslinkers

[0123] [ka]

[0124] Chondroitin sulfate (CS, 5 g, 20 kDa) was dissolved in 50 mL of 0.01 mol / L MES buffer (pH = 5.2) and stirred and sonicated until completely dissolved. EDC (1.54 g, 10 mmol) and NHS (1.14 g, 10 mmol) were added and stirred at room temperature for 30 min. Compound 5 (350 mg, 1 mmol, r = 2, s = 6) was weighed and dissolved in 10 mL of DMSO, added to the reaction system, and stirred at room temperature for 24 h. After the reaction was complete, the pH was adjusted to 8-9, stirred for 1 h, and dialyzed against deionized water for 2-3 d (MWCO 3500). The product was collected, and then 10% trifluoroacetic acid was added. The reaction mixture was reacted for 1 h and further adjusted to neutral. The reaction mixture was then dialyzed against deionized water for 1 d (MWCO 3500). The product was collected and lyophilized to obtain component A-7.1. The labeling of compound 5 on CS was identified by nuclear magnetic resonance spectroscopy. The amount of remaining compound 5 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 5 was calculated to be approximately 1.87% (w / w).

[0125] The above examples provide exemplary structures of ortho-phthalaldehyde-modified chondroitin sulfate-based polymer crosslinkers and methods for synthesizing the same. Based on the disclosure of the present application, those skilled in the art can understand that other ortho-phthalaldehyde-modified chondroitin sulfate-based polymer crosslinkers can be synthesized using ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0126] Example 9 Synthesis of A-8.1, a representative component of xanthan gum-based crosslinkers modified with ortho-phthalaldehyde groups

[0127] [ka]

[0128] Xanthan gum (XG, 5 g, 1.2 MDa) was dissolved in 500 mL of 1% NaOH solution (pH = 5.2) and stirred until completely dissolved. Compound 12 (302 mg, 1.2 mmol) was added to the above system and reacted at 40 °C for 12 h. After the reaction was complete, the pH was neutralized to 7 with dilute hydrochloric acid, and 10 volumes of absolute ethanol were slowly poured into the system, and the white precipitate was collected. The white precipitate was redissolved in 500 mL of deionized water, and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 h, and the pH was further adjusted to neutral. The reaction system was then dialyzed against deionized water (MWCO 14000) for 1 d. The product was collected and lyophilized to obtain component A-8.1. The label of compound 12 on XG was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of compound 12 remaining in the supernatant was measured by high performance liquid chromatography. The labeling rate of compound 12 was calculated to be about 2.10% (w / w).

[0129] The above structural formula and examples provide exemplary structures of xanthan gum-based polymeric crosslinkers modified with ortho-phthalaldehyde groups and methods for synthesizing the same. Based on the disclosure of this application, a person skilled in the art can understand that the xanthan gum-based polymeric crosslinkers modified with ortho-phthalaldehyde groups can be synthesized using other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0130] Example 10 Synthesis of A-9.1, a representative component of gellan gum-based crosslinkers modified with ortho-phthalaldehyde groups

[0131] [ka]

[0132] Gellan gum (GG, 5 g, 500 kDa) was dissolved in 500 mL of 0.01 mol / L MES buffer solution (pH = 5.2), heated to 80°C, and stirred until completely dissolved. Compound 2 (266 mg, 1 mmol, s = 2) was weighed and dissolved in 10 mL of DMSO, and then added to the reaction solution. DMTMM (7 g, 5 mmol) was weighed and added to the reaction system in five portions at 2-h intervals, dissolved in 3 mL of MES buffer solution each time, and the reaction was carried out at 60°C for 24 hours. After the reaction was complete, the pH was adjusted to 8-9 and the mixture was stirred at 60°C for 1 hour. Ten volumes of absolute ethanol were added during the stirring process. The precipitate was collected, followed by the addition of 500 mL of deionized water. The mixture was heated to 60°C and stirred until completely dissolved. 10% trifluoroacetic acid was added and the mixture was allowed to react for 1 hour. The pH was adjusted to neutral, and then ten volumes of absolute ethanol were added during the stirring process. The precipitate was collected and dried in a vacuum oven to obtain component A-9.1. The labeling of compound 2 on GG was identified by nuclear magnetic resonance spectroscopy. The amount of remaining compound 2 in the supernatant was measured by high-performance liquid chromatography. The labeling rate of compound 2 was calculated to be approximately 1.98% (w / w).

[0133] The above structural formula and examples provide exemplary structures of gellan gum-based polymeric crosslinkers modified with ortho-phthalaldehyde groups and methods for synthesizing the same. Based on the disclosure of this application, those skilled in the art can imagine that the gellan gum-based polymeric crosslinkers modified with ortho-phthalaldehyde groups can be synthesized using other ortho-phthalaldehyde precursor derivatives in which the aldehyde groups have been previously protected.

[0134] Example 11 Hydrogel ingredient combination According to the method of the present invention, different polysaccharide-based two-component hydrogel precursor solutions were prepared (component A and component B were dissolved in phosphate buffer solution, pH=7.4).

[0135] [Table 1]

[0136] In Table 1, Component A-... refers to Components A-3.1 to A-4.4 in Examples 4 and 5, and Component B-... refers to Component B-4: polylysine (molecular weight 5 kDa), Component B-5: collagen, and Component B-6: amino-modified 4-arm polyethylene glycol (molecular weight 40 kDa). The range of 0.1 to 40 wt% in Table 1 indicates the preferred mass concentration range of the hydrogel precursor solution.

[0137] In another example of the present invention, the inventors prepared a polysaccharide-based two-component hydrogel according to the component formulation in Table 2, and used it for performance testing and application. The precursor solution and gelation state of the polysaccharide-based two-component hydrogel (formulation corresponding to No. 7) are shown in Figure 3.

[0138] [Table 2]

[0139] Example 12 Hydrogel swelling test To demonstrate that the hydrogels prepared in the present invention have a lower swelling rate than polyethylene glycol-based hydrogels, which have a high swelling rate (swelling rate >100%), the inventors prepared polysaccharide-based two-component hydrogels according to the component formulations in Table 2 and measured their swelling rates. The ortho-phthalaldehyde-modified polyethylene glycol crosslinkers disclosed in Chinese Patents CN111440310A, CN111440334A, CN111574756A, CN111621038A, CN113509591A, CN113694249A, and CN114767920A, CN202010454896.6, and CN202010455951.3 were selected as the control component A of the present invention. [ka]

[0140] Control component A A control two-component hydrogel was prepared according to the component formulation in Table 3 and used to detect the swelling ratio.

[0141] [Table 3]

[0142] The specific detection method is as follows. The solutions of components A and B listed in Tables 2 and 3 were pressed into polytetrafluoroethylene molds using a two-component mixer and cured for 10 minutes, yielding cylindrical gel blocks with a diameter of 10 mm, a thickness of 3 mm, and approximately equal masses. The gel blocks were weighed, the mass recorded as W0, and transferred to 50 mL centrifuge tubes. A pH 7.4 PBS buffer solution (preheated to 37±1°C) was added. The centrifuge tubes were then placed in an incubator at 37±1°C. Samples were removed every two hours, the surface moisture was absorbed with filter paper, and the samples were weighed until the mass no longer increased. The mass at this point was recorded as Wt. After the test was completed, the swelling ratio of the gels was calculated using the following formula (n=3):

[0143] The swelling ratios obtained by the test using the above method are shown in the table below.

number

[0144] The swelling ratios of the hydrogels obtained by the above test are shown in Table 4.

[0145] [Table 4]

[0146] In the above formulations, compared to the control polyethylene glycol-based hydrogels (i.e., formulations 37 to 40), the polysaccharide-based hydrogels of the present invention all have low swelling ratios (<50%) over a wide range of solid contents.

[0147] Example 13 Hydrogel tissue adhesion test The inventors conducted tissue adhesiveness tests on the polysaccharide-based two-component hydrogels of the present invention, selecting commercially available fibrin glue as a control. The specific testing method is as follows: Using a tensile tester (INSTRON, Germany), the shear strength of the hydrogels of the different formulations on pigskin was measured using a standard lap shear test (ASTM F2255). The pigskin was cut into 10 mm and 40 mm rectangles. Transparent polymethyl methacrylate film was used as the tissue hard backing during mechanical testing. The two-component solution of component A and component B or the control fibrin glue in Table 2 was sprayed onto one piece of pigskin using a two-component mixer, resulting in an adhesive area of ​​15 mm x 10 mm. Another piece of pigskin was immediately pressed onto the pigskin onto which the hydrogel precursor solution had been sprayed, and uniform pressure was applied at 100 kPa for 10 minutes. After the hydrogels of each formulation had completely hardened, tensile tests were performed. The shear strength was calculated using the following formula, with Fmax being the maximum force value. To ensure the reliability of the data, three samples were measured under each condition.

number

[0148] The adhesive strength of the hydrogel obtained by the above-mentioned method is shown in Table 5.

[0149] [Table 5]

[0150] As can be seen from the test results in Table 5, compared to commercially available fibrin glue, the polysaccharide-based two-component hydrogels of the present invention have excellent tissue adhesive properties across a wide range of solid contents, making them suitable for use in tissue adhesion or sealing. In Formulations 1 to 5, the tissue adhesive properties of the polysaccharide-based two-component hydrogels improved as the content of Component A increased. It can be seen that increasing the proportion of Component A in the polysaccharide-based two-component hydrogels effectively improves the tissue adhesive properties of the hydrogels.

[0151] Example 14 In vitro hemostatic performance test of hydrogel The inventors conducted an in vitro hemostatic performance test on the polysaccharide-based two-component hydrogel of the present invention, using commercially available fibrin glue as a control. The specific detection method is as follows. The two-component solution of Component A and Component B shown in Table 2, or the control fibrin glue, was sprayed onto a clean 96-well plate using a two-component mixer, allowed to harden for 10 minutes, and then preheated to 37°C. 20 μL of fresh EDTA-anticoagulated sheep blood kept at 37°C was dropped onto the surface of the material. For the blank control group, blood was dropped directly onto the bottom of the 96-well plate and allowed to stand at 37°C for 5 minutes. The blood or clots on the surface of the hydrogel were then washed with 1 mL of deionized water, gently shaken, and the size of the blood clots remaining on the hydrogel surface was observed. The supernatant was aspirated, the absorbance at 540 nm was measured, and the thrombogenicity index was calculated using the following formula. The results are shown in Table 6.

number

[0152] The thrombogenicity index of the hydrogel obtained by the above-mentioned method is shown in Table 6.

[0153] [Table 6]

[0154] As can be seen from the test results in Table 6, compared to commercially available fibrin glue, the polysaccharide-based two-component hydrogels of the present invention have excellent hemostatic performance across a wide range of solids contents, making them suitable for use in hemostasis. In Formulations 1 to 5, the in vitro clotting performance of the polysaccharide-based two-component hydrogels improved as the content of Component A increased. It can be seen that increasing the proportion of Component A in the polysaccharide-based two-component hydrogels effectively improves the hemostatic effect of the hydrogels.

[0155] Example 15 Based on the product formulations obtained in the above examples, and taking into consideration the swelling property, tissue adhesiveness and hemostatic performance comprehensively, two-component hydrogels of formulations 5, 6, 7, 10, 14, 22 and 30 (i.e., formulations corresponding to numbers 5, 6, 7, 10, 14, 22 and 30, respectively) were selected for further specific biomedical applications.

[0156] Example 16 Use of polysaccharide-based two-component hydrogels in skin wound repair Skin defects on the backs of SD rats were selected and divided into two groups: Formulation 5 (group a) and suture (group b). A skin wound repair experiment was conducted. A 10 mm-long skin defect was created on the backs of SD rats. In group a, solutions of component A and component B were injected into the skin defect using a two-component mixer. After the solution had fully filled and penetrated, it was allowed to harden for 10 minutes, allowing the hydrogel to firmly adhere to the wound. In group b, the defect was sutured using non-absorbable surgical suture 4-0 alone. The wounds of each group were observed, and the repair effect was recorded within 7 days. The experimental results showed that group a showed a faster repair rate and no significant scar tissue formation compared to group b. Figure 4 shows photographs of the 7d skin tissue repair experiment using the polysaccharide-based two-component hydrogel (group a) and suture (group b). This demonstrates that the polysaccharide-based two-component hydrogel can be used for skin wound repair.

[0157] Other formulations of polysaccharide-based two-component hydrogels of the present invention can be similarly used for skin wound repair.

[0158] Example 17 Use of polysaccharide-based two-component hydrogels in abdominal wall wound repair An abdominal wall abrasion model was selected in SD rats and divided into two groups: Formulation 5 (group a) and a blank group (group b). The rats were anesthetized and immobilized, their abdominal fur shaved to expose the abdominal cavity, and the left and right abdominal walls were scraped back and forth several times with the back of a scalpel until bleeding occurred, resulting in abdominal wall abrasion models approximately 10 mm in diameter. Solutions of component A and component B were injected into the abdominal wall abrasion site using a two-component mixer. After the solution had fully filled and penetrated, it was allowed to harden for 10 minutes. The abdominal wall wound surface of rats in group b was washed with saline alone. The wound surface and hydrogel were observed, and the healing effect of each group was recorded after 7 days. The experimental results showed that the hydrogel in group a firmly adhered to the wound surface. Compared to group b, the hydrogel-treated abdominal wall wound in group a showed no significant adhesion to intraperitoneal organs and showed a faster healing rate. It can be seen that the above polysaccharide-based two-component hydrogel can be used for abdominal wall wound repair.

[0159] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used for abdominal wall wound repair.

[0160] Example 18 Use of polysaccharide-based two-component hydrogels in closure of pancreatic leaks A pancreatic leakage model was selected in SD rats, and the rats were divided into two groups: Formulation 5 (group a) and a blank group (group b) for the pancreatic leakage closure experiment. SD rats were anesthetized and immobilized. The abdominal fur was shaved and disinfected with iodine. The abdominal cavity was exposed, the retina and pancreas were separated, and some of the splenic blood vessels were ligated to prevent unnecessary bleeding during pancreatic resection. The pancreatic and duodenal terminal sections were resected with a scalpel to prepare the pancreatic leakage model. In group a, solutions of component A and component B were injected into the pancreatic stump using a two-liquid mixer. After sufficient filling and penetration, the solution was allowed to harden for 10 minutes. In group b, the abdomen was closed directly without any treatment. Seven days after surgery, ascites from the SD rats was collected and its protease content was analyzed. Seven days after surgery, the SD rats were euthanized and the abdominal cavity adhesions were observed. The experimental results showed that protease levels in Group B SD rats exceeded normal levels within 7 days after surgery, demonstrating the successful establishment of a pancreatic leakage model. Figure 5 shows a photograph taken 7 days after surgery in which a polysaccharide-based two-component hydrogel (Formulation 5) was used on the pancreatic stump to seal the pancreatic leakage. Compared to Group B, Group A rats had no obvious fluid accumulation or adhesions in their abdominal cavities, and hydrogel material was still observed on the pancreatic stump. After lavaging the peritoneal cavity with saline, ascites was collected to detect protease levels. The experimental results showed that the trypsin content in the abdominal cavities of Group A rats was within normal levels. This indicates that the polysaccharide-based two-component hydrogel can be used to seal pancreatic leakage.

[0161] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used to seal pancreatic leaks.

[0162] Example 19 Use of polysaccharide-based two-component hydrogels in cerebrospinal fluid leak closure A cerebrospinal fluid (CSF) leak closure experiment was performed on SD rats, divided into two groups: one receiving formulation 5 (group a) and the other receiving bone wax (group b). SD rats were anesthetized and immobilized. The back hair was shaved, and the skin and fascia were sequentially incised around the 13th thoracic vertebra and the 1st lumbar vertebra to expose the muscles in the surgical area. On one side, an incision was made from the 12th thoracic vertebra to the 2nd lumbar vertebra, extending to the articular process, by shearing the muscles and tendons connected to the spinous process using ophthalmic scissors. Another parallel incision, approximately 0.5 cm lateral to the first incision, was made with a length and depth similar to the previous incision. The muscle between the two incisions was then resected, exposing three pairs of articular processes (the 12th to 13th thoracic vertebrae, the 13th to 1st lumbar vertebrae, and the 1st to 2nd lumbar vertebrae). The lateral wall of the spinal canal and the intervertebral foramen were simultaneously exposed, the two lateral walls of the spinal canal were cut parallel to expose the spinal cord, and a wound was formed. When bleeding accompanied by cerebrospinal fluid leakage was observed, the model was successfully established. Subsequently, in Group A, a solution of Component A and a solution of Component B were immediately injected into the cerebrospinal fluid leakage site using a two-component mixer. After the solution had fully filled and penetrated, it was allowed to harden for 10 minutes. In Group B, bone wax was immediately applied to the cerebrospinal fluid leakage site. During use, leakage still occurred in Group B. Photographs of the use of a polysaccharide-based two-component hydrogel (Formulation 5, Group A) and bone wax (Group B) to close cerebrospinal fluid leakage in this example are shown in Figure 6. The wounds of the rats in Group A and B were sutured. Two days after surgery, all rats in group B died, while all rats in group A survived. Seven days after surgery, the hydrogel was firmly attached to the spinal injury site of the rats in group A, and no obvious leakage was observed. This indicates that the polysaccharide-based two-component hydrogel can be used to seal cerebrospinal fluid leaks.

[0163] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used to seal cerebrospinal fluid leaks.

[0164] Example 20 Use of polysaccharide-based two-component hydrogels for closure of pulmonary parenchymal air leaks A pulmonary parenchymal air leak model was selected in New Zealand white rabbits, and the rabbits were divided into two groups, Compound 6 (Group A) and Blank (Group B), with three rabbits in each group. 1% sodium pentobarbital was intravenously injected into the ear margin at a dose of 3 mL / kg, and the trachea was intubated under general anesthesia, with ventilation assisted by a ventilator (VT 80 mL, R 30 bpm). Then, a thoracotomy was performed, and the marginal tissue of the lung lobe was excised, leaving a wound surface of approximately 1 cm. 2 The wound surface was thin and at least one bronchiolar stump approximately 1 mm in diameter was visible. In group b, the chest was closed directly without wound preparation. In group a, solutions of components A and B were injected into the lung defect site using a two-component mixer. After the solution was fully filled and penetrated, it was allowed to harden for 10 minutes. After confirming that the hydrogel had adhered to the wound surface, the chest was closed layer by layer, residual air was removed, and the lungs were fully re-expanded. Three white rabbits in group b died of respiratory failure due to persistent air leaks within 4 hours after surgery, while three white rabbits in group a showed no air leaks on the lung wound surface. Dissections on days 7 and 14 after surgery showed that the hydrogel material in group a adhered tightly to the wound surface, with no fluid accumulation or adhesions. This indicates that the polysaccharide-based two-component hydrogel can be used to close pulmonary parenchymal air leaks.

[0165] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used to seal pulmonary parenchymal air leaks.

[0166] Example 21 Use of polysaccharide-based two-component hydrogels in hepatic hemostasis A liver cross-sectional wound model was selected in SD rats, and liver hemostasis experiments were conducted in three groups: Formulation 14 (group a), Formulation 7 (group b), and gauze (group c). After anesthetizing SD rats, the abdominal cavity was opened, the liver was lifted, and placed on pre-weighed filter paper. A 10 mm diameter cross-section was then created in the liver to induce bleeding. For groups a and b, solutions of component A and component B, respectively, were injected into the bleeding site using a two-liquid mixer. As the solutions penetrated, they rapidly gelled in the presence of blood, closing the bleeding opening. For group c, pre-weighed gauze was gently pressed against the cross-sectional wound. The amount of blood loss was measured by the weight increase of the filter paper (and gauze), and the time to hemostasis was recorded using a timer. During the experiment, the blood loss in groups a and b was significantly reduced compared to group c (group a: 0.2 g, group b: 0.1 g, group c: 1.0 g), and the time to hemostasis was significantly shortened (group a: 7 s, group b: 5 s, group c: 50 s). This indicates that the polysaccharide-based two-component hydrogel can be used to stop liver hemostasis.

[0167] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used for hepatic hemostasis.

[0168] Example 22 Use of polysaccharide-based two-component hydrogels for femoral artery hemostasis A femoral artery hemostasis experiment was conducted in SD rats using a femoral artery hemostasis model. The rats were divided into three groups: Formulation 14 (group a), Formulation 10 (group b), and a gauze group (group c). After anesthetizing, the right hind leg and lower abdomen were shaved to expose the femoral artery, vein, and nerve. The femoral artery was carefully dissected, and both ends of the artery were gently closed with hemostats. A 32G needle was used to puncture the artery. For groups a and b, once all arterial blood had drained, the components A and B solutions were immediately injected into the bleeding site using a two-component mixer. For group c, gauze was immediately applied to the bleeding site. The experimental results showed that in groups a and b, the hydrogel rapidly gelled in the presence of blood. After the hemostats were removed, the hydrogel remained firmly attached to the bleeding site, achieving stable closure and preventing further bleeding. The rat in group c died from excessive blood loss and failed to achieve hemostasis. It can be seen that the above polysaccharide-based two-component hydrogel can be used for femoral artery hemostasis.

[0169] Other formulations of the polysaccharide-based two-component hydrogels of the present invention can be similarly used for femoral artery hemostasis.

[0170] Example 23 Manufacture of polysaccharide membrane materials Taking Compound 5 as an example, components A and B were dissolved in a phosphate buffer solution (pH = 7.4), respectively, to obtain a solution of component A and a solution of component B. Both were applied to a clean, flat polytetrafluoroethylene plate using a two-component mixer, cured for 10 minutes, and then swollen in a phosphate buffer solution (pH = 7) containing 5% glycerin until equilibrium was reached. The polysaccharide-based membrane material was then obtained by oven-drying at a constant temperature of 30°C.

[0171] Precursor solutions of other formulations in the present invention can also produce polysaccharide-based membrane materials of different compositions.

[0172] Example 24 Use of polysaccharide-based membrane materials in sutureless closure of surgical wounds A dorsal wound model was selected in SD rats, and the rats were divided into two groups: a membrane material (Formulation 5, group a) and a suture group (group b) in Example 23 for a surgical wound non-suture closure experiment. After anesthetizing the SD rats, the back hair was shaved, and a 1 cm long skin incision was made on the back using a scalpel. For group a, the membrane material was applied to the incision and the incision was closed. For group b, the incision was aligned and sutured with 4-0 non-absorbable surgical suture. The incision healing was observed on days 3 and 10 after surgery. No obvious shedding of the membrane material was observed in group a, and the incision rapidly epithelialized, demonstrating a fast healing rate, while group b showed a slower healing rate. Figure 7 shows a comparative photograph of the H&E staining results 10 days after surgery for the surgical wound non-suture closure group (group a) using the polysaccharide-based membrane material and the suture group (group b) in this example. As can be seen from the H&E staining results, the wound surface in group a was completely healed, with the newly formed tissue close to the skin tissue, while the wound surface in group b was not completely repaired. This indicates that the polysaccharide-based membrane material can be used for non-suture closure of surgical wounds.

[0173] Other formulations of polysaccharide-based membrane materials of the present invention can be similarly used for sutureless closure of surgical wounds.

[0174] Example 25 Use of polysaccharide-based membrane materials as patches in abdominal hernia repair An acute abdominal hernia model was selected in SD rats, and an abdominal hernia repair experiment was conducted using two groups: one receiving the membrane material described in Example 23 (Formulation 5, group a) and the other receiving a Separmesh absorbable composite patch (Bard, group b). The rats were anesthetized, immobilized, and their skin was prepared and disinfected. 1.2 cm x 1.2 cm squares were marked 2 cm from the midline on both sides of the lower abdomen. The abdominal wall was incised layer by layer to create a complete abdominal wall defect with a diameter of 1.2 cm. A 2 cm x 2 cm square patch was placed on each side. The patch in group a was left unsewn, while the patch in group b was intermittently sutured with 4-0 nonabsorbable surgical sutures. The skin was disinfected with 75% ethanol and then intermittently sutured with 3-0 nonabsorbable surgical sutures. Thirty days after surgery, the abdominal cavity was opened to observe adhesion between the patch and the intraperitoneal organs where the patch was located. The experimental results show that there was no significant displacement of the patch in group A, no intraperitoneal hernia, and no obvious adhesion between the patch and the abdominal organs in group A, and there was no significant displacement of the patch in group B, and there was slight adhesion between the suture site and the abdominal retina, indicating that the polysaccharide-based membrane material can be used as a patch for abdominal hernia repair.

[0175] Other formulations of polysaccharide-based film materials of the present invention can similarly be used as patches for abdominal hernia repair.

[0176] Example 26 Manufacturing of polysaccharide powder materials Taking Formulation 22 as an example, components A and B were dissolved in phosphate buffer solution (pH = 7.4), respectively, to obtain solutions of components A and B. These solutions were then applied to a clean, flat polytetrafluoroethylene plate using a two-component mixer and cured for 10 minutes. After this, the mixture was swollen in phosphate buffer solution (pH = 7) to reach equilibrium, and then oven-dried at a constant temperature of 30°C. The dried material was then placed in a ball mill and sieved to obtain a fine powder (mesh size: 60-300 mesh), i.e., a polysaccharide-based powder material. In this example, the microscopic morphology of polysaccharide-based powder materials with different particle sizes is shown in Figure 8. Alternatively, one volume of phosphate buffer solution (pH = 7) was added to the cured hydrogel material, and the hydrogel mass was broken into small-particle microgels by high-speed shear stirring. After the microgels were separated, three volumes of 50%, 75%, and absolute ethanol were added sequentially to replace the water content in the microgels. The microgel was separated, dried in a vacuum oven at a constant temperature of 30°C for 12 hours, and sieved to obtain a powder of 60 mesh to 300 mesh, i.e., a polysaccharide powder material.

[0177] Precursor solutions of other formulations in the present invention can also produce polysaccharide-based powder materials of different compositions.

[0178] Example 27 Use of polysaccharide-based powder materials in minimally invasive surgical hemostasis A minimally invasive surgical hemostatic experiment was conducted in a Bama pig model of acute upper gastrointestinal arterial bleeding. The pigs were divided into two groups: one receiving the powder material (Formulation 22, group a) from Example 26 and the other receiving absorbable hemostatic particles (ARISTA, group b). Adult female Bama pigs (20-25 kg) were anesthetized, and a cannula was inserted into the right femoral artery to monitor blood pressure. The baseline blood pressure (systolic blood pressure) of all animals was then monitored within 5 minutes and adjusted to 90-100 mm Hg. Heparin (10 mg / kg) was then administered intravenously to achieve systemic heparinization. Under endoscopic guidance, the mucosa was dissected and the pulsating artery beneath the stomach mucosa was identified. The artery was then incised with an endoscopic scalpel to induce acute upper gastrointestinal bleeding, and spontaneous bleeding was maintained for 4 minutes. The hemostatic powders from groups a and b were applied to the bleeding site via a catheter, respectively. The bleeding site was observed, and the hemostatic procedure was repeated until hemostasis was achieved. The time to hemostasis and the mass of the material used were recorded. The experimental results showed that during surgery, the hemostatic powder of group A quickly adhered to the wound surface, formed a gel, and was able to stop the bleeding. The hemostatic time was short (group A: 80 seconds, group B: 160 seconds), and the amount of material used was small (group A: 1.1 g, group B: 2 g). This shows that the above polysaccharide-based powder material can be used for minimally invasive surgical hemostasis.

[0179] Other formulations of polysaccharide-based powder materials of the present invention can be similarly used for minimally invasive surgical hemostasis.

[0180] Example 28 Use of polysaccharide-based powder materials as antibacterial drug carriers in antibacterial wound treatment Using Formulation 14 as an example, components A and B were dissolved in phosphate buffer solution (pH 7.4) according to the example. Then, solutions of components A and B were obtained. Both solutions were applied to a clean, flat polytetrafluoroethylene plate using a two-component mixer and allowed to cure for 10 minutes. After that, 1 volume of phosphate buffer solution (pH 7) was added, and the hydrogel mass was broken into small-particle microgels using high-speed shear mixing. The microgel was dispersed in phosphate buffer solution (pH 7.4) containing 0.1% benzalkonium chloride and stirred for 1 hour. The cation-containing benzalkonium chloride adsorbed to the microgel surface by electrostatic adsorption. After the microgel was separated, 3 volumes of 50%, 75%, and anhydrous ethanol were added sequentially to displace the water content in the microgel. The separated microgel was then vacuum-dried at 30°C for 12 hours and sieved to obtain a polysaccharide powder material loaded with benzalkonium chloride.

[0181] A full-thickness incision wound infection model was selected for the backs of BALB / C mice, and the mice were divided into three groups: a polysaccharide powder material with benzalkonium chloride supported on its surface (group a), a benzalkonium chloride solution group (group b), and a blank group (group c) for a body surface wound antibacterial experiment. A 20 mm full-thickness longitudinal incision was made on the backs of 8- to 10-week-old female BALB / C mice, and 50 μL of E. coli 25922 (1 × 10 8 CFU mL- 1 ) was dripped onto the wound site to establish an in-situ acute pathogen infection model. In group a, the powder was evenly sprayed onto the wound. As wound exudate drained, the powder gradually gelled and firmly adhered to the wound surface. In group b, the wound was disinfected with 0.01% benzalkonium chloride solution. In group c, no treatment was performed. The experimental results showed that in group c, the infection began to spread, blood circulation was poor, and symptoms such as ischemia and suppuration gradually occurred, resulting in obvious tissue damage. In group b, the infection spread was limited initially, but symptoms such as suppuration and ischemia also occurred by the seventh day after surgery. In group a, the recovery effect was excellent, bacterial growth was inhibited, and there was no suppuration or ischemia on the wound surface. This indicates that the polysaccharide powder material loaded with benzalkonium chloride can be used for wound antibacterial treatment.

[0182] The polysaccharide powder materials of the other formulations of the present invention can also be used as carriers for antibacterial drugs in antimicrobial wound treatment.

[0183] Example 29 Use of polysaccharide-based powder materials as anti-inflammatory drug carriers in the treatment of inflammation Glucocorticoid drugs have a wide range of indications and have strong regulatory effects on various physiological functions of the body. Clinically, they are widely used to treat various autoimmune diseases, leukemia, asthma, allergic reactions, etc. However, long-term and nonspecific use of hormonal drugs can induce various diseases and cause serious side effects. Therefore, short-term, low-dose topical use is clinically recommended. Using Formulation 5 as an example, components A and B were dissolved in phosphate buffer solution (pH = 7.4) to obtain solutions of components A and B. Both solutions were applied to a clean, flat polytetrafluoroethylene plate using a two-component mixer and allowed to harden for 10 minutes. After this, one volume of phosphate buffer solution (pH = 7) was added, and the bulk hydrogel was then broken into small-particle microgels using high-speed shear mixing. The microgel was dispersed in a phosphate buffer solution (pH 7.4) containing budesonide (20 μg / mL) and stirred for 1 hour. Budesonide was adsorbed onto the surface of the microgel through hydrogen bonding and hydrophilic-hydrophobic interactions. After separating the microgel, three volumes of 50%, 75%, and absolute ethanol were added sequentially to displace the water content of the microgel. The separated microgel was then vacuum-dried at 30°C for 12 hours and sieved to obtain the polysaccharide powder material loaded with budesonide.

[0184] An acute enteritis model was selected in Balb / C mice. Three groups were treated with the polysaccharide powder material coated with budesonide (group A), the budesonide enema solution (group B), and a blank group (group C). 3% DSS drinking water was prepared with sterile water and filtered through a 0.22 μm filter. Mice were given this water for 7 consecutive days. Localized edema and hyperemia, as well as obvious inflammation, were observed via enteroscopy, indicating successful establishment of the model. Group A was sprayed with the powder at the inflamed site via enteroscopy; Group B was administered an enema with budesonide enema solution; and Group C was left untreated. After 7 and 14 days, the mice were observed for inflammation and dissected to determine the colon length of each group. As is clear from the experimental results, in group a, the powder material firmly adhered to the damaged, inflamed wound surface, and the edema and ulceration of the wound surface were alleviated compared to groups b and c. In this example, a statistical graph of the colon lengths of normal mice and mice treated for acute enteritis using the polysaccharide powder material with budesonide loaded on its surface (group a), the budesonide enema solution (group b), and the blank group (group c) is shown in Figure 9. As is clear from the statistical results, the colon lengths of mice in group a were the longest compared to groups b and c. This indicates that the above-mentioned polysaccharide powder material loaded with budesonide can be used to treat inflammation.

[0185] The polysaccharide powder materials of other formulations of the present invention can also be used as carriers for anti-inflammatory drugs in the treatment of inflammation.

[0186] Example 30 Production of polysaccharide-based sponge materials Taking formulation 6 as an example, components A and B were dissolved in phosphate buffer solution (pH = 7.4), respectively, to obtain solutions A and B. Both were sprayed onto a mold using a two-liquid mixer and cured for 10 minutes. After that, the mixture was swollen in phosphate buffer solution (pH = 7) to reach equilibrium, frozen at -20°C for 6 hours, and then freeze-dried at -60°C for 48 hours to obtain a polysaccharide sponge material.

[0187] Precursor solutions of other formulations in the present invention can also produce polysaccharide-based sponge materials of different compositions.

[0188] Example 31 Use of polysaccharide sponge materials as wound dressings in wound healing A wound dressing experiment was conducted on a back defect model using SD rats, divided into three groups: the sponge material described in Example 30 (Formulation 14, group a), a commercially available gelatin sponge group (group b), and a blank group (group c). A circular defect with a diameter of 10 mm was created on the back of each SD rat. The sponges in groups a and b were evenly applied to the wound, while group c was left untreated. Each group was then covered with gauze and medical tape. The experimental results showed that the sponge material in group a was able to firmly adhere to the tissue defect site. Compared to group c, groups a and b showed a faster wound healing rate, with newly formed tissue similar to skin tissue and no obvious scar tissue formation. This indicates that the polysaccharide-based sponge material can be used as a wound dressing for wound treatment.

[0189] The polysaccharide sponge materials of other formulations of the present invention can similarly be used in wound treatment as wound dressings.

[0190] Example 32 Use of polysaccharide-based sponge materials in splenic hemostasis A splenic hemostasis experiment was conducted using New Zealand white rabbits as a splenic hemorrhage model. The rabbits were divided into two groups: one using the sponge material described in Example 30 (Formulation 6, group a) and one using a commercially available absorbable hemostatic gauze (group b). The rabbits were anesthetized and immobilized. The abdominal fur was shaved and disinfected with iodine, and the abdominal cavity was opened to expose the spleen. A scalpel was used to create an 8 mm long, 1 mm deep incision in the spleen, forming a bleeding wound. The hemostatic material was gently pressed against the bleeding site, and the wound surface was observed approximately every 10 seconds. The time to hemostasis was recorded immediately after complete cessation of bleeding. Figure 10 shows a comparative photograph of the polysaccharide-based sponge material (group a) and the commercially available absorbable hemostatic gauze (group b) used for splenic hemostasis in this example. The hemostatic material was weighed before and after use; the difference was the total blood loss. During the experiment, both the materials in Group A and Group B were able to firmly adhere to the surface of the bleeding wound in the spleen. Compared to Group B, Group A achieved shorter hemostasis time (Group A: 120 s, Group B: 180 s) and less blood loss (Group A: 3 g, Group B: 3.5 g). This indicates that the polysaccharide sponge material can be used to stop bleeding in the spleen.

[0191] Other formulations of polysaccharide sponge materials of the present invention can be used for splenic hemostasis as well.

[0192] Example 33 Use of polysaccharide-based sponge materials in cardiac hemostasis. A cardiac hemostasis experiment was conducted using SD rats as a cardiac hemostasis model. The rats were divided into two groups: one receiving the sponge material (Formulation 30, group a) from Example 30 and the other receiving gauze (group b). SD rats were anesthetized and immobilized, and the chest hair was shaved. The thoracic cavity was opened to expose the heart, and the pericardium was removed using fine forceps. A biopsy punch was used to create a 2 mm diameter injury in the wall of the left or right ventricle of the heart. The hemostatic material was then immediately applied to the bleeding site, and the same pressure was applied. The wound surface was observed approximately every 10 seconds, and the time to hemostasis was recorded immediately after complete cessation of bleeding. Figure 11 shows a photograph of the polysaccharide-based sponge material (group a) used for cardiac hemostasis in this example. The hemostatic material was weighed before and after use, and the difference was the total blood loss. For groups in which bleeding could not be stopped within 300 seconds, rats were euthanized by exsanguination. During the experiment, the sponge in group a firmly adhered to the ventricular wound (Figure 11). Compared to group b, group a achieved hemostasis in a shorter time (group a: 60 seconds, group b rats failed to achieve hemostasis within 300 seconds), lost the least blood (group a: 1.2 g, group b: 2.5 g within 300 seconds), and the rats in group a survived the surgery. This indicates that the polysaccharide sponge material can be used for cardiac hemostasis.

[0193] Other formulations of polysaccharide sponge materials of the present invention can be used for cardiac hemostasis as well.

[0194] Example 34 Use of polysaccharide-based sponge materials for non-compressive hemostasis of penetrating wounds A liver perforation model was used in New Zealand white rabbits. The rabbits were divided into two groups: one receiving the sponge material (Formulation 6, group a) from Example 30 and the other receiving gauze (group b). A non-compression penetration wound hemostasis experiment was conducted. After anesthetizing the rabbits, the abdominal cavity was opened, and the liver was lifted and placed on pre-weighed gauze. A 6 mm diameter penetration wound was then created in the liver, allowing bleeding to occur. The hemostatic materials from each group were then packed into the penetration wound. Blood loss was measured based on the weight gain of the hemostatic material and gauze. During the experiment, group a showed significantly reduced blood loss (group a: 2.6 g, group b: 4.5 g) and significantly reduced hemostasis time (group a: 35 s, group b: 96 s) compared with group b. This demonstrates that the polysaccharide-based sponge material can be used for non-compression penetration wound hemostasis.

[0195] Other formulations of polysaccharide sponge materials of the present invention can similarly be used for non-compressive penetrating wound hemostasis.

[0196] The above Examples 16 to 22 demonstrate the use of specific polysaccharide-based two-component hydrogel formulations in tissue repair, closure of tissue fluid leaks, closure of tissue air leaks, hemostasis, etc., and these uses primarily utilize the tissue adhesiveness and hemostatic performance of the polysaccharide-based two-component hydrogels. As can be seen from the descriptions of Examples 12 to 14, all of the polysaccharide-based two-component hydrogel formulations 1 to 36 according to the examples of the present invention have good tissue adhesiveness, hemostatic performance, and low swelling, and therefore all of the polysaccharide-based two-component hydrogel formulations 1 to 36 according to the examples of the present invention can be used for tissue repair, closure of tissue fluid leaks, closure of tissue air leaks, hemostasis, etc.

[0197] The above Examples 23 to 34 demonstrate the preparation of specific formulations of polysaccharide-based membrane materials, polysaccharide-based powder materials, and polysaccharide-based sponge materials, and the corresponding biomedical applications. Based on the contents of this application, those skilled in the art can also prepare the polysaccharide-based two-component hydrogel formulations 1 to 36 in the examples into polysaccharide-based membrane materials, polysaccharide-based powder materials, and polysaccharide-based sponge materials as needed. Similarly, based on the contents of the present application, a person skilled in the art can use polysaccharide-based membrane materials prepared from the polysaccharide-based two-component hydrogel compositions 1 to 36 according to the examples for sutureless surgical wound closure or patches, if necessary, or can use polysaccharide-based powder materials prepared from the polysaccharide-based two-component hydrogel compositions 1 to 36 according to the examples for hemostasis or as carriers for cells, factors, or drugs, if necessary, or can use polysaccharide-based sponge materials prepared from the polysaccharide-based two-component hydrogel compositions 1 to 36 according to the examples for wound dressing products or hemostatic products, if necessary.

[0198] The above-described description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments, and can apply the general principles described herein to other embodiments without creative effort. Therefore, the present invention is not limited to the above-described embodiments. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should fall within the scope of protection of the present invention.

Claims

1. A polysaccharide-based polymer crosslinking agent modified with an ortho-phthalaldehyde group, The structure is as shown in Formula 1: 【Chemistry 1】 In formula 1, P is a natural polysaccharide polymer or a modified or degraded product thereof, and P is one or more selected from hyaluronic acid, cellulose, cellulose derivatives, alginic acid, dextran, agarose, heparin, chondroitin sulfate, carrageenan, tragacanth gum, xanthan gum, gellan gum, guar gum, gum arabic, locust bean gum, starch, starch hydrolysates, and starch derivatives; In Formula 1, n≧2; In Formula 1, the ortho-phthalaldehyde group and P are linked by a covalent bond. A polysaccharide-based polymer crosslinking agent characterized by:

2. The polysaccharide-based polymer crosslinking agent represented by formula 1 is 【Chemistry 2】 【Transformation 3】 is one selected from the following structures: However, 1≦r≦20, 1≦s≦20, 1≦t≦50, and n≧2. Preferably, 1≦r≦6, 1≦s≦10, 1≦t≦30, and n≧2. The polysaccharide-based polymer crosslinking agent according to claim 1 .

3. P is selected from hyaluronic acid, cellulose derivatives, alginic acid, heparin, chondroitin sulfate, xanthan gum, gellan gum, starch and starch derivatives; the cellulose derivative is selected from methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropylmethyl cellulose; Preferably, the cellulose derivative is selected from carboxymethyl cellulose, the starch derivative is selected from oxidized starch, esterified starch, etherified starch and alkylated starch; Preferably, the starch derivative is selected from carboxymethyl starch. The polysaccharide-based polymer crosslinking agent according to claim 1 .

4. An ortho-phthalaldehyde precursor derivative whose aldehyde group has been protected in advance is covalently linked to an active group of a polysaccharide polymer to obtain a polysaccharide polymer modified with the ortho-phthalaldehyde precursor derivative, and then the aldehyde group of the polysaccharide polymer modified with the ortho-phthalaldehyde precursor derivative is deprotected to obtain a polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group.

4. A method for producing the polysaccharide polymer crosslinking agent according to claim 1, 2 or 3.

5. The ortho-phthalaldehyde precursor derivative in which the aldehyde group is protected in advance comprises an ortho-phthalaldehyde group in which the aldehyde group is protected in advance, and a substituent R capable of linking to an active group of a polysaccharide polymer, The structure is as shown in Formula 2: 【Chemistry 4】 In Formula 2, R is selected from a carboxyl substituent, a vinyl sulfone substituent, an epoxy substituent, a halogenated alkane substituent, an isocyanate substituent, and an amino substituent; In Formula 2, R is directly connected to the benzene ring or is connected to the benzene ring via one or more alkylene chains or alkoxy chains; the plurality of alkylene chains and alkoxy chains are connected by an ether bond, an amide bond, an ester bond, a urethane bond, or a urea bond; 5. The method for producing the polysaccharide polymer crosslinking agent according to claim 4.

6. The ortho-phthalaldehyde precursor derivative in which the aldehyde group is previously protected is 【Transformation 5】 【Transformation 6】 is one selected from the compounds However, 1≦r≦20, 1≦s≦20, and 1≦t≦50, and preferably 1≦r≦6, 1≦s≦10, and 1≦t≦30.

6. The method for producing the polysaccharide polymer crosslinking agent according to claim 5.

7. the active group of the polysaccharide polymer is an active group of the polysaccharide polymer itself or a reactive group of the polysaccharide polymer after modification or degradation; Preferably, the active groups of the polysaccharide polymer are selected from hydroxyl groups and carboxy groups.

5. The method for producing the polysaccharide polymer crosslinking agent according to claim 4.

8. The covalent bond between the ortho-phthalaldehyde precursor derivative in which the aldehyde group has been protected in advance and the active group of the polysaccharide polymer is selected from the group consisting of an ether bond, an amide bond, an ester bond, a urethane bond, and a urea bond, and is preferably an ether bond, an amide bond, an ester bond, or a urethane bond.

5. The method for producing the polysaccharide polymer crosslinking agent according to claim 4.

9. When the active group of the polysaccharide polymer is selected from a hydroxyl group, the covalent bond is based on an ether bond between a vinyl sulfone substituent, an epoxy substituent, or a halogenated alkane substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer, an ester bond between a carboxyl substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer, or a urethane bond between an isocyanate substituent of the orthophthalaldehyde precursor derivative and a hydroxyl group of the polysaccharide polymer; When the active group of the polysaccharide polymer is selected from a carboxyl group, the covalent bond is based on an amide bond between the amino substituent of the ortho-phthalaldehyde precursor derivative and the carboxyl group of the polysaccharide polymer. The method for producing the polysaccharide polymer crosslinking agent according to claim 4 .

10. Deprotecting the aldehyde group of the polysaccharide polymer modified with an ortho-phthalaldehyde precursor derivative involves deprotecting the ortho-phthalaldehyde functional group, which has been previously protected and is linked to the polysaccharide polymer, under acidic conditions to obtain a polysaccharide polymer crosslinker modified with an ortho-phthalaldehyde group.

5. The method for producing the polysaccharide polymer crosslinking agent according to claim 4.

11. Component A and component B are dissolved in a solvent to obtain a solution of component A and a solution of component B, and the solution of component A and the solution of component B are mixed to obtain a polysaccharide-based two-component hydrogel; The component A is the ortho-phthalaldehyde group-modified polysaccharide polymer crosslinking agent according to any one of claims 1 to 3, Component B is a water-soluble small molecule, a water-soluble synthetic polymer, or a water-soluble natural polymer containing one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto functional groups contained in a single molecule is 2 or more; The water-soluble natural polymers include proteins, nucleic acids and polysaccharides. A method for producing a polysaccharide-based two-component hydrogel, comprising:

12. Component B is one or more selected from the group consisting of polyethylene glycol derivatives, polyethyleneimines, polyamino acids, proteins, modified proteins, denatured proteins, protein degradation products, polysaccharides, modified polysaccharides, and polysaccharide degradation products; The molecular structure of the substance selected as component B contains one or more groups selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, and mercapto groups; the number of primary amine, hydrazine, hydrazide, hydroxylamine or mercapto functional groups contained in a single molecule is 2 or more; Preferably, the polyethylene glycol derivative is a polyethylene glycol derivative modified with a primary amine, a hydrazine, a hydrazide, a hydroxylamine, or a mercapto group; Preferably, the proteins include collagen, serum proteins, fibrinogen and fibrin; The protein degradation product includes gelatin or a polypeptide, Preferably, the polysaccharide, modified polysaccharide, or degraded polysaccharide is selected from chitosan, modified chitosan, and degraded chitosan; hyaluronic acid, alginic acid, chondroitin sulfate, heparin, cellulose, chitin, and their respective modified and degraded products, each modified with a primary amine, hydrazine, hydrazide, hydroxylamine, or mercapto group. The method for producing the polysaccharide-based two-component hydrogel according to claim 11 .

13. Component B is selected from the group consisting of amino-modified polyethylene glycol derivatives, hydrazide-modified hyaluronic acid, collagen, serum proteins, gelatin, polypeptides, polyamino acids, and chitosan. The method for producing the polysaccharide-based two-component hydrogel according to claim 11 .

14. In the solution of component A, the solid content of component A is 0.1 to 40 wt %, preferably 0.5 to 20 wt %, and more preferably 0.5 to 10 wt %; In the solution of component B, the solid content of component B is 0.1 to 40 wt %, preferably 0.5 to 20 wt %, and more preferably 0.5 to 10 wt %. The method for producing the polysaccharide-based two-component hydrogel according to claim 11 .

15. A polysaccharide-based two-component hydrogel produced by the method according to claim 11, 12, 13 or 14.

16. A polysaccharide-based membrane material obtained by drying the polysaccharide-based two-component hydrogel according to claim 15. a polysaccharide-based powder material obtained by further mechanically ball milling the polysaccharide-based membrane material; or a polysaccharide-based powder material obtained by mixing a solution of component A and a solution of component B for producing the polysaccharide-based two-component hydrogel according to claim 11, followed by producing microgel particles by an emulsification method, a microfluidic method or a mechanical grinding method, and then drying and sieving the microgel particles; a polysaccharide sponge material obtained by mixing a solution of component A, a solution of component B, and a pore-forming agent to produce the polysaccharide two-component hydrogel according to claim 11, to obtain a polysaccharide hydrogel material having a pore structure, and then drying the obtained polysaccharide sponge material; A polysaccharide-based biomaterial characterized by:

17. When the polysaccharide-based biomaterial is selected from a polysaccharide-based sponge material, the pore-forming agent is an additive that forms a pore structure in the material, and is selected from a substance that easily decomposes into gas, polymer microspheres, polyethylene glycol, polyvinylpyrrolidone, a surfactant, and water.

17. The polysaccharide-based biomaterial of claim 16.

18. Use of said polysaccharide-based two-component hydrogel in the manufacture of a tissue repair product. Use of the polysaccharide-based two-component hydrogel in the manufacture of a tissue fluid leakage closure product. Use of the polysaccharide-based two-component hydrogel in the manufacture of a tissue air leak closure product; and and the use of said polysaccharide-based two-component hydrogel in the manufacture of a hemostatic product, the tissue repair includes skin repair and abdominal wall repair; The tissue fluid leakage closure includes a pancreatic fluid leakage closure, a cerebrospinal fluid leakage closure, an intestinal leakage closure, and a gastric leakage closure; the tissue air leak closure includes pulmonary parenchymal air leak closure; The hemostasis includes hepatic hemostasis, renal hemostasis, splenic hemostasis, pancreatic hemostasis, bone cross section hemostasis, arterial hemostasis, and cardiac hemostasis; Use of the polysaccharide-based two-component hydrogel according to claim 15.

19. When the polysaccharide-based biomaterial is a polysaccharide-based membrane material, the use of the polysaccharide-based membrane material includes: Use of said polysaccharide-based membrane material in the manufacture of a sutureless surgical wound closure product; and and the use of said polysaccharide-based film material in the manufacture of a patch product, When the polysaccharide-based biomaterial is a polysaccharide-based powder material, the use of the polysaccharide-based powder material includes: Use of said polysaccharide-based powder material in the manufacture of a hemostatic product; and and the use of said polysaccharide-based powder material in the manufacture of cell, factor, or drug carrier products; The hemostasis includes hemostasis during minimally invasive surgery, hemostasis of the liver, kidney, spleen, pancreas, bone cross-section, arterial, and cardiac hemostasis; The cells, factors, and drug carriers include stem cell carriers, growth promoting factor carriers, antibacterial drug carriers, and anti-inflammatory drug carriers; When the polysaccharide-based biomaterial is a polysaccharide-based sponge material, the use of the polysaccharide-based sponge material may include: Use of said polysaccharide-based sponge material in the manufacture of a wound dressing product; and the use of said polysaccharide-based sponge material in the manufacture of a hemostatic product, The hemostasis includes hepatic hemostasis, renal hemostasis, splenic hemostasis, pancreatic hemostasis, bone cross section hemostasis, arterial hemostasis, cardiac hemostasis, and non-compressive penetrating wound hemostasis. Use of the polysaccharide-based biomaterial according to claim 16.

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