Polysaccharide-based polymer cross-linking agent, polysaccharide-based biomaterial, and preparation method and application thereof

Abstract

The present invention relates to a polysaccharide-based polymer cross-linking agent, a polysaccharide-based biomaterial, and a preparation method and application. The polysaccharide-based polymer cross-linking agent in the present invention is a polysaccharide-based polymer cross-linking agent modified with an o-phthalaldehyde group, and the polysaccharide-based polymer cross-linking agent does not rely on an artificially synthesized hydrophilic polymer skeleton and complex chemical synthesis steps. The polysaccharide-based polymer cross-linking agent provided by the present invention and a water-soluble small molecule or polymer containing one or more of primary amine, diamine, hydrazide, hydroxylamine or thiol groups are combined in an aqueous medium to form a polysaccharide-based two-component hydrogel material, which has the advantages of being injectable, low swelling, and having strong tissue adhesion. In addition, the polysaccharide-based two-component hydrogel material provided by the present invention can also be further prepared into polysaccharide-based biomaterials in the form of films, powders, sponges, etc., which can be used as tissue adhesives, sealants, hemostatic agents, tissue engineering materials, etc., and have broad biomedical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular to a polysaccharide-based polymer cross-linking agent, a polysaccharide-based biomaterial, and a preparation method and application thereof. Background Art

[0002] Hydrogels are highly hydrated polymer materials with three-dimensional cross-linked networks and tunable physical and chemical properties. Their high water content, biocompatibility, and versatility have led to their widespread application in many biomedical fields. Two-component hydrogels, which can be formed and cured in situ, are clinically used as hemostatic agents, tissue adhesives, and sealants. For example, DuraSeal glue produced by Confluent Surgical Company in the United States and CoSeal glue produced by Baxter Company in the United States use polyethylene glycol as the backbone and achieve cross-linking based on the reaction of amino or thiol groups on the end groups with active esters of carboxylic acids. At the same time, the active esters of carboxylic acids react with active groups such as amino groups on the tissue surface to adhere to the tissue and seal the wounded tissue. For example, Chinese patents CN111440310A, CN111440334A, CN 111574756A, CN 111621038 A, CN113509591A, CN113694249A and CN114767920A disclose a series of polyethylene glycol cross-linkers modified with o-phthalaldehyde groups. The two-component hydrogels prepared by cross-linking o-phthalaldehyde with amino-containing polymers can be used as tissue sealants. However, due to the extremely strong hydrophilicity of the polyethylene glycol backbone, the swelling rate of such hydrogels is generally high (>100%). This high swelling not only greatly reduces the mechanical properties of the hydrogel, but also causes compression of surrounding tissues due to the volume expansion. At the same time, there is a risk of gel shedding, which makes such hydrogels a safety hazard in clinical applications. For example, polyethylene glycol sealants represented by DuraSeal and CoSeal are commonly used in neurosurgery to supplement the sealing of the dura mater after suturing to prevent cerebrospinal fluid leakage. However, in actual clinical applications, the volume expansion of the hydrogel after absorbing body fluids has caused nerve compression, leading to multiple adverse events leading to quadriplegia (D Thavarajah, P De Lacy, R Hussain, R M Redfern, Spine. 2010, 35, 25-26.).

[0003] To address the swelling problem, Chinese patent CN114907558A discloses a method for preparing polyethylene glycol-based low-swelling hydrogels by reducing the hydrophilicity of the polyethylene glycol polymer backbone through hydrophobic modification. However, this method is based on complex modifications of the polyethylene glycol backbone and involves multi-step chemical synthesis. Summary of the Invention

[0004] In order to solve the problem of high swelling rate commonly found in polyethylene glycol-based hydrogels in the prior art, the present invention provides a polysaccharide-based polymer cross-linking agent, a polysaccharide-based biomaterial, and a preparation method and application thereof.

[0005] Specifically, the present invention provides a polysaccharide-based polymer cross-linking agent, a method for preparing the polysaccharide-based polymer cross-linking agent, a polysaccharide-based biomaterial, a method for preparing the polysaccharide-based biomaterial, and an application of the polysaccharide-based biomaterial.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect, the present invention provides a polysaccharide-based polymer cross-linking agent.

[0008] The polysaccharide-based polymer crosslinking agent is a polysaccharide-based polymer crosslinking agent modified with an o-phthalaldehyde group, and its structure is shown in Formula 1:

[0009]

[0010] In Formula 1, P is a natural polysaccharide polymer or a modified or degraded product thereof, and P is selected from one or more of 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, or hydrolyzates or derivatives thereof;

[0011] In formula 1, n≥2, which means that the average number of o-phthalaldehyde functional groups on a single polysaccharide polymer chain is greater than or equal to 2;

[0012] In Formula 1, the o-phthalaldehyde group and P are connected via a covalent bond.

[0013] In some embodiments of the present invention, P is selected from hyaluronic acid, cellulose derivatives, alginic acid, heparin, chondroitin sulfate, xanthan gum, gellan gum, starch, or hydrolyzates or derivatives thereof.

[0014] Wherein, the cellulose derivative can be selected from cellulose ethers, including but not limited to methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose;

[0015] Preferably, the cellulose derivative is selected from carboxymethyl cellulose.

[0016] The starch derivative may be selected from oxidized starch, esterified starch, etherified starch or alkylated starch.

[0017] Preferably, the starch derivative is selected from carboxymethyl starch.

[0018] In some specific embodiments of the present invention, the polysaccharide-based polymer crosslinking agent modified with o-phthalaldehyde groups is selected from one of the following structures:

[0019]

[0020] Wherein, 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.

[0021] The second aspect of the present invention provides a method for preparing the polysaccharide-based polymer cross-linking agent.

[0022] The preparation method of the polysaccharide-based polymer cross-linking agent is as follows:

[0023] The aldehyde group-preprotected o-phthalaldehyde precursor derivative is covalently linked to the active group on the polysaccharide polymer to obtain the o-phthalaldehyde precursor derivative-modified polysaccharide polymer, and then the aldehyde group of the o-phthalaldehyde precursor derivative-modified polysaccharide polymer is deprotected to obtain the o-phthalaldehyde group-modified polysaccharide polymer crosslinker.

[0024] The aldehyde-preprotected o-phthalaldehyde precursor derivative comprises an aldehyde-preprotected o-phthalaldehyde group and a substituent R that can be connected to an active group on a polysaccharide polymer, and the structure is shown in Formula 2:

[0025]

[0026] 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.

[0027] In Formula 2, optionally, R may be directly connected to the benzene ring, or may be connected to the benzene ring through a single or multiple alkylene chains or alkoxy chains, wherein the multiple alkylene chains or alkoxy chains are connected through ether bonds, amide bonds, ester bonds, carbamate bonds or urea bonds.

[0028] In some specific embodiments of the present invention, the aldehyde group pre-protected o-phthalaldehyde precursor derivative is selected from one of the following compounds:

[0029]

[0030]

[0031] Wherein, 1≤r≤20, 1≤s≤20, 1≤t≤50. Preferably, 1≤r≤6, 1≤s≤10, 1≤t≤30.

[0032] The active groups of the polysaccharide polymer can be either inherent to the polysaccharide polymer or reactive groups formed after the polysaccharide polymer has been modified or degraded.

[0033] In some embodiments of the present invention, the active groups of the polysaccharide polymer are selected from hydroxyl groups and carboxyl groups.

[0034] The aldehyde group pre-protected o-phthalaldehyde precursor derivative is covalently linked to the active group on the polysaccharide polymer in a manner selected from ether bond, amide bond, ester bond, carbamate bond or urea bond, preferably ether bond, amide bond, ester bond or carbamate bond.

[0035] More specifically, when the active group of the polysaccharide polymer is selected from hydroxyl groups, the covalent bond connection is based on an ether bond connection between a vinyl sulfone substituent, an epoxy substituent or a halogenated alkane substituent on the o-phthalaldehyde precursor derivative and a hydroxyl group on the polysaccharide polymer, based on an ester bond connection between a carboxyl substituent on the o-phthalaldehyde precursor derivative and a hydroxyl group on the polysaccharide polymer, and based on a carbamate bond connection between an isocyanate substituent on the o-phthalaldehyde precursor derivative and a hydroxyl group on the polysaccharide polymer;

[0036] When the active groups of the polysaccharide polymer are selected from carboxyl groups, the covalent bond connection is based on an amide bond connection between the amino substituents on the o-phthalaldehyde precursor derivative and the carboxyl groups on the polysaccharide polymer.

[0037] In some specific embodiments of the present invention, the covalent bond connection is preferably selected from an ether bond connection between a vinyl sulfone substituent on an o-phthalaldehyde precursor derivative and a hydroxyl group on a polysaccharide polymer, an ether bond connection between an epoxy substituent on an o-phthalaldehyde precursor derivative and a hydroxyl group on a polysaccharide polymer, an ester bond connection between a carboxyl substituent on an o-phthalaldehyde precursor derivative and a hydroxyl group on a polysaccharide polymer, and an amide bond connection between an amino substituent on an o-phthalaldehyde precursor derivative and a carboxyl group on a polysaccharide polymer.

[0038] Deprotecting the aldehyde group of the polysaccharide polymer modified with the o-phthalaldehyde precursor derivative means deprotecting the pre-protected o-phthalaldehyde functional group of the aldehyde group connected to the polysaccharide polymer under acidic conditions to obtain a polysaccharide-based polymer crosslinker modified with the o-phthalaldehyde group.

[0039] The structures of the o-phthalaldehyde group-modified polysaccharide-based polymer crosslinkers provided in Formulas 1-1 to 1-12 of the first aspect of the present invention are merely exemplary illustrations of the o-phthalaldehyde group-modified polysaccharide-based polymer crosslinkers that can be obtained by combining the above-mentioned aldehyde group-preprotected o-phthalaldehyde precursor derivatives and polysaccharide polymers through a certain preparation method. Based on the disclosure of this application, those skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivative structures can be used to achieve the o-phthalaldehyde group-modified polysaccharide-based polymer crosslinkers.

[0040] A third aspect of the present invention provides a method for preparing a polysaccharide-based two-component hydrogel, comprising: dissolving component A and component B in a solvent to obtain a component A solution and a component B solution, respectively; the component A solution and the component B solution being the two components of the polysaccharide-based two-component hydrogel; and mixing the component A solution and the component B solution to obtain the polysaccharide-based two-component hydrogel.

[0041] Wherein, the component A is a polysaccharide-based polymer crosslinking agent modified with an o-phthalaldehyde group, as shown in Formula 1, where n≥2;

[0042] The component B is a water-soluble small molecule, a water-soluble artificial synthetic polymer, or a water-soluble natural polymer (such as a protein, a nucleic acid, and a polysaccharide) containing one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl functional groups contained in a single molecule is not less than 2.

[0043] In some embodiments of the present invention, preferably, the component B is selected from one or more of the following substances: polyethylene glycol derivatives, polyethyleneimine, polyamino acids, proteins, protein modifications, protein modifications, protein degradation products, polysaccharides, polysaccharide modifications, or polysaccharide degradation products; and the molecular structure of the substance selected from the component B contains one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or thiol groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or thiol functional groups contained in a single molecule is not less than 2;

[0044] Preferably, the polyethylene glycol derivative is a polyethylene glycol derivative modified with a primary amine, a hydrazine, a hydrazide, a hydroxylamine or a thiol group.

[0045] Preferably, the protein includes collagen, serum protein, fibrinogen and fibrin, and the protein degradation product includes gelatin or polypeptide;

[0046] Preferably, the polysaccharide, polysaccharide modification or polysaccharide degradation product is selected from: chitosan and chitosan modifications and chitosan degradation products; primary amine, diamine, hydrazide, hydroxylamine or thiol-modified hyaluronic acid, alginate, chondroitin sulfate, heparin, cellulose, chitin and their respective modifications and degradation products.

[0047] More preferably, the component B is selected from amino-modified polyethylene glycol derivatives; hydrazide-modified hyaluronic acid; collagen; serum protein; gelatin; polypeptide; polyamino acid; and chitosan.

[0048] In some embodiments of the present invention, the solvent is selected from water, physiological saline, buffer solution, decellularized matrix or cell culture medium solution.

[0049] In some embodiments of the present invention, preferably, in the Component A solution, the solid content of Component A is 0.1-40 wt%, preferably 0.5-20 wt%, and more preferably 0.5-10 wt%; and in the Component B solution, the solid content of Component B is 0.1-40 wt%, preferably 0.5-20 wt%, and more preferably 0.5-10 wt%. Increasing the proportion of Component A in the polysaccharide-based two-component hydrogel can effectively improve the tissue adhesion and hemostatic properties of the hydrogel.

[0050] In some embodiments of the present invention, the preparation temperature for preparing the hydrogel by mixing the component A solution and the component B solution is 0-80° C.; and the preparation pH is 1-12.

[0051] The fourth aspect of the present invention provides a polysaccharide-based two-component hydrogel prepared by the method according to the third aspect of the present invention.

[0052] In a fifth aspect, the present invention provides a variety of polysaccharide-based biomaterials.

[0053] In some embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based membrane material obtained by drying the polysaccharide-based two-component hydrogel provided by the fourth aspect of the present invention.

[0054] In some embodiments of the present invention, the drying method includes natural drying and oven drying.

[0055] Preferably, the drying condition is 30° C. for 12 hours.

[0056] In some embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based powder material obtained by mechanically ball-milling the polysaccharide-based membrane material. Mechanical ball milling involves placing the polysaccharide-based membrane material into a ball mill. The material is subjected to repeated impact, grinding, shear, and pressure forces, causing it to deform and break into a fine powder. To reduce interparticle adhesion and prevent agglomeration, a liquid surfactant and lubricant, such as ethanol, may be added. Liquid additives must not corrode or chemically react with the particles.

[0057] In other possible embodiments of the present invention, the polysaccharide-based biomaterial is a polysaccharide-based powder material obtained by mixing the component A solution and the component B solution provided in the third aspect of the present invention, preparing microgel particles through emulsification, microfluidics, and mechanical crushing, and further drying and screening.

[0058] Preferably, the microgel particles are prepared by mechanical pulverization: the polysaccharide-based hydrogel obtained by mixing the component A solution and the component B solution is cut into particles with a uniform particle size distribution by a high-speed shear homogenizer. Preferably, the particle size distribution of the microgel particles is 1 to 1000 μm.

[0059] In some specific embodiments of the present invention, the drying method is vacuum freeze drying or oven drying. The preferred freeze drying conditions are freezing at -20°C for 6 hours, followed by freeze drying at -60°C for 48 hours; the drying conditions are drying at 30°C for 12 hours. In some specific embodiments of the present invention, the sieving is an operation process of separating coarse powder and fine powder from powders with a large difference in coarseness after pulverization using a mesh-like tool (such as a vibrating sieve). Preferably, the particle size distribution of the sieved powder is 20 mesh to 500 mesh.

[0060] In some embodiments of the present invention, the polysaccharide-based biomaterial is prepared by mixing the component A solution, the component B solution and the pore-forming agent provided in the third aspect of the present invention to obtain a polysaccharide-based hydrogel material having a pore structure, and further drying the obtained polysaccharide-based sponge material.

[0061] Among them, the component A is a polysaccharide-based polymer crosslinker modified with an o-phthalaldehyde group, and the component B is a water-soluble small molecule, a water-soluble artificial synthetic polymer, or a water-soluble natural polymer (such as a protein, a nucleic acid, and a polysaccharide) containing one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or thiol groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or thiol functional groups contained in a single molecule is not less than 2.

[0062] In some embodiments of the present invention, the pore-forming agent is an additive that creates a pore structure in the material, including substances that are easily decomposed into gas, polymer microspheres, polyethylene glycol, polyvinyl pyrrolidone, surfactants, water, and the like.

[0063] Among them, substances that easily decompose into gas, such as ammonium bicarbonate, when added to the material and heated, will release carbon dioxide and ammonia, which will overflow from the material to form a porous structure. Polymer microspheres such as polymethyl methacrylate microspheres, silica microspheres, and polystyrene microspheres.

[0064] Preferably, the pore-forming agent is water, and the hydrogel forms a porous honeycomb sponge structure through freeze drying.

[0065] Preferably, the freeze-drying conditions are freezing at -20°C for 6 hours, followed by freeze-drying at -60°C for 48 hours.

[0066] In a sixth aspect, the present invention provides an application of the polysaccharide-based two-component hydrogel according to the fourth aspect of the present invention, selected from one of the following applications:

[0067] Application of the polysaccharide-based two-component hydrogel in the preparation of tissue repair products;

[0068] Application of the polysaccharide-based two-component hydrogel in the preparation of tissue fluid leakage blocking products;

[0069] Application of the polysaccharide-based two-component hydrogel in the preparation of tissue leakage sealing products;

[0070] Application of the polysaccharide-based two-component hydrogel in the preparation of hemostatic products.

[0071] Tissue repair includes skin repair and abdominal wall repair. Tissue fluid leak plugging includes pancreatic fluid leak plugging, cerebrospinal fluid leak plugging, intestinal leak plugging, and gastric leak plugging. Tissue air leak plugging includes lung parenchymal air leak plugging. Hemostasis includes liver hemostasis, kidney hemostasis, spleen hemostasis, pancreatic hemostasis, bone fracture hemostasis, arterial hemostasis, and cardiac hemostasis.

[0072] The seventh aspect of the present invention provides the use of the polysaccharide-based biomaterial based on the fifth aspect of the present invention.

[0073] When the polysaccharide-based biomaterial is a polysaccharide-based membrane material, the application of the polysaccharide-based membrane material is selected from one of the following applications:

[0074] Application of the polysaccharide-based membrane material in the preparation of suture-free closure products for surgical wounds;

[0075] Application of the polysaccharide-based membrane material in the preparation of patch products;

[0076] Among them, patch products made of polysaccharide-based membrane materials can be used for abdominal hernia repair, etc.

[0077] When the polysaccharide-based biomaterial is a polysaccharide-based powder material, the application of the polysaccharide-based powder material is selected from one of the following applications:

[0078] Application of the polysaccharide-based powder material in the preparation of hemostatic products;

[0079] Application of the polysaccharide-based powder material in the preparation of cell, factor, and drug carrier products;

[0080] Among them, hemostatic products prepared from polysaccharide-based powder materials can be used for minimally invasive surgical hemostasis, liver hemostasis, kidney hemostasis, spleen hemostasis, pancreatic hemostasis, bone section hemostasis, arterial hemostasis and cardiac hemostasis, etc.; cells, factors, and drug carriers include stem cell carriers, growth factor carriers, antibacterial drug carriers and anti-inflammatory drug carriers, etc.

[0081] When the polysaccharide-based biomaterial is a polysaccharide-based sponge material, the application of the polysaccharide-based sponge material is selected from one of the following applications:

[0082] Application of the polysaccharide-based sponge material in the preparation of wound dressing products;

[0083] Application of the polysaccharide-based sponge material in the preparation of hemostatic products.

[0084] When the polysaccharide-based sponge material is used to prepare hemostatic products, it can be used for liver hemostasis, kidney hemostasis, spleen hemostasis, pancreas hemostasis, bone section hemostasis, artery hemostasis, heart hemostasis and non-compression penetrating wound hemostasis.

[0085] Polysaccharides are a type of natural water-soluble polymer with a wide range of sources and good biocompatibility.

[0086] The present invention provides a class of polysaccharide-based polymer crosslinking agents modified with o-phthalaldehyde groups. The o-phthalaldehyde-modified polysaccharide-based polymer crosslinking agents of the present invention do not rely on artificially synthesized hydrophilic polymer backbones or complex chemical synthesis steps. The polysaccharide-based polymer crosslinking agents provided by the present invention have the advantages of a wide range of raw material sources and a simple preparation method.

[0087] The present invention also combines a polysaccharide-based polymer crosslinker with a small molecule or polymer derivative containing one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl groups in an aqueous medium to form a polysaccharide-based two-component hydrogel material. The hydrogel material is composed of two cross-linked components: one is a polysaccharide-based polymer crosslinker modified with an o-phthalaldehyde group, and the other is a water-soluble small molecule, water-soluble artificial synthetic polymer, water-soluble natural polymer (such as protein, nucleic acid, and polysaccharide) containing one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl groups. Each of the above two components contains no less than 2 corresponding functional groups. The polysaccharide-based two-component hydrogel material provided by the present invention has the advantages of injectability, low swelling, and strong tissue adhesion, and can be used as a tissue adhesive, sealant, hemostatic agent, tissue engineering material, etc.

[0088] The present invention also provides polysaccharide-based biomaterials (such as films, powders, and sponges) in various forms based on polysaccharide-based polymer crosslinkers or hydrogel materials. The two-component hydrogels, films, powders, and sponges provided by the present invention can be used as tissue adhesives, sealants, hemostatic agents, tissue engineering materials, etc., have broad biomedical application prospects, and are of great significance for clinical hemostasis, tissue adhesion, and sealing. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the hyaluronic acid-based crosslinking agent (i.e., component A-1.2) in Example 2.

[0090] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the carboxymethyl cellulose-based crosslinking agent (i.e., component A-2.2) in Example 3.

[0091] Figure 3 These are photos of the precursor solution and the gelled state of the polysaccharide-based two-component hydrogel (Formula 7) in Example 11.

[0092] Figure 4 This is a photo of the polysaccharide-based two-component hydrogel (group a) and suture (group b) in Example 16 after 7 days of application in skin tissue repair.

[0093] Figure 5 This is a photo taken 7 days after the polysaccharide-based two-component hydrogel (group a) in Example 18 was used to block pancreatic juice leakage from the pancreatic stump.

[0094] Figure 6 These are photos of the polysaccharide-based two-component hydrogel (group a) and bone wax (group b) used to block cerebrospinal fluid leakage in Example 19.

[0095] Figure 7 These are comparative photographs of the H&E staining results 10 days after surgery in which the polysaccharide-based membrane material in Example 24 was applied to the surgical wound closure without suture (Group a) and the suture group (Group b).

[0096] Figure 8 The microscopic morphology of the polysaccharide-based powder materials of different particle sizes under a microscope in Example 26 is shown.

[0097] Figure 9 This is a statistical graph of the colon length of normal mice and mice treated with acute enteritis in Example 29, including the polysaccharide-based powder material surface-loaded with budesonide (Group a), the budesonide enema solution (Group b), and the blank group (Group c).

[0098] Figure 10 Comparative photos of the sponge material (group a) in Example 32 and the commercial absorbable hemostatic gauze (group b) used for spleen hemostasis.

[0099] Figure 11 This is a photograph of the sponge material (group a) in Example 33 being used to stop heart bleeding. DETAILED DESCRIPTION

[0100] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0101] Example 1: Synthesis of aldehyde-preprotected o-phthalaldehyde precursor derivatives (i.e., compounds 1 to 13)

[0102] (I) Synthesis of Compounds 1-3 (s=2, t=7)

[0103]

[0104] (1) Synthesis of compound 1: The synthesis process was based on 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 (400MHz, DMSO-d6): δ = 12.74 (brs, 1H), 8.29 (d, J = 7.6Hz, 1H), 8.12 (s, 1H), 7.65 (d, J = 7.6Hz, 1H), 6.30 (s, 1H), 6.11 (s, 1H), 3.40-3.30 (m, 6H).

[0105] (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, and stirred at room temperature for 30 minutes. Ethylenediamine (27.05 g, 0.45 mmol) was dissolved in anhydrous DCM and stirred, and the above reaction system was quickly added dropwise to the ethylenediamine solution, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, most of the solvent was removed, and the residual 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 crude product was purified by silica gel chromatography to obtain Compound 2 (9.0 g, yield 75%). 1 H NMR (400MHz, DMSO-d6): δ=8.18(t,1H),8.07(d,1H),7.80(dd,1H),7.56(dd,1H),6.05(h,2 H),4.59(dt,1H),4.25(dt,1H),3.38(tt,J=5.1Hz,2H),3.34(s,3H),3.03(tt,J=6.3,2H).

[0106] (3) Synthesis of compound 3: α,ω-dicarboxyl polyethylene 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 minutes. Compound 2 (5 g, 18.8 mmol) was dissolved in anhydrous DCM (50 mL) and gradually added dropwise to the above system. The reaction was stirred at room temperature for 12 hours. After the reaction was completed, most of the solvent was removed, and the residual 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 crude product was purified by silica gel column chromatography to obtain compound 3 (10.5 g, yield 79%). 1 H NMR (400MHz, DMSO-d6): δ = 8.86 (s, 1H), 8.21 (d, J = 7.6Hz, 1H), 7.93 (s, 1H), 7.61 ( d,J=7.6Hz,1H),6.38(s,1H),6.30(s,1H),3.80-3.48(m,28H),3.40-3.30(m,6H).

[0107] (II) Synthesis of Compounds 4-8 (r = 2, s = 6, t = 7)

[0108]

[0109] (1) Synthesis of compound 4: The synthesis process was based on 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 HNMR (400MHz, 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).

[0110] (2) Synthesis of compound 5: The synthesis process was similar to that of compound 2. 1H NMR (400MHz, 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=8.2Hz,2H),1.87(t,J=6.5Hz,2H),1.59-1.43(m,4H),1.46-1.30(m,4H).

[0111] (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 completion of the reaction, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain Compound 6 (5.2 g, 80% yield). 1 H NMR (400MHz, 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=8.7Hz,2H),2.55(dd,2H),2.48-2.34(m,4H),1.56-1.43(m,4H),1.40-1.27(m,4H).

[0112] (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 minutes. The above reaction system was gradually added dropwise to the NH2-PEG-NH2 solution and stirred at room temperature for 12 hours. After the reaction was completed, most of the solvent was removed, and the residual 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 crude product was purified by silica gel column chromatography to obtain compound 7 (10.4 g, yield 81%). 1H NMR (400MHz, 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).

[0113] (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 stirred HDI solution. The mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation, and the mixture was redissolved in a small amount of DCM and poured into n-pentane to precipitate a solid. This reaction was repeated twice and then drained to obtain Compound 8 (5.5 g, 92% yield). 1 H NMR (400MHz, 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=5.7,4.7,1.0Hz,2H),2. 90-2.71(m,4H),2.41(t,J=8.3Hz,2H),1.87(t,J=6.5Hz,2H),1.59-1.27(m,8H).

[0114] (III) Synthesis of Compounds 9-13

[0115]

[0116] (1) Synthesis of Compound 9: Intermediate 1 (5 g, 25.5 mmol) was dissolved in anhydrous DCM (40 mL), and 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 reaction was stirred at room temperature for 48 h. Subsequently, the phases were separated, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography to obtain Compound 9 (5.6 g, 85% yield). 1H NMR (400MHz, 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 = 2.2Hz, 2H), 3.81 (t, J = 2.2Hz, 2H), 3.37-3.34 (s, 6H).

[0117] (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 stirred HDI solution, and the reaction was 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. After repeating twice, Compound 10 (9.76 g, 70% yield) was obtained. 1 H NMR (400MHz, 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 = 5.3Hz, 1 H),6.03–5.93(m,2H),3.38-3.31(s,6H),3.20-3.13(m,2H),3.06(q,J=5.5Hz,2H),1.67-1.27(m,8H).

[0118] (3) Synthesis of Compound 11: Under anhydrous and oxygen-free conditions, intermediate 1 (5 g, 25.5 mmol) was dissolved in anhydrous DCM. 2-Chloroethanesulfonyl chloride (5 g, 30.6 mmol) was slowly added dropwise to the solution in an ice bath at 0°C. After a 10-min reaction, anhydrous TEA (4.23 mL, 30.6 mmol) was slowly added dropwise and the reaction was continued at room temperature for 12 h. After completion of the reaction, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to afford compound 11 (6.9 g, 95% yield). 1 H NMR (400MHz, 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=2.9,1.7Hz,2H),3.41-3.33(s,6H).

[0119] (4) Synthesis of Compound 12: Intermediate 1 (5 g, 25.5 mmol) was suspended in 30 mL of water and a 1.5 M aqueous NaOH solution (10 mL) was added. The system gradually became clear. The mixture was stirred for 40 minutes, and epichlorohydrin (7.1 g, 76.5 mmol) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, most of the solvent was removed, and the residual 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 crude product was purified by silica gel column chromatography to obtain Compound 12 (5.8 g, 90% yield). 1 H NMR (400MHz, DMSO-d6): δ = 7.45 (s, 1H), 7.14 (s, 1H), 6.91 (dd, J = 8.3, 1H), 6.07-5.9 9(m,2H),4.11(d,J=3.3Hz,2H),3.42(s,1H),3.38-3.32(m,6H),2.91-2.78(m,2H).

[0120] Example 2: Synthesis of Representative Components A-1.1 to A-1.4 of Hyaluronic Acid-Based Crosslinkers Modified with O-Phthalaldehyde Groups

[0121]

[0122] (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-morpholino)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 added to the above reaction solution. 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 1.4 g, 5 mmol) was weighed and added to the above reaction system in three portions, each time dissolved in 3 mL of MES buffer solution, with an interval of 1 h. The reaction was carried out at 37°C for 24 h. After the reaction, the pH was adjusted to 8-9, stirred for 1 hour, and dialyzed against deionized water (MWCO 14,000) for 2-3 days. The product was collected and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 hour, and the solution was adjusted to neutral. The reaction system was then dialyzed against deionized water (MWCO 14,000) for 1 day, and the product was collected and freeze-dried to yield component A-1.2. The labeling of compound 2 on HA was confirmed by H NMR spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 2 in the supernatant was determined by HPLC. The labeling yield of compound 2 was calculated to be approximately 6.78% (w / w).

[0123]

[0124] (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). 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 MWCO 7000 was used for dialysis. The rest of the synthesis process was similar to that of component A-1.1. The H NMR spectrum of the representative hyaluronic acid-based crosslinker (i.e., component A-1.2) in Example 2 is shown in FIG. Figure 1 As shown, the labeling of compound 5 on HA was identified by H NMR spectrum. The supernatant of the reaction system was collected, and the amount of residual compound 5 in the supernatant was determined by HPLC. The labeling rate of compound 5 was calculated to be approximately 4.21% (w / w).

[0125]

[0126] (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, the pH was neutralized to 7 with dilute hydrochloric acid and dialyzed against deionized water for 2-3 days (MWCO 14000). The product was collected and 10% trifluoroacetic acid was added. The reaction was continued for 1 h and then adjusted to neutrality. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-1.3. The labeling of compound 11 on HA was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 11 in the supernatant was determined by HPLC. The labeling rate of compound 11 was calculated to be approximately 4.12% (w / w).

[0127]

[0128] (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, the pH was neutralized to 7 with dilute hydrochloric acid and dialyzed against deionized water for 2-3 days (MWCO 14000). The product was collected and 10% trifluoroacetic acid was added. The reaction was continued for 1 h and then adjusted to neutrality. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-1.4. The labeling of compound 12 on HA was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 12 in the supernatant was determined by HPLC. The labeling rate of compound 12 was calculated to be approximately 4.01% (w / w).

[0129] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified hyaluronic acid-based polymer crosslinker. Based on the disclosure of this application, one skilled in the art will appreciate that other o-phthalaldehyde protected derivatives can be used to synthesize the o-phthalaldehyde group-modified hyaluronic acid-based polymer crosslinker.

[0130] Example 3: Synthesis of Representative Components A-2.1 to A-2.4 of O-Phthalaldehyde-Modified Carboxymethyl Cellulose-Based Crosslinking Agents

[0131]

[0132] (1) Synthesis of component A-2.1: Carboxymethyl cellulose (CMC, 5 g, viscosity (2%, 25°C) 1200 mPa·s, carboxymethyl substitution degree 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 dimethylaminopyridine p-toluenesulfonate (DPTS, 3 g, 12 mmol) were added to the above solution in sequence. The reaction was allowed to proceed at room temperature for 24 h. After the reaction was completed, the mixture was dialyzed against a sodium chloride aqueous solution containing dilute hydrochloric acid (pH = 4) for 2-3 days (MWCO 14000). The product was collected and 10% trifluoroacetic acid was added. The reaction was allowed to proceed for 1 h and then adjusted to neutral. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-2.1. The labeling of compound 1 on CMC was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 1 in the supernatant was determined by HPLC. The labeling rate of compound 1 was calculated to be approximately 2.48% (w / w).

[0133]

[0134] (2) Synthesis of component A-2.2: CMC (5 g, viscosity (2%, 25°C) 675 mPa·s, carboxymethyl substitution degree 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. The rest of the synthesis process was referred to the synthesis of component A-1.2. The H NMR spectrum of the representative carboxymethyl cellulose-based crosslinking agent (i.e. component A-2.2) in Example 3 is shown in FIG. Figure 2 As shown, the labeling of compound 5 on CMC was identified by H NMR spectrum. The supernatant of the reaction system was collected, and the amount of residual compound 5 in the supernatant was determined by HPLC. The labeling rate of compound 5 was calculated to be approximately 3.31% (w / w).

[0135]

[0136] (3) Synthesis of component A-2.3: CMC (5 g, viscosity (2%, 25°C) 675 mPa·s, carboxymethyl substitution degree 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 directly added to the reaction system; a dialysis bag with MWCO 7000 was selected for dialysis. The rest of the synthesis process was referred to the synthesis of component A-2.1. The labeling of compound 7 on CMC was identified by nuclear magnetic resonance spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 7 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 7 was calculated to be approximately 3.67% (w / w).

[0137]

[0138] (4) Synthesis of component A-2.4: CMC (5 g, viscosity (2%, 25°C) 2000 mPa·s, carboxymethyl substitution degree 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, the pH was neutralized to 7 with dilute hydrochloric acid and dialyzed against deionized water for 2-3 days (MWCO 14000). The product was collected and added with 10% trifluoroacetic acid, reacted for 1 h, and then adjusted to neutrality. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-2.4. The labeling of compound 9 on CMC was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 9 in the supernatant was determined by HPLC. The labeling rate of compound 9 was calculated to be approximately 3.01% (w / w).

[0139] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified carboxymethyl cellulose-based polymer crosslinking agent. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified carboxymethyl cellulose-based polymer crosslinking agent.

[0140] Example 4: Synthesis of Representative Components A-3.1 to A-3.3 of the O-Phthalaldehyde-Modified Alginate-Based Crosslinking Agent

[0141]

[0142] (1) Synthesis of Component A-3.1: Alginic acid (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 to the above solution in sequence. A dialysis bag with a MWCO of 3500 was used for dialysis. The rest of the synthesis process was similar to that of Component A-2.1. The labeling of Compound 3 on Alg was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual Compound 3 in the supernatant was determined by HPLC. The labeling rate of Compound 3 was calculated to be approximately 2.63% (w / w).

[0143]

[0144] (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 minutes. Compound 5 (350 mg, 1 mmol, r = 2, s = 6) was weighed and added to the above reaction system, and stirred at room temperature for 24 hours. After the reaction was completed, the pH was neutralized to 7 with dilute hydrochloric acid, and the mixture was dialyzed with deionized water for 2-3 days (MWCO 7000). After the product was collected, 10% trifluoroacetic acid was added and the mixture was reacted for 1 hour. The mixture was then adjusted to neutrality. The reaction system was then dialyzed with deionized water for 1 day (MWCO 7000), and the product was collected and freeze-dried to obtain component A-3.2. The labeling of compound 5 on Alg was identified by H NMR spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 5 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 5 was calculated to be approximately 2.57% (w / w).

[0145]

[0146] (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. The remaining synthesis process was referred to the synthesis of component A-1.3. The labeling of compound 11 on Alg was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 11 in the supernatant was determined by HPLC. The labeling rate of compound 11 was calculated to be approximately 2.20% (w / w).

[0147] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified alginate-based polymer crosslinker. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde-group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified alginate-based polymer crosslinker.

[0148] Example 5: Synthesis of Representative Components A-4.1 and A-4.2 of Carboxymethyl Starch-Based Crosslinking Agents Modified with O-Phthalaldehyde Groups

[0149]

[0150]

[0151] (1) Synthesis of component A-4.1: Disperse carboxymethyl starch sodium (CMS, type A, 5 g, pH 5.0-7.5) in 250 mL of deionized water. Add compound 4 (0.3 g, 1.2 mmol, r = 2), EDC (1.86 g, 12 mmol) and DPTS (3 g, 12 mmol) to the above system in sequence. React at room temperature for 24 h. After the reaction is completed, reprecipitate with 10 times the volume of anhydrous ethanol to precipitate a white powder. Collect the solid and redisperse it in 250 mL of deionized water. Add 10% trifluoroacetic acid and react for 1 h. Adjust the pH to neutral. Reprecipitate with 10 times the volume of anhydrous ethanol to precipitate a white powder, namely component A-4.1. Identify the marker of compound 4 on CMS by hydrogen nuclear magnetic spectrum. The supernatant of the reaction system was collected, and the amount of residual compound 4 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 4 was calculated to be approximately 2.15% (w / w).

[0152]

[0153] (2) Synthesis of Component A-4.2: CMS (Type A, 5 g, pH 5.0-7.5) was dissolved in 40 mL of 0.25 M NaOH solution; compound 12 (0.3 g, 1.2 mmol) was added to the above system; and finally, the mixture was reprecipitated with 10 volumes of anhydrous ethanol to precipitate a white powder. The rest of the synthesis process was referred to the synthesis of component A-1.4. The labeling of compound 12 on CMS was identified by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 12 in the supernatant was determined by HPLC. The labeling rate of compound 12 was calculated to be approximately 1.78% (w / w).

[0154] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified carboxymethyl starch-based polymer crosslinking agent. Based on the disclosure of this application, those skilled in the art will appreciate that other carboxymethyl starches from different sources or with different structures can also be used to synthesize the o-phthalaldehyde group-modified carboxymethyl starch-based polymer crosslinking agent. Similarly, other aldehyde-group-preprotected o-phthalaldehyde precursor derivatives can also be used to synthesize the o-phthalaldehyde group-modified carboxymethyl starch-based polymer crosslinking agent.

[0155] Example 6: Synthesis of Representative Components A-5.1 and A-5.2 of Hydroxyethyl Cellulose-Based Crosslinking Agents Modified with O-Phthalaldehyde Groups (m≥1)

[0156]

[0157] (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 also added as a catalyst. The reaction was stirred at 70°C for 24 h. After the reaction was completed, the same volume of ice water was slowly added and the mixture was dialyzed against deionized water for 2-3 days (MWCO 14000). The product was collected and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 h and then adjusted to neutral. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain Component A-5.1. The labeling of Compound 8 on HEC was identified by H NMR spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 8 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 8 was calculated to be approximately 4.12% (w / w).

[0158]

[0159] (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 also added as a catalyst. The reaction was stirred at 70°C for 24 h. After the reaction was completed, an equal volume of ice water was slowly added. The mixture was dialyzed against deionized water for 2-3 days (MWCO 14000). The product was collected and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 h and then adjusted to neutral. The reaction system was then dialyzed against deionized water for 1 day (MWCO 7000). The product was collected and freeze-dried to obtain component A-5.2. The marker of compound 10 on HEC was identified by H NMR spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 10 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 10 was calculated to be approximately 3.14% (w / w).

[0160] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified hydroxyethyl cellulose-based polymer crosslinking agent. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified hydroxyethyl cellulose-based polymer crosslinking agent.

[0161] Example 7: Synthesis of Representative Component A-6.1 of Heparin-Based Crosslinker Modified with O-Phthalaldehyde Groups

[0162]

[0163] 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 react at room temperature for 24 h. After completion of the reaction, the mixture was dialyzed against a sodium chloride aqueous solution containing dilute hydrochloric acid (pH = 4) for 2-3 days (MWCO 14000). The product was collected and added with 10% trifluoroacetic acid. The reaction was allowed to react for 1 h and then adjusted to neutral. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-6.1. The labeling of compound 6 on Hep was confirmed by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 6 in the supernatant was determined by HPLC. The labeling yield of compound 6 was calculated to be approximately 2.56% (w / w).

[0164] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified heparin-based polymer crosslinker. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified heparin-based polymer crosslinker.

[0165] Example 8: Synthesis of Representative Component A-7.1 of Chondroitin Sulfate-Based Crosslinker Modified with O-Phthalaldehyde Groups

[0166]

[0167] Chondroitin sulfate (CS, 5 g, 20 kDa) was dissolved in 50 mL of 0.01 mol / L MES buffer (pH = 5.2), 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 minutes. Compound 5 (350 mg, 1 mmol, r = 2, s = 6) was weighed and dissolved in 10 mL of DMSO and added to the reaction system. The reaction was stirred at room temperature for 24 hours. After completion of the reaction, the pH was adjusted to 8-9, stirred for 1 hour, and dialyzed against deionized water for 2-3 days (MWCO 3500). The product was collected and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 hour, then adjusted to neutral. The reaction system was then dialyzed against deionized water for 1 day (MWCO 3500). The product was collected and freeze-dried to obtain component A-7.1. The labeling of compound 5 on CS was identified by H NMR spectroscopy. The supernatant of the reaction system was collected, and the amount of residual compound 5 in the supernatant was determined by high performance liquid chromatography. The labeling rate of compound 5 was calculated to be approximately 1.87% (w / w).

[0168] The above examples provide the structures and synthesis methods of exemplary o-phthalaldehyde group-modified chondroitin sulfate-based polymer crosslinkers. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified chondroitin sulfate-based polymer crosslinkers.

[0169] Example 9: Synthesis of Representative Component A-8.1 of the O-Phthalaldehyde-Modified Xanthan Gum-Based Crosslinking Agent

[0170]

[0171] 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, the pH was neutralized to 7 with dilute hydrochloric acid. The above system was slowly poured into 10 volumes of anhydrous ethanol, 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 then adjusted to neutrality. The reaction system was then dialyzed against deionized water for 1 day (MWCO 14000). The product was collected and freeze-dried to obtain component A-8.1. The labeling of compound 12 on XG was confirmed by H NMR spectroscopy. The supernatant of the reaction system was collected and the amount of residual compound 12 in the supernatant was determined by HPLC. The labeling rate of compound 12 was calculated to be approximately 2.10% (w / w).

[0172] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified xanthan gum polymer crosslinker. Based on the disclosure of this application, one skilled in the art will appreciate that other aldehyde group-preprotected o-phthalaldehyde precursor derivatives can be used to synthesize the o-phthalaldehyde group-modified xanthan gum polymer crosslinker.

[0173] Example 10: Synthesis of Representative Component A-9.1 of Gellan Gum-Based Crosslinking Agent Modified with O-Phthalaldehyde Groups

[0174]

[0175] Gellan gum (GG, 5 g, 500 kDa) was dissolved in 500 mL of 0.01 mol / L MES buffer (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 added to the above reaction solution. DMTMM (7 g, 5 mmol) was weighed and added to the above reaction system in five portions, each dissolved in 3 mL of MES buffer, with 2-h intervals. The reaction was continued at 60°C for 24 h. After the reaction, the pH was adjusted to 8-9, and the mixture was stirred at 60°C for 1 h. During the stirring process, 10 volumes of anhydrous ethanol were added. The precipitate was collected, and 500 mL of deionized water was added. The mixture was heated to 60°C, stirred until completely dissolved, and 10% trifluoroacetic acid was added. The reaction was allowed to react for 1 h, and then the mixture was adjusted to neutral. Subsequently, 10 volumes of anhydrous ethanol were added while stirring. The precipitate was collected and dried under vacuum to obtain component A-9.1. The labeling of compound 2 on GG was identified by H NMR spectrum. The supernatant of the reaction system was collected and the amount of residual compound 2 in the supernatant was determined by HPLC. The labeling rate of compound 2 was calculated to be approximately 1.98% (w / w).

[0176] The above structural formula and examples provide the structure and synthesis method of an exemplary o-phthalaldehyde group-modified gellan gum-based polymer crosslinking agent. Based on the disclosure of this application, one skilled in the art would be able to conceive of using other aldehyde group-preprotected o-phthalaldehyde precursor derivatives to synthesize the o-phthalaldehyde group-modified gellan gum-based polymer crosslinking agent.

[0177] Example 11: Hydrogel component ratio

[0178] According to the method of the present invention, a two-component hydrogel precursor solution containing different polysaccharides was prepared (components A and B were dissolved in phosphate buffer solution, pH = 7.4).

[0179] Table 1

[0180]

[0181] Components A-... in Table 1 refer to components A-3.1 through A-4.4 described in Examples 4 and 5; components B-... in Table 1 refer to components B-4 (polylysine, molecular weight 5 kDa), B-5 ​​(collagen), and B-6 (amine-modified four-arm polyethylene glycol, molecular weight 40 kDa). References to 0.1-40 wt% in Table 1, etc., indicate preferred concentration ranges for the hydrogel precursor solution.

[0182] In the remaining embodiments of the present invention, the inventors prepared polysaccharide-based two-component hydrogels according to the ratios in Table 2 for performance testing and application. Figure 3 shown.

[0183] Table 2

[0184]

[0185]

[0186] Example 12: Hydrogel swelling performance test

[0187] To demonstrate that the hydrogels prepared in the present invention exhibit low swelling properties compared to highly swollen polyethylene glycol-based hydrogels (swelling ratio > 100%), the inventors prepared polysaccharide-based two-component hydrogels according to the ratios shown in Table 2 and tested their swelling ratios. Polyethylene glycol crosslinkers modified with o-phthalaldehyde groups, as disclosed in Chinese patents CN111440310A, CN111440334A, CN111574756A, CN 111621038 A, CN113509591A, CN113694249A, CN114767920A, CN202010454896.6, and CN202010455951.3, were selected for use as control component A of the present invention.

[0188]

[0189] A two-component hydrogel of the control group was prepared according to the ratio in Table 3 for swelling rate testing.

[0190] Table 3

[0191] Serial number formula 37 Control group A / B-1 (3wt% / 1wt%) 38 Control group A / B-2 (3wt% / 2wt%) 39 Control group A / B-3 (3wt% / 2wt%) 40 Control group A / B-7 (3wt% / 1wt%)

[0192] The specific detection method is as follows: squeeze the component A solutions and component B solutions mentioned in Table 2 and Table 3 into a polytetrafluoroethylene mold through a double liquid mixer. After curing for 10 minutes, a cylindrical gel block with a diameter of 10 mm and a thickness of 3 mm of similar mass is obtained. Weigh the above gel block, record the mass as W0, and transfer it to a 50mL centrifuge tube, add a PBS buffer solution with a pH of 7.4 (the solution has been heated to 37±1°C in advance), then place the centrifuge tube in a 37±1°C incubator, take out the sample every 2 hours, absorb the surface moisture with filter paper, and weigh it until the mass no longer increases. Record the mass at this time as Wt. End the experiment and calculate the gel swelling rate (n=3) according to the following formula:

[0193] The swelling ratios obtained by the above method are shown in the following table:

[0194]

[0195] The swelling ratio of the hydrogel obtained by the above method is shown in Table 4:

[0196] Table 4

[0197]

[0198]

[0199] In the above formulations, compared to the polyethylene glycol-based hydrogels in the control group (ie, formulations 37-40), the polysaccharide-based hydrogels of the present invention have a low swelling ratio (<50%) over a wider solid content range.

[0200] Example 13: Hydrogel Tissue Adhesion Performance Test

[0201] The inventors tested the tissue adhesion properties of the polysaccharide-based two-component hydrogels of the present invention, using commercial Fibrin glue as a control group. The specific testing method is as follows: The shear strength of the hydrogels of different formulations on pigskin was measured using a standard lap shear test (ASTM F2255) using a tensile testing machine (INSTRON, Germany). The pigskin was cut into 10 mm and 40 mm rectangles. During the mechanical testing, a transparent polymethyl methacrylate film served as a hard backing for the tissue. The solutions of Component A and Component B listed in Table 2, or the two-component solution of Fibrin glue as a control group, were sprayed onto a piece of pigskin measuring 15 mm x 10 mm using a dual mixer. Immediately, another piece of pigskin was placed on the pigskin sprayed with the hydrogel precursor solution and pressed evenly with a force of 100 kPa for 10 minutes. After the hydrogels of each formulation had fully solidified, tensile testing was performed. The shear strength was calculated using the following formula, where Fmax is the maximum force.

[0202] To ensure the reliability of the data, three samples were measured under each condition.

[0203]

[0204] The adhesion strength of the hydrogels tested by the above method is shown in Table 5:

[0205] Table 5

[0206]

[0207]

[0208] The test results in Table 5 demonstrate that, compared to commercially available fibrin glue, the polysaccharide-based two-component hydrogels of the present invention exhibit excellent tissue adhesion across a wide range of solids content, enabling tissue adhesion or sealing applications. In Formulations 1 through 5, increasing the proportion of Component A enhances the tissue adhesion of the polysaccharide-based two-component hydrogels. This indicates that increasing the proportion of Component A in the polysaccharide-based two-component hydrogels effectively improves their tissue adhesion.

[0209] Example 14: In vitro hemostatic performance test of hydrogel

[0210] The inventors conducted in vitro hemostatic testing on the polysaccharide-based two-component hydrogels of the present invention, using commercial fibrin glue as a control group. The specific testing method was as follows: Component A and Component B solutions (or a control two-component solution of fibrin glue) listed in Table 2 were sprayed onto a clean 96-well plate using a dual mixer, allowed to cure for 10 minutes, and then preheated at 37°C. 20 μL of fresh 37°C EDTA-anticoagulated sheep blood was dripped onto the surface of the material. For the blank control group, blood was dripped directly onto the bottom of the 96-well plate and allowed to stand at 37°C for 5 minutes. Subsequently, the blood or thrombus on the hydrogel surface was washed with 1 mL of deionized water and gently shaken. The size of the remaining clots on the hydrogel surface was observed. The supernatant was aspirated and the absorbance at 540 nm was measured. The thrombosis index was calculated according to the following formula. The results are shown in Table 6.

[0211]

[0212] The hydrogel thrombosis index obtained by the above method is shown in Table 6:

[0213] Table 6

[0214]

[0215]

[0216]

[0217] The test results in Table 6 demonstrate that, compared to commercially available fibrin glue, the polysaccharide-based two-component hydrogels of the present invention exhibit excellent hemostatic properties across a wide range of solids content, making them suitable for hemostatic applications. In Formulations 1 through 5, increasing the proportion of Component A enhances the in vitro coagulation properties of the polysaccharide-based two-component hydrogels. This indicates that increasing the proportion of Component A in the polysaccharide-based two-component hydrogels effectively improves their hemostatic efficacy.

[0218] Example 15: Based on the product formulas obtained in the above examples, taking into account the swelling properties, tissue adhesion and hemostatic properties, formulas 5, 6, 7, 10, 14, 22 and 30 (i.e., the formulas corresponding to serial numbers 5, 6, 7, 10, 14, 22 and 30, respectively) of two-component hydrogels were selected to further implement specific biomedical applications.

[0219] Example 16: Application of polysaccharide-based two-component hydrogel in skin wound repair

[0220] The skin defect wounds on the back of SD rats were selected and divided into two groups for skin wound repair experiments: Formula 5 (Group a) and Suture Group (Group b). A 10 mm long skin defect wound was constructed on the back of SD rats. For Group a, the component A solution and the component B solution were injected into the skin defect through a double liquid mixer, and the solution was waited for full filling and penetration. After solidification for 10 minutes, the hydrogel adhered firmly to the wound. For Group b, only 4-0 non-absorbable surgical sutures were used to suture the defect. The wounds of each group were observed, and the repair effect was recorded within 7 days. The experimental results showed that compared with Group b, Group a had a faster repair rate and no obvious scar tissue was generated. In this embodiment, the photos of the polysaccharide-based two-component hydrogel (Group a) and suture (Group b) used for skin tissue repair for 7 days are shown in the figure below. Figure 4 As shown, the above-mentioned polysaccharide-based two-component hydrogel can be used for skin wound repair.

[0221] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used for skin wound repair.

[0222] Example 17: Application of polysaccharide-based two-component hydrogel in abdominal wall wound repair

[0223] An abdominal wall abrasion model was developed in SD rats, and two groups were selected for an abdominal wall wound repair experiment: Formula 5 (Group a) and a blank group (Group b). The SD rats were anesthetized and immobilized, and their abdominal hair was shaved to expose the abdominal cavity. The left and right abdominal walls were scraped back and forth several times with the back of a scalpel until bleeding occurred, creating an abdominal wall abrasion model with a diameter of approximately 10 mm. The solutions of Component A and Component B were injected into the abraded area of ​​the abdominal wall using a dual-mixer. The solutions were allowed to fully fill and penetrate, and solidified for 10 minutes. The abdominal wall wounds of Group b rats were cleaned with only physiological saline. The wounds and hydrogels were observed, and the repair effects of the wounds in each group were recorded after 7 days. The experimental results showed that the hydrogel in Group a adhered firmly to the wound surface. Compared with the hydrogel in Group b, the abdominal wall wounds treated with the hydrogel in Group a showed no significant adhesion to the abdominal viscera and repaired at a faster rate. This indicates that the polysaccharide-based two-component hydrogel can be used for abdominal wall wound repair.

[0224] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used for repairing abdominal wall wounds.

[0225] Example 18: Application of polysaccharide-based two-component hydrogel in blocking pancreatic juice leakage

[0226] A pancreatic leakage model was established in SD rats, and two groups were tested for pancreatic leakage plugging: Formula 5 (Group a) and a blank group (Group b). SD rats were anesthetized and immobilized, their abdominal hair shaved, and disinfected with iodine. The abdominal cavity was exposed, the greater omentum and pancreas excised, and some splenic vessels ligated to prevent unnecessary bleeding during pancreatic resection. The pancreatic-duodenal distal end was resected with a scalpel to create a pancreatic leakage model. In Group a, Component A and Component B solutions were injected into the pancreatic stump using a double-mixer. The solutions were allowed to fully fill and permeate the pancreas and solidify for 10 minutes. In Group b, no treatment was performed, and the abdomen was closed. Seven days after surgery, the peritoneal fluid of the SD rats was collected and analyzed for protease content. Seven days after surgery, the rats were euthanized and their peritoneal adhesions observed. The results showed that protease levels in the SD rats in Group b exceeded normal levels seven days after surgery, confirming the successful establishment of the pancreatic leakage model. In this example, a polysaccharide-based two-component hydrogel (Formula 5) was applied to the pancreatic stump to block pancreatic fluid leakage 7 days after surgery. Figure 5 Compared to group B, rats in group A showed no significant intraperitoneal effusion or adhesions, and the hydrogel material was still visible on the pancreatic stump. After peritoneal lavage with saline, the peritoneal fluid was collected for protease analysis. The results showed that trypsin levels in the peritoneal cavities of rats in group A were normal. This indicates that the polysaccharide-based two-component hydrogel can be used to block pancreatic fluid leakage.

[0227] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used to block pancreatic juice leakage.

[0228] Example 19: Application of polysaccharide-based two-component hydrogel in blocking cerebrospinal fluid leakage

[0229] A SD rat cerebrospinal fluid (CSF) leakage closure experiment was conducted in two groups: Formulation 5 (Group a) and a bone wax group (Group b). The rats were anesthetized and immobilized, and the back hair was shaved. The skin and fascia were then incised sequentially, centered around T13 and L1, to expose the muscles in the surgical area. On one side, ophthalmic scissors were used to cut the muscles and tendons connected to the spinous process closely adjacent to it, creating an incision approximately 0.5 cm in length from T12 to L2 and deep enough to reach the articular process. Another parallel incision was made approximately 0.5 cm lateral to this incision, with a length and depth similar to the previous one. The muscle between the two incisions was removed, exposing three pairs of articular processes (T12-T13, T13-L1, and L1-L2). The lateral walls of the spinal canal and intervertebral foramen were exposed simultaneously. The spinal cord was exposed by transecting the lateral walls of the spinal canal parallel to each other, creating a wound surface. Successful modeling was considered successful when bleeding and cerebrospinal fluid leakage were observed. Subsequently, for group a, component A solution and component B solution were immediately injected into the cerebrospinal fluid leakage site through a dual liquid mixer, and the solution was allowed to fully fill and penetrate and solidify for 10 minutes; for group b, bone wax was immediately applied to the cerebrospinal fluid leakage site. During use, group b still had leakage. In this example, the polysaccharide-based two-component hydrogel (Formula 5, group a) and bone wax (group b) were used to block cerebrospinal fluid leakage. Figure 6 As shown. The wounds of the two groups of rats were sutured. Two days after surgery, all rats in group B had died, while all rats in group A had survived. Seven days after surgery, the hydrogel was observed to be firmly adhered to the spinal injury site in the rats in group A, with no apparent leakage. This demonstrates that the polysaccharide-based two-component hydrogel can be used to block cerebrospinal fluid leakage.

[0230] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used for blocking cerebrospinal fluid leakage.

[0231] Example 20: Application of polysaccharide-based two-component hydrogel in blocking lung parenchymal air leakage

[0232] A lung parenchymal air leakage model was used in New Zealand white rabbits. Two groups of three rabbits were used for the lung parenchymal air leakage occlusion experiment: Formulation 6 (Group a) and a blank group (Group b). The rabbits were injected with 1% sodium pentobarbital (3 mL / kg) intravenously at the ear margin. General anesthesia was followed by endotracheal intubation and mechanical ventilation (VT 80 mL, R 30 bpm). Thoracotomy was then performed, and the marginal tissue of the lung lobe was resected. The wound surface was approximately 1 cm 2, at least one bronchial stump with a diameter of about 1 mm was visible. For group b, the wound was not treated and the chest was closed directly. For group a, the component A solution and the component B solution were injected into the lung defect through a double liquid mixer, and the solution was allowed to fully fill and penetrate, solidify for 10 minutes, and after confirming that the hydrogel adhered to the wound, the chest was closed layer by layer to remove residual air and allow the lung to fully re-expand. Three rabbits in group b died of respiratory failure due to continuous air leakage within 4 hours after surgery, while no air leakage was observed in the lung wounds of the three rabbits in group a. After autopsy 7 and 14 days after surgery, the hydrogel material in group a was tightly attached to the wound, and no effusion or adhesion was observed. It can be seen that the above-mentioned polysaccharide-based two-component hydrogel can be used for sealing air leakage in the lung parenchyma.

[0233] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used to block lung parenchymal air leakage.

[0234] Example 21: Application of polysaccharide-based two-component hydrogel in liver hemostasis

[0235] A liver transverse section trauma model was selected for SD rats, and the liver hemostasis experiment was carried out in three groups: formula 14 (group a), formula 7 (group b) and gauze group (group c). After the SD rats were anesthetized, their abdominal cavity was opened, the liver was lifted and placed on a pre-weighed filter paper, and then a transverse section with a diameter of 10 mm was established on the liver to cause bleeding. For group a and group b, the component A solution and the component B solution were respectively injected into the bleeding site through a double liquid mixer. As the solution penetrated, group a and group b quickly gelled in the presence of blood, blocking the bleeding site. For group c, a pre-weighed gauze was lightly pressed on the transverse wound. The amount of blood loss was measured by the weight gain of the filter paper (and gauze), and the hemostasis time was recorded by a timer. During the experiment, compared with Group C, blood loss in Groups a and b was significantly reduced (0.2 g in Group a, 0.1 g in Group b, and 1.0 g in Group c), and hemostasis time was significantly shortened (7 seconds in Group a, 5 seconds in Group b, and 50 seconds in Group c). This indicates that the polysaccharide-based two-component hydrogel can be used for liver hemostasis.

[0236] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used for liver hemostasis.

[0237] Example 22: Application of polysaccharide-based two-component hydrogel in femoral artery hemostasis

[0238] A femoral artery bleeding model was established in SD rats, and femoral artery hemostasis experiments were conducted in three groups: Formulation 14 (Group a), Formulation 10 (Group b), and a gauze group (Group c). After anesthesia, the rats' right hind legs and lower abdomen were shaved to expose the femoral artery, vein, and nerve. The femoral artery was carefully dissected, and the artery was gently clamped at both ends with hemostats. A 32G needle was then used to puncture the artery. In Groups a and b, once the blood had drained from the artery, the component A and component B solutions were immediately injected into the bleeding site using a double mixer. In Group c, gauze was immediately applied to the bleeding site. The experimental results showed that the hydrogels in Groups a and b rapidly gelled in the presence of blood. Even after the hemostats were removed, the hydrogels remained firmly adhered to the bleeding site, achieving stable occlusion and preventing further bleeding. However, the rats in Group c died from excessive blood loss, and hemostasis was not achieved. This demonstrates that the polysaccharide-based two-component hydrogel can be used for femoral artery hemostasis.

[0239] The polysaccharide-based two-component hydrogels of other formulations in the present invention can also be used for femoral artery hemostasis.

[0240] Example 23: Preparation of polysaccharide-based membrane material

[0241] Taking formula 5 as an example, components A and B were respectively dissolved in phosphate buffer solution (pH = 7.4) to obtain component A solution and component B solution, which were then applied to a clean, flat polytetrafluoroethylene plate through a double liquid mixer. After curing for 10 minutes, the solution was swelled in a phosphate buffer solution (pH = 7) containing 5% glycerol to equilibrium, and dried at a constant temperature of 30°C to obtain a polysaccharide-based membrane material.

[0242] Precursor solutions of other formulations in the present invention can also be used to prepare polysaccharide-based membrane materials with different components.

[0243] Example 24: Application of polysaccharide-based membrane materials in suture-free closure of surgical wounds

[0244] A SD rat back wound model was selected and divided into two groups for a suture-free closure of surgical wound experiment: the membrane material (formula 5, group a) in Example 23 and the suture group (group b). After the SD rats were anesthetized, the hair on their backs was shaved, and a 1 cm long skin incision was made on their backs with a scalpel. For group a, the membrane material was attached to the incision to close it. For group b, the incisions were aligned and sutured with 4-0 non-absorbable surgical sutures. The healing of the incisions was observed 3 days and 10 days after surgery: the membrane material in group a did not show obvious shedding, the incision epithelialized faster, and the healing speed was fast; the healing speed of group b was slow. In this embodiment, the polysaccharide-based membrane material was used for suture-free closure of surgical wounds (group a) and the suture group (group b). The H&E staining results 10 days after surgery are shown in the following photos. Figure 7H&E staining results showed that the wounds in group a healed completely, with the newly formed tissue resembling skin tissue, while the wounds in group b were not fully repaired. This suggests that the polysaccharide-based membrane material can be used for suture-free closure of surgical wounds.

[0245] The polysaccharide-based membrane materials of other formulations in the present invention can also be used for suture-free closure of surgical wounds.

[0246] Example 25: Application of polysaccharide-based membrane material as a patch for abdominal hernia repair

[0247] An acute abdominal wall hernia model was established in SD rats, and two groups were used for abdominal wall hernia repair experiments: the membrane material described in Example 23 (Formula 5, Group a) and the Separmesh absorbable composite patch (Bard, Group b). After anesthesia, the rats were fixed, their skin prepared, and disinfected. A 1.2 cm x 1.2 cm square was marked 2 cm on either side of the midline of the lower abdomen. The abdominal wall was then 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 not sutured; the patch in Group b was sutured with interrupted 4-0 non-absorbable surgical sutures on all sides. The skin was disinfected with 75% ethanol and then sutured with interrupted 3-0 non-absorbable surgical sutures. Thirty days after surgery, the abdominal cavity was opened and the adhesions between the viscera and the patch were observed. The experimental results showed that the patch in Group A showed no significant displacement, no intra-abdominal hernia, and no significant adhesion between the patch and the abdominal viscera. In Group B, the patch showed no significant displacement, but there was minimal adhesion between the sutured area and the greater omentum. This suggests that the polysaccharide-based membrane material can be used as a patch for abdominal hernia repair.

[0248] The polysaccharide-based membrane materials of other formulations in the present invention can also be used as patches for abdominal hernia repair.

[0249] Example 26: Preparation of polysaccharide-based powder material

[0250] Taking formula 22 as an example, components A and B are dissolved in phosphate buffer solution (pH = 7.4) to obtain component A solution and component B solution, which are then applied to a clean, flat polytetrafluoroethylene plate through a double liquid mixer. After curing for 10 minutes, the solution is swollen in a phosphate buffer solution (pH = 7), dried at a constant temperature of 30°C, and the dried material is placed in a ball mill. After sieving, a fine powder (mesh size of 60 to 300 mesh) is obtained, i.e., a polysaccharide-based powder material. In this embodiment, the microscopic morphology of the polysaccharide-based powder materials of different particle sizes under a microscope is as follows: Figure 8Alternatively, the solidified hydrogel material is added with one volume of phosphate buffer solution (pH = 7), and high-speed shear stirring is used to cut the block hydrogel into smaller microgels. After separating the microgels, 3 volumes of 50%, 75%, and anhydrous ethanol are added sequentially to displace the water in the microgels. The separated microgels are vacuum-dried at 30°C for 12 hours and sieved to obtain a powder with a mesh size of 60 to 300, i.e., the polysaccharide-based powder material.

[0251] Precursor solutions of other formulations in the present invention can also be used to prepare polysaccharide-based powder materials with different components.

[0252] Example 27: Application of polysaccharide-based powder materials in minimally invasive surgery for hemostasis

[0253] The Bama pig acute upper gastrointestinal arterial bleeding model was selected and divided into two groups for minimally invasive surgical hemostasis experiments: the powder material in Example 26 (Formula 22, Group a) and absorbable hemostatic granules (ARISTA, Group b). Adult female Bama pigs (weighing 20-25 kg) were anesthetized and cannulated in the right femoral artery to monitor blood pressure. Subsequently, within 5 minutes, the baseline blood pressure (systolic pressure) of all animals was monitored and adjusted to 90-100 mm Hg, and then heparin (10 mg / kg) was administered intravenously to achieve systemic heparinization. Under endoscopy, the mucosa was peeled off and the submucosal pulsating artery in the stomach was identified. The artery was then cut with an endoscopic knife to induce acute upper gastrointestinal bleeding and maintain natural bleeding for 4 minutes. The hemostatic powders of Group a and Group b were applied to the bleeding site through a catheter respectively, and the bleeding site was observed. Unless hemostasis was achieved, the hemostasis procedure was repeated, and the hemostasis time and the quality of the materials used were recorded. The experimental results showed that during the operation, the hemostatic powder in group a was able to quickly adhere to the wound surface and form a colloid to block bleeding. The hemostasis time was shorter (group a: 80s, group b: 160s), and less material was used (group a: 1.1g, group b: 2g). It can be seen that the above-mentioned polysaccharide-based powder material can be used for hemostasis in minimally invasive surgery.

[0254] The polysaccharide-based powder materials of other formulations in the present invention can also be used for hemostasis in minimally invasive surgery.

[0255] Example 28: Polysaccharide-based powder materials used as antibacterial drug carriers for wound antibacterial treatment

[0256] Taking Formulation 14 as an example, components A and B were dissolved in phosphate buffer solution (pH = 7.4) to obtain component A solution and component B solution, respectively. The two solutions were then applied to a clean, flat polytetrafluoroethylene plate using a dual mixer. After curing for 10 minutes, a volume of phosphate buffer solution (pH = 7) was added. High-speed shear stirring was used to cut the block hydrogel into microgels with smaller particle sizes. The microgels were dispersed in a phosphate buffer solution (pH = 7.4) containing benzalkonium chloride (0.1%) and stirred for 1 hour. Due to electrostatic adsorption, the cationic benzalkonium chloride was adsorbed on the surface of the microgels. After the microgels were separated, 3 volumes of 50%, 75%, and anhydrous ethanol were added in sequence to replace the water in the microgels. The separated microgels were vacuum-dried at 30°C for 12 hours and sieved to obtain a polysaccharide-based powder material loaded with benzalkonium chloride.

[0257] A full-thickness incisional wound infection model was used in Balb / C mice, and three groups were divided into three groups for surface wound antibacterial experiments: the polysaccharide-based powder material loaded with benzalkonium chloride (Group a), the benzalkonium chloride solution group (Group b), and the blank group (Group c). A 20 mm long full-thickness longitudinal incision was made on the back of 8-10 week-old female BALB / C mice, and 50 μL of Escherichia coli 25922 (1×10 8 CFU mL -1 ) was dripped dropwise into the wound site to establish an in situ acute pathogenic bacteria infection model. The powder of group a was evenly sprayed onto the wound. As the wound exudates, the powder gradually gelled and firmly adhered to the wound surface; group b used a 0.01% benzalkonium chloride solution to disinfect the wound, and group c did not receive any treatment. The experimental results showed that the infected area of ​​group c began to spread, blood circulation was poor, ischemia, suppuration and other phenomena gradually appeared, and tissue damage was obvious; the spread rate of the infected area of ​​group b was limited in the early stage, but suppuration, ischemia and other phenomena still appeared 7 days after the operation; the recovery effect of group a was the best, bacterial proliferation was inhibited, and there was no suppuration, ischemia and other phenomena on the wound surface. It can be seen that the above-mentioned polysaccharide-based powder material loaded with benzalkonium chloride can be used for antibacterial treatment of wounds.

[0258] The polysaccharide-based powder materials of other formulations in the present invention can also be used as carriers for loading antibacterial drugs and applied in the antibacterial treatment of wounds.

[0259] Example 29: Polysaccharide-based powder materials used as anti-inflammatory drug carriers for the treatment of inflammation

[0260] Glucocorticoid drugs have a wide range of indications and have a strong regulatory effect on various physiological functions of the whole body. They are widely used clinically for various autoimmune diseases, leukemia, asthma, allergic reactions, etc. However, long-term non-specific use of hormone drugs may induce a variety of diseases and have serious side effects. Therefore, local short-term and small-dose use of hormone drugs is the recommended method of use in clinical practice. Taking formula 5 as an example, components A and B are dissolved in phosphate buffer solution (pH = 7.4) to obtain component A solution and component B solution, and the two are applied to a clean and flat polytetrafluoroethylene plate through a double mixer. After curing for 10 minutes, one volume of phosphate buffer solution (pH = 7) is added, and the block hydrogel is cut into microgels with smaller particle size by high-speed shear stirring. The microgels were dispersed in a phosphate buffer solution (pH 7.4) containing budesonide (20 μg / mL) and stirred for 1 hour. Budesonide adsorbed onto the microgel surface due to hydrogen bonding and hydrophilic-hydrophobic interactions. After the microgels were separated, 3 volumes of 50%, 75%, and anhydrous ethanol were added sequentially to displace the water in the microgels. The separated microgels were then vacuum-dried at 30°C for 12 hours and sieved to obtain a budesonide-loaded polysaccharide powder.

[0261] A Balb / C mouse model of acute enteritis was used for treatment of acute enteritis in three groups: the aforementioned polysaccharide-based powder material loaded with budesonide (Group a), budesonide enema solution (Group b), and a blank control group (Group c). 3% DSS drinking water was prepared with sterile water and filtered through a 0.22μm filter membrane. The mice were then given this solution for seven consecutive days. Colonoscopy revealed localized edema and congestion in the mouse tissues, indicating a successful model. Group a had the powder sprayed onto the inflamed area via colonoscopy, while Group b received budesonide enema solution. Group c received no treatment. After 7 and 14 days, the mice were observed for signs of inflammation and the colon lengths of each group were dissected and calculated. The results showed that the powder material in Group a adhered firmly to the damaged inflamed wound surface, and compared to Groups b and c, wound edema and ulceration were reduced in Group a. In this example, the colon length statistics of normal mice, polysaccharide-based powder material loaded with budesonide (group a), budesonide enema (group b), and blank group (group c) after treatment of acute enteritis in mice are shown in the figure below: Figure 9 Statistical results showed that the colon length of mice in group a was the longest compared to those in groups b and c. This suggests that the polysaccharide-based powder material loaded with budesonide can be used in the treatment of inflammation.

[0262] The polysaccharide-based powder materials of other formulations in the present invention can also be used as carriers for loading anti-inflammatory drugs and applied to the treatment of inflammation.

[0263] Example 30: Preparation of polysaccharide-based sponge material

[0264] Taking formula 6 as an example, components A and B were respectively dissolved in phosphate buffer solution (pH = 7.4) to obtain solution A and solution B, which were sprayed into the mold through a double liquid mixer. After curing for 10 minutes, they were swelled and balanced in phosphate buffer solution (pH = 7), frozen at -20°C for 6 hours, and then freeze-dried at -60°C for 48 hours to obtain a polysaccharide-based sponge material.

[0265] Precursor solutions of other formulations in the present invention can also be used to prepare polysaccharide-based sponge materials with different components.

[0266] Example 31: Polysaccharide-based sponge material used as wound dressing for wound treatment

[0267] Select SD rat back defect model, divide two groups and carry out wound dressing experiment: the sponge material (formula 14, group a) in embodiment 30, commercial gelatin sponge group (group b) and blank group (group c).Make a circular defect wound with a diameter of 10mm on the back of SD rat.Evenly cover the sponges of group a and group b on the wound respectively, group c does not do any treatment, and each group is covered with gauze and medical tape subsequently.The experimental results show that the sponge material of group a can firmly adhere to the tissue defect site, and compared to group c, the wound recovery rate of group a and group b is faster, and the new tissue is similar to skin tissue, without obvious scar tissue generation.As can be seen, the above-mentioned polysaccharide-based sponge material can be used as wound dressing and applied to wound treatment.

[0268] The polysaccharide-based sponge materials of other formulations in the present invention can also be used as wound dressings for wound treatment.

[0269] Example 32: Polysaccharide-based sponge material is used to stop hemorrhage in the spleen;

[0270] A New Zealand white rabbit spleen bleeding model was selected and the spleen hemostasis experiment was carried out in two groups: the sponge material in Example 30 (Formula 6, Group a) and commercial absorbable hemostatic gauze (Group b). The New Zealand white rabbit was anesthetized and fixed, its abdominal hair was shaved, and the abdominal cavity was opened after disinfection with iodine to expose its spleen. An 8 mm long and 1 mm deep incision was made on its spleen with a scalpel to create a bleeding wound. The hemostatic material was gently pressed on the bleeding area, and the wound was observed about every 10 seconds. The hemostasis time was recorded immediately after the bleeding stopped completely. In this embodiment, a photographic comparison of the polysaccharide-based sponge material (Group a) and the commercial absorbable hemostatic gauze (Group b) used for spleen hemostasis is shown in Figure 2. Figure 10 The hemostatic materials were weighed before and after use, and the difference was the total blood loss. During the experiment, both Group A and Group B materials adhered firmly to the splenic bleeding wound. Compared with Group B, Group A achieved shorter hemostasis time (Group A: 120 seconds, Group B: 180 seconds) and less blood loss (Group A: 3g, Group B: 3.5g). This demonstrates that the polysaccharide-based sponge material can be used for splenic hemostasis.

[0271] The polysaccharide-based sponge materials of other formulations in the present invention can also be used for spleen hemostasis.

[0272] Example 33: Application of polysaccharide-based sponge material to cardiac hemostasis;

[0273] A SD rat heart bleeding model was selected and divided into two groups for cardiac hemostasis experiments: the sponge material in Example 30 (Formula 30, Group a) and the gauze group (Group b). The SD rats were anesthetized and fixed, and the chest hair was shaved. The chest cavity was opened and the heart was exposed, and the pericardium was removed using fine tweezers. A biopsy punch was used to create a 2mm diameter injury to the left ventricle or right ventricle wall of the heart. Subsequently, the hemostatic material was immediately applied to the bleeding site and the same pressure was applied. The wound was observed approximately every 10 seconds, and the hemostasis time was recorded immediately after the bleeding stopped completely. In this embodiment, photos of the polysaccharide-based sponge material (Group a) being applied to cardiac hemostasis are shown in the figure. Figure 11 As shown. The hemostatic material was weighed before and after use, and the difference was the total blood loss. For the groups that failed to achieve hemostasis within 300 seconds, the rats were euthanized by bleeding. During the experiment, the sponge of group a firmly adhered to the ventricular wound surface (such as Figure 11 Compared with group B, group A had a shorter hemostasis time (60 seconds, while group B rats failed to achieve hemostasis within 300 seconds) and the least blood loss (1.2 g, while group B lost 2.5 g within 300 seconds). Furthermore, group A rats successfully survived the surgery. This indicates that the polysaccharide-based sponge material can be used for cardiac hemostasis.

[0274] The polysaccharide-based sponge materials of other formulations in the present invention can also be used for cardiac hemostasis.

[0275] Example 34: Application of polysaccharide-based sponge material in hemostasis of non-compressive penetrating wounds;

[0276] A New Zealand white rabbit liver perforation model was selected and divided into two groups for a non-compressive penetrating wound hemostasis experiment: the sponge material (Formula 6, Group a) in Example 30 and the gauze group (Group b). After the New Zealand white rabbit was anesthetized, its abdominal cavity was opened, the liver was lifted and placed on a pre-weighed gauze, and then a penetrating wound with a diameter of 6 mm was established on the liver to cause bleeding. Subsequently, the hemostatic materials of different groups were stuffed into the penetrating wound. The amount of blood loss was measured by the weight gain of the hemostatic material and gauze. During the experiment, compared with group b, the amount of blood loss in group a was significantly reduced (Group a: 2.6g, Group b: 4.5g), and the hemostasis time was significantly shortened (Group a: 35s, Group b: 96s). It can be seen that the above-mentioned polysaccharide-based sponge material can be used for non-compressive penetrating wound hemostasis.

[0277] The polysaccharide-based sponge materials of other formulations in the present invention can also be used to stop bleeding from non-compressive penetrating wounds.

[0278] The above Examples 16 to 22 provide applications of polysaccharide-based two-component hydrogels with specific formulas in tissue repair, tissue fluid leakage blocking, tissue gas leakage blocking, hemostasis, etc. These applications mainly utilize the tissue adhesion and hemostatic properties of the polysaccharide-based two-component hydrogels. According to the records of Examples 12 to 14, the polysaccharide-based two-component hydrogel formulas 1-36 provided in the embodiments of the present invention all have good tissue adhesion, hemostatic properties and low swelling properties. Therefore, the polysaccharide-based two-component hydrogel formulas 1-36 provided in the embodiments of the present invention can be used in tissue repair, tissue fluid leakage blocking, tissue gas leakage blocking, hemostasis, etc.

[0279] The above Examples 23 to 34 provide the preparation of polysaccharide-based film materials, polysaccharide-based powder materials, and polysaccharide-based sponge materials with specific formulas and their corresponding biomedical applications. According to the contents of this application, those skilled in the art can prepare the polysaccharide-based two-component hydrogel formulas 1-36 provided in the examples into polysaccharide-based film materials, polysaccharide-based powder materials, and polysaccharide-based sponge materials as needed. Similarly, according to the contents of this application, those skilled in the art can use the polysaccharide-based two-component hydrogel formulas 1-36 provided in the examples as needed for suture-free closure and patching of surgical wounds; or use the polysaccharide-based powder materials prepared from the polysaccharide-based two-component hydrogel formulas 1-36 provided in the examples as needed for hemostasis, or as a cell, factor, or drug carrier; or use the polysaccharide-based sponge materials prepared from the polysaccharide-based two-component hydrogel formulas 1-36 provided in the examples as wound dressing products or hemostatic products as needed.

[0280] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 be within the scope of protection of the present invention.

Claims

1. A polysaccharide-based polymer crosslinking agent, characterized in that: Select one of the following structures: 、 、 Formula 1-1 Formula 1-2 、 Formula 1-3 、 、 Formula 1-4 Formula 1-5 、 Formula 1-6 、 Formula 1-7 、 Formula 1-8 、 、 Formula 1-9 Formula 1-10 、 Formula 1-11 Formula 1-12 wherein P is selected from one or more of 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, or starch derivatives; 1≤r≤20, 1≤s≤20, 1≤t≤50, n≥2.

2. The polysaccharide-based polymer cross-linking agent according to claim 1, characterized in that 1≤r≤6, 1≤s≤10, 1≤t≤30, n≥2.

3. The polysaccharide-based polymer cross-linking agent according to claim 1, characterized in that P is selected from hyaluronic acid, cellulose derivatives, alginic acid, heparin, chondroitin sulfate, xanthan gum, gellan gum, starch or starch derivatives.

4. The polysaccharide-based polymer cross-linking agent according to claim 3, characterized in that The cellulose derivative is selected from methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose or hydroxypropyl methyl cellulose; The starch derivative is selected from oxidized starch, esterified starch, etherified starch or alkylated starch.

5. The method for preparing the polysaccharide-based polymer cross-linking agent according to claim 1, characterized in that: The aldehyde group-preprotected o-phthalaldehyde precursor derivative is covalently linked to the active group on the polysaccharide polymer to obtain the o-phthalaldehyde precursor derivative-modified polysaccharide polymer, and then the aldehyde group of the o-phthalaldehyde precursor derivative-modified polysaccharide polymer is deprotected to obtain the o-phthalaldehyde group-modified polysaccharide polymer crosslinker.

6. The method for preparing a polysaccharide-based polymer cross-linking agent according to claim 5, characterized in that: The aldehyde group pre-protected o-phthalaldehyde precursor derivative is selected from one of the following compounds: 、 、 、 、 、 、 、 、 、 、 。 7. The method for preparing a polysaccharide-based polymer cross-linking agent according to claim 5, characterized in that: The active groups of the polysaccharide polymer are active groups of the polysaccharide polymer itself, or reactive groups of the polysaccharide polymer after being modified or degraded.

8. The method for preparing a polysaccharide-based polymer cross-linking agent according to claim 5, characterized in that: Deprotecting the aldehyde group of the polysaccharide polymer modified with the o-phthalaldehyde precursor derivative means that the o-phthalaldehyde functional group of the aldehyde group connected to the polysaccharide polymer is deprotected under acidic conditions to obtain a polysaccharide polymer crosslinker modified with o-phthalaldehyde groups.

9. A method for preparing a polysaccharide-based two-component hydrogel, characterized in that: Dissolving component A and component B in a solvent respectively to obtain a component A solution and a component B solution, and mixing the component A solution and the component B solution to obtain the polysaccharide-based two-component hydrogel; Wherein, the component A is a polysaccharide-based polymer cross-linking agent as described in any one of claims 1 to 4; The component B is a water-soluble small molecule, a water-soluble synthetic polymer, or a water-soluble natural polymer containing one or more of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine, or sulfhydryl functional groups contained in a single molecule is not less than 2; The water-soluble natural macromolecules include proteins, nucleic acids and polysaccharides.

10. The method for preparing a polysaccharide-based two-component hydrogel according to claim 9, characterized in that: The component B is selected from one or more of the following substances: polyethylene glycol derivatives, polyethyleneimine, polyamino acids, proteins, protein modifications, polysaccharides, polysaccharide modifications or polysaccharide degradation products; and the molecular structure of the substance selected from component B contains one or more of primary amine, hydrazine, hydrazide, hydroxylamine or thiol groups, and the number of primary amine, hydrazine, hydrazide, hydroxylamine or thiol functional groups contained in a single molecule is not less than 2.

11. The method for preparing a polysaccharide-based two-component hydrogel according to claim 10, characterized in that: The polyethylene glycol derivative is a polyethylene glycol derivative modified with primary amine, hydrazine, hydrazide, hydroxylamine or thiol. The protein includes collagen, serum protein, fibrinogen and fibrin, and the protein modification includes gelatin or polypeptide; The polysaccharide, polysaccharide modification or polysaccharide degradation product is selected from: chitosan and chitosan modification and chitosan degradation product; primary amine, diamine, hydrazide, hydroxylamine or thiol modified hyaluronic acid, alginic acid, chondroitin sulfate, heparin, cellulose, chitin and their respective modifications and degradation products.

12. The method for preparing a polysaccharide-based two-component hydrogel according to claim 9, characterized in that: The component B is selected from: Amine-modified polyethylene glycol derivatives, hydrazide-modified hyaluronic acid, collagen, serum protein, gelatin, polypeptide, polyamino acid or chitosan.

13. The method for preparing a polysaccharide-based two-component hydrogel according to claim 9, characterized in that: In the component A solution, the solid content of component A is 0.1-40 wt %, and in the component B solution, the solid content of component B is 0.1-40 wt %.

14. The method for preparing a polysaccharide-based two-component hydrogel according to claim 13, characterized in that: In the component A solution, the solid content of component A is 0.5-20 wt %, and in the component B solution, the solid content of component B is 0.5-20 wt %.

15. The method for preparing a polysaccharide-based two-component hydrogel according to claim 14, characterized in that: In the component A solution, the solid content of component A is 0.5-10 wt %, and in the component B solution, the solid content of component B is 0.5-10 wt %.

16. A polysaccharide-based two-component hydrogel prepared by the preparation method according to any one of claims 9 to 15.

17. A polysaccharide-based biomaterial, characterized in that Selected from one of the following biological materials: A polysaccharide-based membrane material obtained by drying the polysaccharide-based two-component hydrogel according to claim 16; A polysaccharide-based powder material obtained by drying the polysaccharide-based two-component hydrogel according to claim 16 and further mechanically ball-milling; or a polysaccharide-based powder material obtained by drying and sieving microgel particles prepared by mixing the component A solution and the component B solution used to prepare the polysaccharide-based two-component hydrogel according to claim 9 and then using an emulsification method, a microfluidics method, or a mechanical pulverization method; A polysaccharide-based sponge material is obtained by mixing the component A solution, the component B solution and the pore-forming agent for preparing the polysaccharide-based two-component hydrogel according to claim 9, and further drying the mixture.

18. The polysaccharide-based biomaterial according to claim 17, characterized in that: When the polysaccharide-based biomaterial is selected from a polysaccharide-based sponge material, the pore-forming agent is an additive that creates a pore structure in the material, and is selected from substances that are easily decomposed into gas, polymer microspheres, polyethylene glycol, polyvinyl pyrrolidone, surfactants or water.

19. Use of the polysaccharide-based two-component hydrogel according to claim 16, characterized in that: Select one of the following applications: Application of the polysaccharide-based two-component hydrogel in the preparation of tissue repair products; Application of the polysaccharide-based two-component hydrogel in the preparation of tissue fluid leakage blocking products; Application of the polysaccharide-based two-component hydrogel in the preparation of tissue leakage sealing products; Application of the polysaccharide-based two-component hydrogel in the preparation of hemostatic products; Among them, the tissue repair includes skin repair and abdominal wall repair; the tissue fluid leakage blocking includes pancreatic fluid leakage blocking, cerebrospinal fluid leakage blocking, intestinal leakage blocking, and gastric leakage blocking; the tissue air leakage blocking includes lung parenchyma air leakage blocking; the hemostasis includes liver hemostasis, kidney hemostasis, spleen hemostasis, pancreatic hemostasis, bone section hemostasis, arterial hemostasis and heart hemostasis.

20. Use of the polysaccharide-based biomaterial according to claim 17, characterized in that: When the polysaccharide-based biomaterial is a polysaccharide-based membrane material, the application of the polysaccharide-based membrane material is selected from one of the following applications: Application of the polysaccharide-based membrane material in the preparation of suture-free closure products for surgical wounds; Application of the polysaccharide-based membrane material in the preparation of patch products; When the polysaccharide-based biomaterial is a polysaccharide-based powder material, the application of the polysaccharide-based powder material is selected from one of the following applications: Application of the polysaccharide-based powder material in the preparation of hemostatic products; Application of the polysaccharide-based powder material in the preparation of cell, factor, and drug carrier products; The hemostasis includes hemostasis during minimally invasive surgery, hemostasis of the liver, hemostasis of the kidneys, hemostasis of the spleen, hemostasis of the pancreas, hemostasis of bone sections, hemostasis of arteries and hemostasis of the heart; the cells, factors and drug carriers include stem cell carriers, growth factor carriers, antibacterial drug carriers and anti-inflammatory drug carriers; When the polysaccharide-based biomaterial is a polysaccharide-based sponge material, the application of the polysaccharide-based sponge material is selected from one of the following applications: Application of the polysaccharide-based sponge material in the preparation of wound dressing products; Application of the polysaccharide-based sponge material in the preparation of hemostatic products; The hemostasis includes liver hemostasis, kidney hemostasis, spleen hemostasis, pancreas hemostasis, bone section hemostasis, artery hemostasis, heart hemostasis and non-compression penetrating wound hemostasis.

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