A high molecular adhesive material, its preparation method and application

Abstract

The application relates to the technical field of medical materials, in particular to a high-molecular adhesive material and a preparation method and application thereof. The preparation method of the high-molecular adhesive material comprises the following steps: adding alkali into acrylic acid to obtain neutralized acrylic acid; adding the neutralized acrylic acid, sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate into an inorganic solvent, then adding potassium persulfate (KPS), and heating to react to obtain a gel; and freeze-drying the gel to obtain the high-molecular adhesive material, namely, a CMC / PAA-NHS adhesive modified by N-hydroxysuccinimide. The application also provides the high-molecular adhesive material prepared by the preparation method and application of the high-molecular adhesive material as a medical material. The application solves the problem of poor adhesion of existing hemostatic sponges and hemostatic powders, and also solves the problem of limited hemostatic performance of hydrogel adhesives.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a polymer adhesive material, its preparation method, and its application. Background Technology

[0002] Globally, traumatic hemorrhage causes more than 2 million deaths annually. Whether from war wounds, traffic accidents, or surgery, uncontrollable massive bleeding remains the leading cause of death. Statistics show that most trauma-related deaths occur within one hour of injury, primarily due to hemorrhagic shock. If timely and rapid hemostasis is achieved, approximately 50% of these patients can be saved. However, in severe trauma, the body's own clotting system cannot control massive bleeding. Therefore, rapid and effective intervention using hemostatic materials is of crucial clinical significance in prolonging the treatment window and improving survival rates.

[0003] Currently, commonly used hemostasis methods in clinical practice include surgical suturing and the application of hemostatic materials. While traditional surgical suturing techniques are effective, they are complex, time-consuming, prone to causing secondary damage to surrounding tissues, increasing patient pain, and difficult to implement in emergency situations. In recent years, with advancements in materials science and engineering, various hemostatic materials in different formulations have been developed using different cross-linking methods, various raw materials, and chemical modification methods. These materials can be mainly classified into the following three categories: Hemostatic sponges: Gelatin sponges and other hemostatic sponges concentrate blood components by rapidly absorbing liquid. Their porous structure can accumulate red blood cells and platelets, promoting coagulation cascade reactions and blood clot formation. However, they have disadvantages such as insufficient mechanical strength, poor adhesion, and easy detachment.

[0004] Hemostatic powder: Its main components include zeolite and other substances, and it has the advantages of high stability, easy storage, and convenient use. Taking zeolite as an example, the microporous structure of zeolite can quickly absorb water from the blood, thereby concentrating clotting factors and platelets in the blood and accelerating the clotting process. However, it also has problems such as exothermic burns and weak tissue adhesion.

[0005] Hydrogel adhesives: These achieve physical closure of bleeding through physical adhesion, forming a mechanical barrier to stop the bleeding and exhibiting excellent tissue adhesion and sealing performance. However, their hemostatic mechanism is singular, their ability to control active massive bleeding is limited, and some products have biocompatibility or degradation issues.

[0006] While existing technologies meet clinical needs to some extent, they still suffer from problems such as insufficient hemostatic efficiency, poor tissue adhesion, low mechanical strength, and poor biocompatibility. Therefore, developing a novel hemostatic material that combines rapid hemostasis, strong tissue adhesion, excellent mechanical properties, and good biocompatibility is of great significance for improving the level of trauma treatment. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a polymer adhesive material, its preparation method and application, to solve the problem of poor adhesion of existing hemostatic sponges and hemostatic powders, and also to solve the problems of limited hemostatic performance, poor biocompatibility and difficulty in degradation of hydrogel adhesives.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a polymer adhesive material includes the following steps: S1. Add alkali to acrylic acid to obtain neutralized acrylic acid; S2. Neutralized acrylic acid, sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate are added to an inorganic solvent, then potassium persulfate (KPS) is added, and the mixture is heated to react and obtain a gel. S3. Freeze-dry the gel to obtain a polymeric adhesive material, namely N-hydroxysuccinimide-modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS).

[0009] Based on the aforementioned technical methods, by adding sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate to alkali-neutralized acrylic acid, the acrylic acid retains some -COOH and introduces -COONa during copolymerization. Simultaneously, potassium persulfate (KPS) is added as an initiator, creating a three-dimensional network structure. This network carries a suitable amount of negative charge and forms electrostatic-hydrogen bond synergistic entanglement with the -COONa on the carboxymethyl cellulose chains. N-hydroxysuccinimide acrylate is attached to the gel backbone via double bonds, "suspending" the highly active NHS ester on the surface of the three-dimensional network. Therefore, when this polymeric adhesive material is used as a hemostatic material, upon contact with blood or tissue fluid, the NHS ester rapidly forms covalent amides with protein amino groups, while the residual carboxylic acid and sodium carboxylate are bridged with tissue polysaccharides and calcium ions through hydrogen bonds, achieving dual adhesion of "chemical anchoring + physical adsorption." Simultaneously, freeze-drying imparts a porous microstructure, allowing for instantaneous absorption of water and concentration of clotting factors. This covalent-non-covalent synergistic effect gives the material a wet interface adhesion force far exceeding that of traditional hemostatic sponges / powders, significantly shortening hemostasis time and reducing the risk of rebleeding. This effectively solves the problem of poor adhesion in existing hemostatic sponges and powders, and also addresses the limited hemostatic performance of hydrogel adhesives. Furthermore, sodium carboxymethyl cellulose and polyacrylic acid are both biocompatible natural / near-natural polysaccharides and synthetic polymers, avoiding the chronic inflammation or secondary damage caused by the poor biocompatibility of traditional hemostatic materials. This also resolves the problem of poor biocompatibility found in existing hydrogel adhesives.

[0010] Preferably, step S2 includes: adding neutralized acrylic acid, sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate to an inorganic solvent, then adding N'N-methylenebisacrylamide (MBA) and potassium persulfate (KPS), heating to react, and obtaining a gel.

[0011] The addition of crosslinking agent N'N-methylenebisacrylamide (MBA) and initiator potassium persulfate (KPS) makes the resulting three-dimensional network structure more compact.

[0012] Preferably, the temperature of the heating reaction is 60~70℃.

[0013] The reaction is carried out by heating at a temperature of 60~70℃. If the temperature is too low, the reaction is controllable but the efficiency is extremely low and the product residue is high. If the temperature is too high, the exothermic reaction increases sharply in the early stage of the reaction, which can easily lead to local overheating, explosive polymerization, system loss of control, and increased side reactions. Preferably, the temperature of the heating reaction is 65°C.

[0014] Preferably, the alkali is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution, sodium carbonate solution, and potassium carbonate solution.

[0015] Preferably, the sodium hydroxide solution is selected from an aqueous sodium hydroxide solution, wherein the sodium hydroxide aqueous solution contains 30% sodium hydroxide by mass.

[0016] Preferably, the degree of neutralization of the neutralized acrylic acid is 65-75%. If the neutralization is too low, the local polymerization reaction will be violent, resulting in uneven polymerization and affecting the product performance. If the neutralization is too high, the solution viscosity will be high, heat dissipation will be hindered, and explosive polymerization will easily occur.

[0017] Preferably, the mass percentages of neutralized acrylic acid, sodium carboxymethyl cellulose, N-hydroxysuccinimide acrylate, N'N-methylenebisacrylamide (MBA), and potassium persulfate (KPS) in the polymer adhesive material are 10-30 wt%, 0.5-3 wt%, 0.5-3 wt%, 0-0.05 wt%, and 0.05-0.5 wt%, respectively.

[0018] Preferably, the inorganic solvent is selected from water. Specifically, the water is deionized water.

[0019] Preferably, the freeze-drying is carried out under vacuum conditions, and the freeze-drying temperature is -75℃ to -85℃.

[0020] Preferably, the freeze-drying temperature is -80°C.

[0021] Preferably, the particle size of the polymer adhesive material is 100~500µm.

[0022] Preferably, the particle size of the polymer adhesive material is 200~400µm.

[0023] The present invention also provides a polymer adhesive material prepared by the preparation method described herein.

[0024] This invention also provides the application of polymer adhesives as medical materials.

[0025] Preferably, the polymer adhesive is used as a hemostatic material.

[0026] The beneficial effects of this invention are: The preparation method of the polymer adhesive material of the present invention involves adding sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate to alkali-neutralized acrylic acid. During copolymerization, the acrylic acid retains some -COOH and introduces -COONa. Simultaneously, potassium persulfate (KPS) is added as an initiator to create a three-dimensional network structure. This network carries a suitable amount of negative charge and forms electrostatic-hydrogen bond synergistic entanglement with the -COONa on the carboxymethyl cellulose chains. The N-hydroxysuccinimide acrylate is attached to the gel backbone through double bonds, "suspending" the highly active NHS ester on the surface of the three-dimensional network. Therefore, when this polymer adhesive material is used as a hemostatic material, upon contact with blood or tissue fluid, the NHS ester rapidly forms covalent amides with protein amino groups, while the residual carboxylic acid and sodium carboxylate are bridged with tissue polysaccharides and calcium ions through hydrogen bonds, achieving dual adhesion of "chemical anchoring + physical adsorption." Simultaneously, freeze-drying imparts a porous microstructure, allowing for instantaneous absorption of water and concentration of clotting factors. Covalent-non-covalent synergistic effects endow the material with wet interfacial adhesion far exceeding that of traditional hemostatic sponges / powders, significantly shortening hemostasis time and reducing the risk of rebleeding. Furthermore, sodium carboxymethyl cellulose and polyacrylic acid are both biocompatible natural / near-natural polysaccharides and synthetic polymers, avoiding the chronic inflammation or secondary damage problems caused by the poor biocompatibility of traditional hemostatic materials.

[0027] The present invention discloses a method for preparing a novel hemostatic powder, CMC / PAA-NHS, synthesized from sodium carboxymethyl cellulose and polyacrylic acid grafted with N-hydroxysuccinimide ester. The prepared CMC / PAA-NHS absorbs water from the blood, forming a strongly adhesive hydrogel that seals bleeding points, prevents bacterial contamination, and accelerates the hemostasis process. Research results show that CMC / PAA-NHS possesses strong adhesive strength, excellent mechanical strength, and good biocompatibility. In rat models of liver injury bleeding, rat models of complete femoral vein transection bleeding, and rat models of complete femoral artery and vein transection bleeding, CMC / PAA-NHS exhibits excellent hemostatic effects and can be applied to vascular wounds. Furthermore, CMC / PAA-NHS has the potential to activate intrinsic coagulation pathways, promote erythrocyte aggregation, and accelerate blood clot formation. Based on in vivo and in vitro data, CMC / PAA-NHS shows great potential as a hemostatic agent and has significant application value in the field of medical materials technology. Attached Figure Description

[0028] Figure 1 Fourier transform infrared spectrum; Figure 2 Scanning electron microscope image of dried CMC / PAA-NHS-1; Figure 3 The image shows a scanning electron microscope (SEM) image of CMC / PAA-NHS after swelling equilibrium. Figure 4 This is a graph showing the results of in vitro adhesion testing; Figure 5 The diagram shows the results of the compressive stress analysis. Figure 6 The result of the contact angle measurement is shown in the figure. Figure 7 The graph shows the results of the liquid absorption rate measurement. Figure 8 Plot showing the relative proliferation rate of CMC / PAA-NHS cells; Figure 9 Image showing the results of live / dead staining of CMC / PAA-NHS cells; Figure 10 A graph showing the in vitro hemolysis rate of CMC / PAA-NHS; Figure 11 This is a graph showing the relative activity of platelet LDH. Figure 12 The graph shows the changes in body weight of mice over 14 days. Figure 13 This is a graph showing the results of blood biochemistry. Figure 14 Image showing the results of H&E staining; Figure 15 This is a graph showing the results of in vitro coagulation time. Figure 16Figure showing the results of treatment in a rat liver injury and hemorrhage model; Figure 17 This is a diagram showing the results of treatment in a rat model of complete femoral vein transection and bleeding. Figure 18 This is a diagram showing the results of treatment in a rat model of complete transection of the femoral artery and vein causing bleeding. Figure 19 This is a graph showing the results of red blood cell adsorption. Figure 20 The results are for APTT and PT. Detailed Implementation

[0029] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0030] The materials used in the following embodiments are as follows: Sodium carboxymethyl cellulose (CMC) and sodium hydroxide were purchased from Sinopharm Group; acrylic acid, N-hydroxysuccinimide acrylate (AA-NHS), potassium persulfate (KPS), and N'N-methylenebisacrylamide (MBA) were purchased from Maclean Biotech; phosphate-buffered saline (PBS), physiological saline, MEM culture medium, and fetal bovine serum were purchased from Thermo Fisher Scientific (Shanghai, China); aspartate aminotransferase (AST / GOT) test kit, protein quantification (TP) assay kit, alkaline phosphatase (AKP) test kit, alanine aminotransferase (ALT / GPT) test kit, and creatinine (CRE) assay kit were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; and APTT and PT assay kits were purchased from Shenzhen Raydu Life Science Co., Ltd.

[0031] Male Sprague Dawley SD rats, weighing 180–220 g, and healthy Kunming mice, weighing 18–20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Example 1

[0032] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve acrylic acid (AA), sodium carboxymethyl cellulose (CMC), and N-hydroxysuccinimide acrylate (AA-NHS) with a neutralization degree of 70% in deionized water and mix thoroughly at 500 r / min. Then add N'N-methylenebisacrylamide (MBA) and potassium persulfate (KPS), mix thoroughly, adjust the speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-1). In the prepared polymer adhesive material CMC / PAA-NHS-1, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30 wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 2 wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1 wt%, the mass percentage of N'N-methylenebisacrylamide (MBA) is 0.018 wt%, and the mass percentage of potassium persulfate (KPS) is 0.075 wt%. Example 2

[0033] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve acrylic acid (AA), sodium carboxymethyl cellulose (CMC), and N-hydroxysuccinimide acrylate (AA-NHS) with a neutralization degree of 70% in deionized water and mix thoroughly at 500 r / min. Then add N'N-methylenebisacrylamide (MBA) and potassium persulfate (KPS), mix thoroughly, adjust the speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-2). In the prepared polymer adhesive material CMC / PAA-NHS-2, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30 wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 1 wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1 wt%, the mass percentage of N'N-methylenebisacrylamide (MBA) is 0.018 wt%, and the mass percentage of potassium persulfate (KPS) is 0.075 wt%. Example 3

[0034] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve acrylic acid (AA), sodium carboxymethyl cellulose (CMC), and N-hydroxysuccinimide acrylate (AA-NHS) with a neutralization degree of 70% in deionized water and mix thoroughly at 500 r / min. Then add N'N-methylenebisacrylamide (MBA) and potassium persulfate (KPS), mix thoroughly, adjust the speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-3). In the prepared polymer adhesive material CMC / PAA-NHS-3, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30 wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 0.5 wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1 wt%, the mass percentage of N'N-methylenebisacrylamide (MBA) is 0.018 wt%, and the mass percentage of potassium persulfate (KPS) is 0.075 wt%. Example 4

[0035] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve acrylic acid (AA), sodium carboxymethyl cellulose (CMC), and N-hydroxysuccinimide acrylate (AA-NHS) with a neutralization degree of 70% in deionized water and mix thoroughly at 500 r / min. Then add an appropriate amount of potassium persulfate (KPS), mix evenly, adjust the rotation speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-4). In the prepared polymer adhesive material CMC / PAA-NHS-4, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 2wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1wt%, and the mass percentage of potassium persulfate (KPS) is 0.075wt%. Example 5

[0036] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve acrylic acid (AA), sodium carboxymethyl cellulose (CMC), and N-hydroxysuccinimide acrylate (AA-NHS) with a neutralization degree of 70% in deionized water and mix thoroughly at 500 r / min. Then add an appropriate amount of potassium persulfate (KPS), mix thoroughly, adjust the rotation speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-5). In the prepared polymer adhesive material CMC / PAA-NHS-5, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 1wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1wt%, and the mass percentage of potassium persulfate (KPS) is 0.075wt%. Example 6

[0037] A method for preparing various polymer adhesive materials includes the following steps: S1. Acrylic acid is neutralized with a 30% (w / w) aqueous hydroxide solution to obtain acrylic acid (AA) with a neutralization degree of 70%. S2. Dissolve 70% neutralized acrylic acid (AA), 0.5 wt% sodium carboxymethyl cellulose (CMC), and 1 wt% N-hydroxysuccinimide acrylate (AA-NHS) in deionized water and mix thoroughly at 500 r / min. Then add an appropriate amount of potassium persulfate (KPS), mix thoroughly, adjust the speed to 100 r / min, and heat the reaction at 65℃. Stop heating when the reaction solution has completely formed a gel to obtain a gel. S3. After cooling the gel to room temperature, cut it into pieces measuring 5×2×0.5cm. 3 The block-shaped material is placed in a refrigerator at -80°C for vacuum freeze-drying, and then pulverized into powder particles of 40-100 mesh to obtain a polymer adhesive material, namely N-hydroxysuccinimide modified sodium carboxymethyl cellulose / polyacrylic acid adhesive (CMC / PAA-NHS-6). In the prepared polymer adhesive material CMC / PAA-NHS-6, the mass percentage of acrylic acid (AA) with a neutralization degree of 70% is 30wt%, the mass percentage of sodium carboxymethyl cellulose (CMC) is 0.5wt%, the mass percentage of N-hydroxysuccinimide acrylate (AA-NHS) is 1wt%, and the mass percentage of potassium persulfate (KPS) is 0.075wt%.

[0038] Detection and Analysis 1) Fourier transform infrared spectroscopy analysis Sodium carboxymethyl cellulose (CMC) and N-hydroxysuccinimide acrylate (AA-NHS) from Example 1, as well as the prepared CMC / PAA-NHS-1 powder, were mixed uniformly with potassium bromide (at a ratio of 1:100 w / w) and thoroughly ground. Using the potassium bromide tableting method, CMC, AA-NHS, and CMC / PAA-NHS-1 were mixed with KBr and pressed into uniform transparent sheets. These sheets were then tested using a Nicolet IS5 infrared spectrometer from Nicolet Corporation, USA, with the test wavelength set to 4000–400 cm⁻¹. -1 The number of scans was 16. The results are as follows: Figure 1 As shown.

[0039] from Figure 1 Analysis shows that 3421cm -1 The broad and strong stretching vibration peak nearby is the absorption peak of the OH stretching vibration of the sugar unit, 2921 cm⁻¹. -1 The nearby peak represents the absorption peak of the saturated CH stretching vibration in the glycogen unit, while the above peak represents the characteristic elution position of sodium carboxymethyl cellulose. 1736 cm⁻¹ -1 The nearby peak is the C=O stretching vibration peak in AA-NHS, at 1210 cm⁻¹. -1 The nearby peaks represent the vibrational peaks of CNC, which are characteristic peaks of AA-NHS. As can be seen from the figure, CMC / PAA-NHS-1 contains characteristic spectral bands of both CMC and AA-NHS, confirming the successful preparation of CMC / PAA-NHS-1.

[0040] 2) Scanning electron microscopy analysis The surface morphology of the CMC / PAA-NHS-1 powder particles prepared in Example 1 was observed using a Hitachi Regulus 8230 scanning electron microscope. The CMC / PAA-NHS-1 powder particles were adhered to conductive adhesive for testing, and then gold was sputtered onto the surface. The results are as follows: Figure 2 As shown.

[0041] from Figure 2 Analysis shows that CMC / PAA-NHS-1 is relatively uniformly pulverized with a particle size of 200-400 μm. In addition, the surface of CMC / PAA-NHS powder particles is rough, which facilitates the absorption of liquid, increases the contact area with blood, achieves adhesion and seals the bleeding point, and is conducive to rapid hemostasis.

[0042] CMC / PAA-NHS-1 was fully swollen in deionized water until swelling equilibrium was reached, then frozen in liquid nitrogen, vacuum dried, and analyzed by scanning electron microscopy. The results are as follows: Figure 3 As shown.

[0043] from Figure 3The CMC / PAA-NHS-1 sample exhibits a porous honeycomb structure, indicating that it can absorb liquid in the liquid and is a gel containing liquid, which is conducive to absorbing blood and applying physical pressure to the bleeding point.

[0044] 3) In vitro adhesion assay Adhesion of CMC / PAA-NHS in wet tissues The specific operating steps are as follows: wet the pig skin with PBS, add 100mg of CMC / PAA-NHS prepared in Examples 1 to 6 to the surface of the pig skin tissue, and observe whether it can adhere.

[0045] Figure 4 The figure 'a' represents a photograph of CMC / PAA-NHS adhesion to the surface of pig skin. From... Figure 4 Analysis of sample a shows that moistened pigskin forms a stable and robust adhesion interface with CMC / PAA-NHS, which does not detach after rinsing with water. Furthermore, it can tightly adhere to biological tissues such as the rat heart and spleen. When CMC / PAA-NHS comes into contact with moist tissue, it rapidly forms physical cross-links. Because CMC / PAA-NHS contains a large number of hydroxyl and carboxyl groups, it forms hydrogen bonds with the tissue surface. Then, the NHS ester groups in CMC / PAA-NHS form covalent cross-links with the abundant primary amine groups in the biological tissue, achieving strong adhesion.

[0046] To further quantitatively characterize the bioadhesion properties of the powder, overlap shearing test, 180° peel test, and tensile test were conducted.

[0047] The specific operating steps of the overlap shear test are as follows: Using fresh pig heart as the material, the fresh pig heart was cleaned and cut into six 50×20×5mm pieces. The overlap shear was performed on the epicardium of the pig heart. Then, 200mg of CMC / PAA-NHS prepared in Examples 1 to 6 was placed at one end of the epicardium of the pig heart, evenly distributed on the overlap area, which was set to 20mm×20mm. Another piece of pig heart epicardium was then overlapped on top. After a period of interaction, the pieces were transferred to a universal tensile testing machine. The testing speed was set at 50mm / min, and the test was conducted at room temperature. The test was repeated three times, with pig fibrin glue as a blank control. The shear strength was calculated as the ratio of the maximum force to the overlap area.

[0048] The material used in the 180° dissection experiment was a fresh pig heart, measuring 60×20×5mm.

[0049] The specific operating steps of the 180° peel test are as follows: 200 mg of CMC / PAA-NHS prepared in Examples 1 to 6 are placed at one end of the epicardium of a pig heart, evenly distributed on the adhesive area, which is set to 20 mm × 20 mm. Another piece of pig epicardium is placed on top, with CMC / PAA-NHS acting as an adhesive between the two pieces of pig epicardium. After interaction for a period of time, a universal tensile testing machine is used to measure the force at a speed of 50 mm / min at room temperature. The test is repeated three times, with pig fibrin glue as a blank control. When the peel process reaches a stable state, the measured force reaches a steady state. The interfacial toughness is calculated from the tensile force and the adhesive width, and the calculation formula is shown in Equation (I).

[0050] In formula (I), Indicates interface toughness, unit: ; The value indicates that after the peeling process enters a stable stage, the universal tensile testing machine measures a tensile force value that remains basically constant, in N; W represents the adhesive width, in meters.

[0051] The material used in the tensile test was a fresh pig heart, with the adhesive surface being the epicardium of the pig heart. The pig heart was cut to a size of 20×20×5 mm.

[0052] The specific operating steps of the tensile test are as follows: A pig heart is bonded to a T-shaped mold using cyanoacrylate. 100 mg of CMC / PAA-NHS prepared in Examples 1 to 6 is placed on the epicardium of the pig heart. After the pig heart interacts with the adhesive for a period of time, it is transferred to a universal tensile testing machine. The system speed is set to 50 mm / min, and the test is conducted at room temperature. Using pig fibrin glue as a control, the test is repeated three times. The tensile strength is calculated from the force and the bonded area, as shown in formula (II).

[0053] In formula (II), This represents tensile strength, expressed in Pa. This indicates the maximum tensile force that the device can withstand when it breaks under tension, expressed in Newtons (N). This indicates the width of the bonding area, in meters (m). This indicates the length of the adhesive area, in meters (m).

[0054] The results for shear strength, interfacial toughness, and tensile strength are as follows: Figure 4 As shown in b~h.

[0055] Figure 4 b represents a photograph of the adhesion of CMC / PAA-NHS-1 to the surfaces of rat lung, kidney, heart, and spleen tissues. Figure 4 The 'c' represents a schematic diagram of the overlap shearing experiment of CMC / PAA-NHS on the surfaces of rat lung, kidney, heart, and spleen tissues. Figure 4 The figure 'd' represents a comparison of the shear strength of porcine fibrin glue and CMC / PAA-NHS. Figure 4 The 'e' represents a schematic diagram of a 180° peeling experiment. Figure 4 f represents a comparison of the interfacial toughness between porcine fibrin glue and CMC / PAA-NHS. Figure 4 The symbol g represents a schematic diagram of a tensile test. Figure 4 The figure with 'h' represents a comparison of the tensile strength of porcine fibrin glue and CMC / PAA-NHS.

[0056] from Figure 4 As shown in sections b to h, when the CMC / PAA-NHS prepared in Examples 1 to 6 is used as an adhesive and comes into contact with tissue, it can form a strong adhesion with the body tissue, and the adhesion effect is better than that of the positive control porcine fibrin adhesive; in particular, the results show that the shear strength of CMC / PAA-NHS-1 prepared in Example 1 is 5947.5±418.8 Pa, and the interfacial toughness is 21.1±1.7 J / m. 2 The tensile strength was 4450.8±452.0 Pa, and the shear strength of CMC / PAA-NHS-5 prepared in Example 5 was 6517.5±998.5 Pa, with an interfacial toughness of 22.8±1.0 J / m. 2 The tensile strength was 4508.3±102.8 Pa, effectively demonstrating that CMC / PAA-NHS, as an adhesive, significantly outperformed porcine fibrin adhesive in adhesion. CMC / PAA-NHS, as an adhesive, could form strong adhesion with wound tissue, sealing bleeding points, facilitating hemostasis, and reducing wound infection. The results showed that CMC / PAA-NHS-1 and CMC / PAA-NHS-5, as adhesives, had better adhesion effects than the adhesives prepared in Examples 2-4 and Example 6, thus identifying CMC / PAA-NHS-1 and CMC / PAA-NHS-5 as having superior adhesion properties.

[0057] 4) Compression stress analysis Based on shear strength, interfacial toughness, and tensile strength, CMC / PAA-NHS-1 prepared in Example 1 and CMC / PAA-NHS-5 prepared in Example 5, which have high adhesion strength, were selected for the following experiments.

[0058] To investigate the ability of CMC / PAA-NHS as an adhesive to resist high-pressure deformation, compressive stress was measured. Equal masses of deionized water were added to CMC / PAA-NHS-1 powder prepared in Example 1 and CMC / PAA-NHS-5 powder prepared in Example 5 to form gels. These gels were then cut into cylinders with a diameter of 15 mm and a height of 5 mm. Fibrin glue was used as a blank control. A universal tensile testing machine was used to measure the stress of CMC / PAA-NHS at 80% compressive strain at a testing speed of 5 mm / min. The compressive stress at 80% compressive strain was calculated based on the force and the area of ​​compression, using the formula shown in equation (III). The results are as follows: Figure 5 As shown.

[0059] In equation (III), Conpressive stress represents the compressive stress at 80% compressive strain, in kPa; F represents the instantaneous force when the specimen reaches 80% compressive strain, in N; and d represents the diameter of the cylinder, in m.

[0060] from Figure 5 Analysis shows that, under 80% compressive strain, the compressive stress of CMC / PAA-NHS-1 is 485.7±45.5 kPa, and that of CMC / PAA-NHS-5 is 259.1±19.9 kPa. Under 80% compressive strain, the compressive stress of both CMC / PAA-NHS-1 and CMC / PAA-NHS-5 tends to be greater than that of porcine fibrin glue. In particular, the compressive stress of CMC / PAA-NHS-1 is significantly higher than that of the positive control porcine fibrin glue, indicating that the CMC / PAA-NHS-1 prepared by the method of this invention has high compressive strength as an adhesive, which is beneficial for ensuring the structural integrity of the adhesive and can effectively compress blood vessels for hemostasis.

[0061] 5) Contact angle measurement To evaluate the surface wetting properties of deionized water on materials, the hydrophilicity and hydrophobicity of CMC / PAA-NHS materials were compared. CMC / PAA-NHS-1 particles prepared in Example 1 and CMC / PAA-NHS-5 particles prepared in Example 5 were respectively compressed into tablets. Static contact angles were measured at four locations using a Dingsheng-JY-82C microscope, with deionized water as the medium. The results are as follows: Figure 6 As shown.

[0062] from Figure 6 Analysis revealed that the static contact angles of both CMC / PAA-NHS-1 and CMC / PAA-NHS-5 were less than 90°, with CMC / PAA-NHS-1 exhibiting a static contact angle of 36.0 ± 1.5°. This indicates that both material samples are hydrophilic. A smaller static contact angle suggests better wettability of deionized water on the material, indicating better hydrophilicity. Hydrophilic surfaces are generally more conducive to cell adhesion. CMC / PAA-NHS-1 exhibits weaker hydrophilicity than CMC / PAA-NHS-5. This may be because CMC / PAA-NHS-1 forms a more compact three-dimensional network structure. The contact angle test involved compressing CMC / PAA-NHS into tablets, resulting in a more compact tablet that is more difficult for deionized water to wet, thus leading to a larger static contact angle.

[0063] 6) Liquid absorption rate determination Liquid absorption and swelling are important factors affecting hemostasis. Rapid absorption of water from the blood concentrates clotting factors and promotes the coagulation cascade reaction. However, excessive water absorption and swelling capacity can affect the adhesion effect. To analyze the water absorption and swelling capacity of CMC / PAA-NHS as an adhesive, a liquid absorption rate experiment was conducted.

[0064] The specific operating steps are as follows: Wrap the CMC / PAA-NHS (W0) prepared in Examples 1 and 5 respectively with 100-mesh nylon mesh, weigh the total weight (W1) of the nylon mesh and CMC / PAA-NHS, and immerse them in deionized water or saline at room temperature for 0.5 min, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min (t). Remove them from the water, remove the surface liquid with filter paper, and then weigh the total weight (W1) of the nylon mesh containing the absorbed liquid. t Based on the weight of the liquid before and after absorption by the CMC / PAA-NHS, the liquid absorption rate of the CMC / PAA-NHS was calculated using formula (IV). The results are as follows: Figure 7 As shown.

[0065] In equation (IV), W t The value represents the total weight of the CMC / PAA-NHS nylon mesh after the absorbent liquid is removed at time t, where t represents the time of removal, and the unit is g. W0 represents the initial total weight of CMC / PAA-NHS and nylon mesh, in g; W0 represents the initial weight of CMC / PAA-NHS, in g.

[0066] When CMC / PAA-NHS reaches swelling equilibrium, the liquid absorption rate is the swelling rate. From Figure 7 Analysis revealed that the swelling ratio of CMC / PAA-NHS-1 in deionized water was 14409.1±267.4%, while that in physiological saline was 2306.7±68.3%. The liquid absorption rate of CMC / PAA-NHS-5 in deionized water was 1669.8±54.9%, and that in physiological saline was 1490.2±66.5%. These results indicate that the liquid absorption rate of CMC / PAA-NHS-1 in both deionized water and physiological saline was significantly higher than that of CMC / PAA-NHS-5. This may be because the cross-linking agent MBA was added during the preparation of CMC / PAA-NHS-1, forming a denser three-dimensional network structure. This structure facilitates the absorption and containment of more liquid through capillary action and osmotic pressure effects. Furthermore, the significantly higher liquid absorption rate of CMC / PAA-NHS in deionized water compared to physiological saline suggests that the presence of common ion effects in physiological saline affected the osmotic pressure difference between the material's interior and exterior, thus weakening its liquid absorption capacity.

[0067] 7) Cytotoxicity assay Cytotoxicity assays were performed using the MTT assay with thiazolyl blue colorimetric method, with L929 cells as the experimental cells. The extraction solution was prepared by first calculating the total amount of extract absorbed per gram of CMC / PAA-NHS particles. Excluding the amount absorbed by CMC / PAA-NHS, the extract was prepared at a ratio of 0.1 g / mL. The extraction solution was MEM medium.

[0068] 96-well plates were seeded (3500 cells / well). 100 µL of CMC / PAA-NHS extract prepared in Examples 1 to 6 was added to each well. MEM medium was used as the blank control group. The plates were incubated in a 5% CO2 incubator for 24 h. After incubation, the liquid in each well was discarded, and 100 µL of MTT solution (1 mg / mL) was added to each well. The plates were then incubated in a 5% CO2 incubator for 4 h. Then, 100 µL of dimethyl sulfoxide was added to each well, and the plates were shaken for 10 min to ensure complete dissolution of the crystal violet precipitate. The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated using the following formula (V). The results are shown below. Figure 8 As shown.

[0069] In equation (V), Cell Viability Indicates cell viability. This indicates the absorbance value of the CMC / PAA-NHS group. This represents the absorbance value of the blank control group.

[0070] Cell status was observed using the Calcein-AM / PI live / dead cell double staining kit. L929 cells were cultured using the MTT assay (thiazolyl blue staining method), with 3500 cells seeded per well. 100 µL of CMC / PAA-NHS extract prepared in Examples 1 to 6 was added to each well. The control group used MEM medium. Cells were incubated in a 5% CO2 incubator for 24 h. Then, 100 µL of the staining working solution was prepared according to the Calcein-AM / PI live / dead cell double staining kit and added to each well of a 96-well plate. The plates were incubated in the dark for 30 min in a CO2 incubator. After incubation, cell staining was observed using 490 nm excitation light. The results are shown below. Figure 9 As shown.

[0071] from Figure 8 Analysis showed that the cell viability of CMC / PAA-NHS-1 was 101.4±5.3%, CMC / PAA-NHS-2 was 93.9±3.1%, CMC / PAA-NHS-3 was 98.6±7.7%, CMC / PAA-NHS-4 was 104.2±7.8%, CMC / PAA-NHS-5 was 94.8±8.3%, and CMC / PAA-NHS-6 was 109.3±3.0%. The cell viability of all CMC / PAA-NHS groups was greater than 90%, and the results of cell viability greater than 100% also proved that cell proliferation occurred, indicating that CMC / PAA-NHS has good biocompatibility.

[0072] from Figure 9 Analysis showed that, compared with the blank control group, the CMC / PAA-NHS group had similar cell number and morphology, exhibiting good cell compatibility. The results indicated that the cell viability in the CMC / PAA-NHS group was greater than 90%, and the cell morphology was similar to that of the blank control group, demonstrating that CMC / PAA-NHS has good in vitro cell compatibility.

[0073] 8) Hemolysis test A 3.8% (w / v) sodium citrate solution was prepared using physiological saline as an anticoagulant. Whole blood was collected from rats via the abdominal aorta and anticoagulated at a 1:9 ratio with the anticoagulant to obtain fresh anticoagulated whole blood. This fresh anticoagulated whole blood was then centrifuged at 3000 rpm for 5 minutes. The upper layer was platelet-rich plasma, and the lower layer was hematocrit red blood cells. The upper platelet-rich plasma layer was discarded. Physiological saline was added, and the mixture was slowly mixed. The mixture was then centrifuged again, and the supernatant was discarded. The blood was washed three times with physiological saline until the supernatant was clear and transparent. This supernatant was then discarded, yielding the hematocrit red blood cells. These cells were then resuspended in physiological saline to a 5% hematocrit.

[0074] To investigate the blood compatibility of CMC / PAA-NHS, a hemolysis experiment was conducted. Red blood cells were diluted with physiological saline. CMC / PAA-NHS-1 (10 mg) prepared in Example 1 and CMC / PAA-NHS-5 (10 mg) prepared in Example 5, along with 200 μL of red blood cell suspension and 5 mL of physiological saline, were mixed thoroughly. All centrifuge tubes were placed in a 37°C water bath for 1 hour. The negative control was physiological saline, and the positive control was deionized water. The mixture was centrifuged at 3000 rpm for 10 minutes. 200 μL of each sample was transferred to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader. The hemolysis rate was calculated according to the following formula (VI): In formula (VI), Indicates the hemolysis rate. This indicates the optical density value of the CMC / PAA-NHS group. This represents the optical density value of the saline group. This represents the optical density value of the deionized water group.

[0075] The results are as follows Figure 10 As shown.

[0076] from Figure 10 Analysis showed that the DW group (deionized water group) had an in vitro hemolysis rate generally considered to be 100%, while the in vitro hemolysis rate of the CMC / PAA-NHS group was less than 5%, meeting the national medical device biological evaluation standards. Observation of the supernatant color after the experiment revealed that the supernatant of the CMC / PAA-NHS group was similar to that of the physiological saline group, being colorless and transparent, while the deionized water group showed a bright red color. This indicates that the effect of CMC / PAA-NHS on erythrocytes is similar to that of physiological saline, with no significant destructive effect.

[0077] 9) Platelet compatibility test A 3.8% (w / v) sodium citrate solution was prepared using physiological saline as an anticoagulant. Whole blood was then collected from rats via the abdominal aorta and anticoagulated at a 1:9 ratio with the anticoagulant to obtain fresh anticoagulated whole blood. Centrifugation was performed to obtain platelet-rich plasma (PRP) and platelet-poor plasma (PPP). The platelet concentration was adjusted to 1 × 10⁻⁶ using PPP and PBS buffer. 8 Platelet count / mL, ready for use. Prepare platelet suspension (1×10⁻⁶ / mL). 8 The sample (number of samples / mL) was mixed thoroughly with an equal volume of CMC / PAA-NHS extract and incubated at 37°C for 30 min. PBS buffer served as the negative control, and 1% Triton X-100 served as the positive control. After incubation, the sample was centrifuged at 400 x g for 5 min, and the supernatant was used to determine LDH activity. Following the instructions of the lactate dehydrogenase (LDH) kit, the absorbance at 440 nm was measured using a microplate reader. LDH activity was calculated according to the kit instructions, and then the relative LDH activity of the sample group was calculated based on the LDH activities of the sample group and the positive control group, as shown in formula (VII). The results are as follows: Figure 11 As shown.

[0078] In equation (VII), Indicates relative activity. This indicates the optical density value of the CMC / PAA-NHS group. This represents the optical density value of the 1% Triton X-100 positive control group.

[0079] from Figure 11 Analysis showed that the relative LDH activity in the PBS group was 32.6±6.2%, in the CMC / PAA-NHS-1 group it was 28.4±14.8%, and in the CMC / PAA-NHS-5 group it was 29.2±7.0%. Platelets treated with CMC / PAA-NHS exhibited similar LDH release to the PBS negative control, indicating that the CMC / PAA-NHS extract as a binder has good biocompatibility with platelets. In summary, this fully demonstrates that the CMC / PAA-NHS of this invention has good biocompatibility as a binder.

[0080] 10) Acute toxicity test The preparation method of the extract involved first calculating the total amount of extract absorbed by each gram of CMC / PAA-NHS particles. Excluding the amount absorbed by CMC / PAA-NHS, the extract was prepared at a ratio of 0.1 g / mL. The extract was physiological saline. Healthy male Kunming mice, weighing 18–22 g, were randomly divided into groups. Animals in the CMC / PAA-NHS experimental group received a single intraperitoneal injection of 50 mL / kg of the sample extract, while the blank control group received 0.9% sodium chloride injection. Immediate reactions and systemic toxicity were observed and recorded after administration. Mice were weighed daily, and blood was collected on day 14 for blood biochemical analysis. Liver and kidney tissues were fixed and H&E stained for observation. Results are as follows: Figures 12 to 14 As shown.

[0081] Figures 12 to 14 In the table, Saline represents the blank control group, CMC / PAA-NHS-1 represents the experimental group receiving a single intraperitoneal injection of 50 mL / kg of the extract of CMC / PAA-NHS-1 prepared in Example 1, and CMC / PAA-NHS-5 represents the experimental group receiving a single intraperitoneal injection of 50 mL / kg of the extract of CMC / PAA-NHS-5 prepared in Example 5.

[0082] from Figure 12 Analysis showed that no deaths or convulsions occurred in the experimental groups receiving intraperitoneal injections of CMC / PAA-NHS-1 and CMC / PAA-NHS-5 extracts. Physiological saline served as a blank control; compared to the blank control group, the experimental groups showed no significant difference in weight, indicating that CMC / PAA-NHS does not induce acute toxicity. Figure 13 Blood biochemical analysis of rats revealed no significant differences in liver and kidney function indicators between the experimental and control groups, indicating that the extracts of CMC / PAA-NHS-1 and CMC / PAA-NHS-5 did not adversely affect liver metabolism or kidney filtration function. Figure 14 H&E staining histological analysis showed no obvious pathological reactions in the liver and kidney tissues of the experimental group. This fully demonstrates that CMC / PAA-NHS-1 and CMC / PAA-NHS-5 have good biocompatibility and do not cause functional damage to major metabolic organs.

[0083] 11) In vitro coagulation time assay The in vitro coagulation time assay aimed to evaluate the effect of CMC / PAA-NHS as a binder on blood coagulation. The specific procedure was as follows: 4 mg of CMC / PAA-NHS-1 prepared in Example 1 and CMC / PAA-NHS-5 prepared in Example 5 were placed in separate EP tubes. 200 μL of fresh anticoagulated whole blood was added, and the binder was thoroughly mixed with the whole blood. 20 μL of 0.2 M calcium chloride solution was added to initiate the coagulation process. The tubes were incubated in a 37°C water bath. Every 15 seconds, the EP tubes were tilted to observe whether a stable blood clot formed, whether the blood was completely coagulated and non-flowing, and whether the inverted EP tube remained stable. The in vitro coagulation time was recorded. Celox was used as a positive control, and Blank was used as a negative control. Each group was measured three times. The results are as follows: Figure 15 As shown.

[0084] from Figure 15 Analysis showed that the in vitro coagulation time in the Celox group was significantly shorter than that in the control group, which had an in vitro coagulation time of 8.26 ± 0.14 min, indicating the successful construction of the in vitro coagulation experiment. Meanwhile, the in vitro coagulation time in the CMC / PAA-NHS group was significantly shorter than that in the control group, and there was no statistically significant difference compared to the Celox group. Specifically, the in vitro coagulation time for CMC / PAA-NHS-1 was 5.68 ± 0.17 min, and for CMC / PAA-NHS-5 it was 6.19 ± 0.11 min. This demonstrates that the CMC / PAA-NHS prepared in this invention can absorb water in whole blood, forming a porous viscous gel, thereby accelerating the whole blood coagulation process.

[0085] 12) In vivo hemostasis experiment To investigate the hemostatic effect of CMC / PAA-NHS, rat models of liver injury and bleeding, rat models of complete femoral vein transection and bleeding, and rat models of complete femoral artery and vein transection and bleeding were established.

[0086] A rat model of liver injury and hemorrhage was established. SD rats were randomly divided into four groups: gauze group, Celox group, CMC / PAA-NHS-1 group, and CMC / PAA-NHS-5 group. The gauze group served as the negative control group, and the Celox group served as the positive control group. Rats were fixed on an operating table, and the skin was incised along the lower right side of the thoracic cavity using surgical scissors to expose the liver tissue. A wound 8 mm wide and 3 mm deep was created using a 5 mL syringe needle, allowing free bleeding for 10 seconds. CMC / PAA-NHS (100 mg) was then placed at the bleeding site, the wound was covered with gauze, and pressure was applied using a 50 g weight. The hemostasis time and bleeding volume were recorded during the hemostasis process, which was repeated five times. Results are as follows: Figure 16 As shown.

[0087] Figure 16In the table, Figure a shows a representative image of hemostatic performance in a rat liver injury model, Figure b is a schematic diagram of the construction of a rat liver injury bleeding model, Figure c is a comparison of hemostasis time in rat liver injury bleeding experiments, and Figure d is a comparison of blood loss in rat liver injury bleeding experiments.

[0088] Celox was used as a positive control group. It is currently a leading marketed hemostatic wound dressing both domestically and internationally, and therefore served as a positive control material. Hemostasis time and blood loss were recorded for each group. Figure 16 Comparative analysis showed that the hemostasis time was significantly shorter in the CMC / PAA-NHS-1 group (49.0±4.9s) and the CMC / PAA-NHS-5 group (54.2±12.5s) compared to the gauze group (136.4±23.1s). The blood loss was significantly reduced in the CMC / PAA-NHS-1 group (0.138±0.013g) and the CMC / PAA-NHS-5 group (0.151±0.022g) compared to the gauze group (0.316±0.045g). Furthermore, the hemostatic effects of the CMC / PAA-NHS group and the Celox group were comparable, consistent with the results of the in vitro coagulation experiment.

[0089] To establish a rat model of total femoral vein transection and hemorrhage, SD rats were selected for the experiment. Five SD rats were included in each of the following groups: gauze group, Celox group, CMC / PAA-NHS-1 group, and CMC / PAA-NHS-5 group. The gauze group served as a negative control, and the Celox group as a positive control. SD rats were anesthetized with isoflurane and fixed on the operating table. The groin area of ​​the rat's thigh was cut open with surgical scissors to expose the femoral vein, which appeared deep red. The femoral vein was then transversely severed (approximately 1 cm proximal to the heart at the junction of the femoral vein, femoral artery, and femoral nerve). Free bleeding was allowed for 5 seconds, with blood flowing out in streams from the bleeding point. The blood loss was collected using gauze. CMC / PAA-NHS (300 mg) was then applied to seal the bleeding point, followed by gauze coverage and pressure applied using a 200g weight. When no blood oozing is observed and bleeding ceases after 1 minute, the wound is considered successfully hemostatically controlled. The hemostasis time is recorded, and the blood loss during the hemostasis process is calculated by the weight difference of the gauze before and after application. Results are as follows: Figure 17 As shown Figure 17 In the table, Figure a shows a representative image of hemostatic performance in a rat femoral vein total transection bleeding model; Figure b is a schematic diagram of the construction of the rat femoral vein total transection bleeding model; Figure c is a comparison of hemostasis time in the rat femoral vein total transection bleeding experiment; and Figure d is a comparison of blood loss in the rat femoral vein total transection bleeding experiment.

[0090] from Figure 17Analysis showed that the blood loss was 2.78±0.24g in the gauze group, 1.20±0.14g in the Celox group, 0.63±0.07g in the CMC / PAA-NHS-1 group, and 0.83±0.08g in the CMC / PAA-NHS-5 group. It was noted that the blood loss was significantly reduced in the CMC / PAA-NHS and Celox groups compared to the gauze group; furthermore, the blood loss in the CMC / PAA-NHS group was significantly less than that in the Celox positive control group. Regarding hemostasis time, the hemostasis time was 335.0±22.1s in the gauze group, 148.2±12.2s in the Celox group, 82.0±12.1s in the CMC / PAA-NHS-1 group, and 113.0±7.4s in the CMC / PAA-NHS-5 group. The results showed that the hemostasis time in the CMC / PAA-NHS group and the Celox group was significantly shorter than that in the gauze group; moreover, the hemostasis time in the CMC / PAA-NHS group was significantly shorter than that in the Celox positive control group; in addition, the hemostasis time in the CMC / PAA-NHS-1 group was shorter than that in the CMC / PAA-NHS-5 group. Compared with the rat liver injury bleeding model, the hemostatic effect of the CMC / PAA-NHS group was better than that of the Celox group. Due to the different model construction, the bleeding volume in the rat femoral vein total transection bleeding model was larger and the bleeding speed was faster. The CMC / PAA-NHS group can absorb water in the blood and form a viscous gel, thereby sealing the bleeding point and exerting a better hemostatic effect of CMC / PAA-NHS. In summary, compared with the gauze group and the Celox group, the CMC / PAA-NHS group showed significantly reduced blood loss and hemostasis time (**p<0.01), and the hemostasis time of CMC / PAA-NHS-1 was shorter than that of CMC / PAA-NHS-5 (*p<0.05). Therefore, the rat femoral artery and vein total transection hemorrhage model will be investigated using CMC / PAA-NHS-1 as the experimental group.

[0091] To further investigate the hemostatic properties of CMC / PAA-NHS, a rat model of complete transection of the femoral artery and vein was established. SD rats were randomly divided into three groups: a gauze group, a Celox group, and a CMC / PAA-NHS-1 group. Rats were fixed on an operating table, and the femoral artery and vein were exposed by incising the groin area of ​​the rat's thigh with surgical scissors. Then, the femoral artery and vein were transversely severed (approximately 1 cm proximal to the heart at the junction of the femoral vein, femoral artery, and femoral nerve), allowing free bleeding for 5 seconds. CMC / PAA-NHS (300 mg) was then applied to seal the bleeding point, followed by gauze coverage and pressure. The hemostasis time and blood loss were recorded, and the process was repeated 5 times. Results are as follows: Figure 18 As shown.

[0092] Figure 18In the table, Figure a shows a representative image of hemostatic performance in a rat model of complete transection of the femoral artery and vein; Figure b shows a comparison of hemostasis time in the rat model of complete transection of the femoral artery and vein; and Figure c shows a comparison of blood loss in the rat model of complete transection of the femoral artery and vein.

[0093] from Figure 18 Analysis showed that, in terms of blood loss during hemostasis, the gauze group lost 3.62±0.11g, the Celox group lost 1.35±0.51g, and the CMC / PAA-NHS-1 group lost 0.75±0.19g. Compared with the gauze group, the blood loss in the Celox and CMC / PAA-NHS-1 groups was significantly reduced, indicating that all three hemostatic materials could effectively control massive bleeding from the femoral artery and vein. Notably, the blood loss in the CMC / PAA-NHS-1 group was significantly lower than that in the Celox positive control group, showing that it has a stronger hemostatic ability in the massive bleeding model. Regarding hemostasis time, the gauze group required 270.6±6.4s to achieve hemostasis, while the Celox group and CMC / PAA-NHS-1 group had hemostasis times of 171.4±17.4s and 142.0±14.3s, respectively, both significantly faster than the gauze group. The hemostasis time of the CMC / PAA-NHS-1 group was significantly shorter than that of the Celox positive control group, indicating that it can achieve effective closure of large blood vessel wounds more quickly.

[0094] In summary, compared with the gauze group and the Celox group, the CMC / PAA-NHS-1 group showed significant advantages in both blood loss and hemostasis time, demonstrating superior hemostatic performance. Compared with the previous rat liver injury bleeding model, CMC / PAA-NHS-1 showed a more pronounced advantage over Celox in the rat femoral artery and vein total transection model, possibly due to the physiological characteristics of this model: large bleeding volume and rapid bleeding rate. Upon contact with large amounts of blood, CMC / PAA-NHS-1 rapidly absorbs water from the blood, forming a highly adhesive porous hydrogel in situ, effectively sealing bleeding points and resisting blood flow impact. Its unique liquid absorption and gel-forming properties are fully utilized in such severe hemorrhage scenarios, thus exhibiting a superior hemostatic effect compared to Celox.

[0095] 13) Red blood cell adsorption experiment To further analyze the effect of CMC / PAA-NHS as a binder on erythrocyte aggregation, an erythrocyte adsorption experiment was conducted, and the morphology of erythrocyte aggregation was observed directly using scanning electron microscopy.

[0096] The specific operating steps are as follows: Prepare a 3.8% (w / v) sodium citrate solution using physiological saline as an anticoagulant. Then, collect whole blood from rats via the abdominal aorta and anticoagulate using the anticoagulant at a ratio of 1:9 to obtain fresh anticoagulated whole blood. Centrifuge the fresh anticoagulated whole blood at 3000 rpm for 5 minutes. The upper layer is platelet-rich plasma, and the lower layer is hematocrit red blood cells. Discard the upper platelet-rich plasma layer, add physiological saline, mix slowly, centrifuge again, and discard the supernatant. Wash three times with physiological saline until the supernatant is clear and transparent after centrifugation. Discard the supernatant to obtain the hematocrit red blood cells. Then, prepare a 5% hematocrit red blood cell suspension using physiological saline. The prepared 5% hematocrit red blood cells are used for absorbance testing with an ELISA reader and observation under a scanning electron microscope.

[0097] Weigh 25 mg of CMC / PAA-NHS-1 prepared in Example 1 and CMC / PAA-NHS-5 prepared in Example 5 into 10 mL centrifuge tubes, respectively. Add 5 mL of 5% hematocrit red blood cell suspension to each tube and incubate in a 37°C water bath for 30 min. After incubation, take 1 mL of the CMC / PAA-NHS-infused suspension into each centrifuge tube and add deionized water at a ratio of 1:4 to lyse the red blood cells. Then, take 200 μL into a 96-well plate and measure the absorbance at 540 nm using a microplate reader. The blank control group is red blood cell suspension without CMC / PAA-NHS binder. Other operations are the same as above. Each group is repeated three times. Results are expressed as Mean ± SD. Figure 19 As shown in a. The erythrocyte aggregation rate (RBCs aggregation rate) is calculated according to the following formula (VIII).

[0098] In equation (VIII), A Samples Indicates the absorbance value of the CMC / PAA-NHS group, A Control This represents the absorbance value of the blank control group.

[0099] CMC / PAA-NHS were placed in EP tubes, and 5% hematocrit red blood cell suspension was added to immerse the CMC / PAA-NHS in the red blood cell suspension. The tubes were incubated at 37°C for 30 min, and then washed three times with PBS to remove physically adhering red blood cells from the surface of the CMC / PAA-NHS. Cell fixation solution was then added, and the tubes were fixed at 4°C for 8 h. After fixation, 25%, 50%, 75%, and 100% ethanol solutions were prepared and used to perform gradient elution of the CMC / PAA-NHS to achieve dehydration. The tubes were then dried at room temperature, sputter-coated with gold, and the morphology was observed using a scanning electron microscope. The results are as follows: Figure 19 As shown in b.

[0100] from Figure 19 As shown in Figure a and the analysis, the erythrocyte aggregation rate of CMC / PAA-NHS-1 is 56.8±4.0%, while that of CMC / PAA-NHS-5 is 50.5±0.6%, indicating that CMC / PAA-NHS has the function of aggregating erythrocytes. This proves that CMC / PAA-NHS of the present invention, as a binder, can absorb blood moisture and concentrate blood cells. Furthermore, the erythrocyte aggregation rate of CMC / PAA-NHS-1 is higher than that of CMC / PAA-NHS-5, demonstrating that CMC / PAA-NHS-1 has a stronger liquid absorption capacity than CMC / PAA-NHS-5. The introduction of a cross-linking agent in CMC / PAA-NHS-1 results in a denser three-dimensional network structure, thereby increasing the contact area with erythrocytes. Figure 19 Figure b shows a scanning electron microscope image of erythrocytes adhering to CMC / PAA-NHS. It can be seen that a large number of morphologically intact erythrocytes adhere to the surface of CMC / PAA-NHS, and a few activated erythrocytes are also present, which also verifies that CMC / PAA-NHS has good blood compatibility.

[0101] 14) APTT and PT To investigate the hemostatic mechanism of CMC / PAA-NHS, fresh anticoagulated whole blood from SD rats was centrifuged at 800 rpm for 10 min. The supernatant was platelet-rich plasma (PRP), which was aspirated. The lower layer was then centrifuged at 3000 rpm for 10 min to obtain a clearer supernatant platelet-poor plasma (PPP). 10 mg of CMC / PAA-NHS-1 prepared in Example 1 and CMC / PAA-NHS-5 prepared in Example 5 were mixed with 5 mL of PPP and incubated at 37°C for 30 min. The blank control group did not contain CMC / PAA-NHS particles. The supernatant serum was analyzed using an automated coagulation analyzer (RAC-30) to determine coagulation parameters. The samples were reacted with APTT and PT reagents, with each sample tested in triplicate. Results are expressed as Mean ± SD. Figure 20 As shown.

[0102] from Figure 20 Analysis showed that, compared with the blank control group, the PT value of the CMC / PAA-NHS group was not significantly different, while the APTT value of the CMC / PAA-NHS group was significantly lower, indicating that CMC / PAA-NHS has the ability to activate the intrinsic pathway, thereby shortening the clotting time and achieving rapid hemostasis.

[0103] In summary, the preparation method of the polymer adhesive material of the present invention involves adding sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate to alkali-neutralized acrylic acid. During copolymerization, the acrylic acid retains some -COOH and introduces -COONa. Simultaneously, potassium persulfate (KPS) is added as an initiator to create a three-dimensional network structure. This network carries a suitable amount of negative charge and forms electrostatic-hydrogen bond synergistic entanglement with the -COONa on the carboxymethyl cellulose chains. The N-hydroxysuccinimide acrylate is attached to the gel backbone via double bonds, "suspending" the highly active NHS ester on the surface of the three-dimensional network. Therefore, when this polymer adhesive material is used as a hemostatic material, upon contact with blood or tissue fluid, the NHS ester rapidly forms a covalent amide with the protein amino groups, while the residual carboxylic acid / sodium salt is bridged with tissue polysaccharides and calcium ions through hydrogen bonds, achieving dual adhesion of "chemical anchoring + physical adsorption." Simultaneously, freeze-drying imparts a porous microstructure, allowing for instantaneous absorption of water and concentration of clotting factors. Covalent-non-covalent synergistic effects endow the material with wet interfacial adhesion far exceeding that of traditional hemostatic sponges / powders, significantly shortening hemostasis time and reducing the risk of rebleeding. Furthermore, sodium carboxymethyl cellulose and polyacrylic acid are both biocompatible natural / near-natural polysaccharides and synthetic polymers, avoiding the chronic inflammation or secondary damage problems caused by the poor biocompatibility of traditional hemostatic materials.

[0104] The present invention discloses a method for preparing a novel hemostatic powder, CMC / PAA-NHS, synthesized from sodium carboxymethyl cellulose and polyacrylic acid grafted with N-hydroxysuccinimide ester. The prepared CMC / PAA-NHS absorbs water from the blood, forming a strongly adhesive hydrogel that seals bleeding points, prevents bacterial contamination, and accelerates the hemostasis process. Research results show that CMC / PAA-NHS possesses strong adhesive strength, excellent mechanical strength, and good biocompatibility. In rat models of liver injury bleeding, rat models of complete femoral vein transection bleeding, and rat models of complete femoral artery and vein transection bleeding, CMC / PAA-NHS exhibits excellent hemostatic effects and can be applied to vascular wounds. Furthermore, CMC / PAA-NHS has the potential to activate intrinsic coagulation pathways, promote erythrocyte aggregation, and accelerate blood clot formation. Based on in vivo and in vitro data, CMC / PAA-NHS shows great potential as a hemostatic agent and has significant application value in the field of medical materials technology.

[0105] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A method for preparing a polymer adhesive material, characterized in that, Includes the following steps: S1. Add alkali to acrylic acid to obtain neutralized acrylic acid; S2. Neutralized acrylic acid, sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate are added to an inorganic solvent, then potassium persulfate is added, and the mixture is heated to react and obtain a gel. S3. Freeze-dry the gel to obtain a polymeric adhesive material, namely N-hydroxysuccinimide-modified sodium carboxymethyl cellulose / polyacrylic acid adhesive.

2. The method for preparing the polymer adhesive material according to claim 1, characterized in that, S2 includes: adding neutralized acrylic acid, sodium carboxymethyl cellulose and N-hydroxysuccinimide acrylate to an inorganic solvent, then adding N'N-methylenebisacrylamide and potassium persulfate, heating to react, and obtaining a gel; And / or, the temperature of the heating reaction is 60~70°C.

3. The method for preparing the polymer adhesive material according to claim 1, characterized in that, The alkali is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution, sodium carbonate solution, and potassium carbonate solution.

4. The method for preparing the polymer adhesive material according to claim 1, characterized in that, The degree of neutralization in the neutralized acrylic acid is 65-75%.

5. The method for preparing the polymer adhesive material according to claim 1, characterized in that, The mass percentages of the neutralized acrylic acid, sodium carboxymethyl cellulose, N-hydroxysuccinimide acrylate, N'N-methylenebisacrylamide, and potassium persulfate in the polymer adhesive material are 10-30 wt%, 0.5-3 wt%, 0.5-3 wt%, 0-0.05 wt%, and 0.05-0.5 wt%, respectively.

6. The method for preparing the polymer adhesive material according to claim 1, characterized in that, The inorganic solvent is selected from water; And / or, the freeze-drying is carried out under vacuum conditions, and the freeze-drying temperature is -75℃ to -85℃.

7. The method for preparing the polymer adhesive material according to claim 1, characterized in that, The powder particle size of the polymer adhesive material is 100~500µm.

8. A polymeric adhesive material prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the polymer adhesive material according to claim 8 as a medical material.

10. The application according to claim 9, characterized in that, The polymer adhesive material is used as a hemostatic material.

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