Anti-freezing hemostatic gel material and preparation method thereof

Abstract

The invention relates to the field of biological materials, and provides an anti-freezing hemostatic gel material and a preparation method thereof, and the preparation method comprises the following steps: S1, preparing an EA compound; the preparation method comprises the following steps: carrying out a reaction on epigallocatechin gallate and 3-acrylamide phenylboronic acid to prepare an EA compound; s2, preparing a gel precursor liquid: heating methylacryloylated gelatin and acrylic acid in deionized water until the methylacryloylated gelatin and the acrylic acid are completely dissolved; adding the anti-freezing compound and uniformly mixing; then, acrylic acid N-succinimide ester is added, and the gel precursor liquid is obtained; the anti-freezing compound is prepared by mixing nano cellulose, proline and glycerol; s3, preparing the anti-freezing hemostatic gel material: mixing the EA compound, the gel precursor fluid, acrylic acid, an initiator and an accelerant for reaction to prepare the anti-freezing hemostatic gel material. The anti-freezing hemostatic gel material disclosed by the invention is good in adhesiveness and high in mechanical strength, and can well meet the use requirements of a plateau environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, and in particular, provides an anti-freezing hemostatic gel material and a preparation method thereof. BACKGROUND

[0002] The plateau region, especially the region above 3000 meters above sea level, presents extremely unique and severe natural environmental characteristics, the most notable of which is low pressure hypoxia and extreme cold. In the combined environment of low pressure hypoxia and extreme cold at high altitudes, once injured and bleeding, the situation will become extremely critical. Low temperature will slow down the physiological function of the human body, blood vessels will constrict, and blood viscosity will increase, making the hemostatic process more difficult. Hypoxia will affect the normal metabolism and function of cells, weaken the body's self-repairing ability, and slow down the healing of wounds, greatly increasing the risk of infection. In addition, due to the harsh environment, rescue is extremely difficult, and the injured often have difficulty in receiving timely and effective treatment in a short period of time, which puts extremely high performance requirements on hemostatic materials.

[0003] An ideal hemostatic material for plateau environment application not only needs to have rapid and efficient hemostatic ability to stop bleeding in a short period of time, but also needs to have good anti-freezing performance to maintain stable properties at extremely low temperatures, ensuring that its hemostatic efficacy is not affected, thereby gaining valuable treatment time for the injured in the harsh plateau environment. Existing hemostatic materials include the following: (1) traditional hemostatic materials such as gauze and bandage: using their physical properties, bleeding is stopped by pressing the wound. However, this method is relatively slow, especially in the face of more serious bleeding, it is often difficult to quickly and effectively stop bleeding. (2) Hemostatic gel: mainly water gel hemostatic materials, which are made into hemostatic sponges, hemostatic powders, medical dressings, and injectable gels for application in the hemostatic field. Due to its unique physical and chemical properties, it can quickly absorb blood and swell, thereby achieving hemostatic function. However, water gel hemostatic materials, due to their main component being a high-molecular material rich in water, in a low-temperature environment, the water molecules inside will crystallize, forming ice crystals. The growth of ice crystals will destroy the network structure of the water gel, causing its mechanical properties to drop sharply, from soft and elastic to hard and fragile, greatly reducing its hemostatic performance. In order to solve this problem, researchers have conducted a lot of research and proposed a variety of methods to improve the anti-freezing performance of hemostatic water gel. One of the common methods is to introduce antifreeze agents such as inorganic salts or organic solvents. For example, in some studies, inorganic salts such as sodium chloride, calcium chloride, etc. are added to the hydrogel system, and these inorganic salt ions can interact with water molecules to lower the freezing point of water, thereby inhibiting the formation of ice crystals; organic solvents such as ethylene glycol, propylene glycol, etc. are also often used as antifreeze agents, which can form hydrogen bonds with water, change the arrangement of water molecules, and improve the antifreeze ability of the hydrogel. However, the presence of inorganic salts or organic solvents can reduce the strength of the intermolecular association in the hydrogel, resulting in poor mechanical properties of the gel, which limits its practical application.

[0004] Therefore, it is of great practical significance and application value to develop a high-performance hydrogel hemostatic material that can function stably in the combined environment of high-altitude low pressure hypoxia and extreme cold, in order to improve the level of trauma treatment in plateau areas and protect the lives and health of people in plateau areas. SUMMARY In view of the above deficiencies in the prior art, the core purpose of the present application is to provide an antifreeze hemostatic gel material and a preparation method thereof, which can prepare a hemostatic gel with good antifreeze performance, and the hemostatic gel has good adhesion and high mechanical strength, and can well meet the use requirements in high-altitude environments.

[0005] The present application is realized by the following technical solutions: A preparation method of an antifreeze hemostatic gel material, comprising the following steps: S1 preparing an EA complex; the EA complex is prepared by reacting epigallocatechin gallate (EGCG) and 3-acrylamidophenylboronic acid (APBA); S2 preparing a gel precursor solution: taking methacrylated gelatin (GelMA) and acrylic acid (AAc) in deionized water and heating to complete dissolution; adding an antifreeze complex and mixing uniformly; then adding acrylic acid N-succinimidyl ester (AAc-NHS) to obtain the gel precursor solution; The antifreeze complex is prepared by mixing nanocellulose, proline and glycerol; S3 preparing an antifreeze hemostatic gel material: mixing and reacting the EA complex, the gel precursor solution, acrylic acid, an initiator and a promoter to prepare the antifreeze hemostatic gel material.

[0006] In order to adapt to the conditions of extreme cold and low pressure hypoxia in high-altitude environments, the present application mainly realizes the application of hemostatic gel materials in such environments from two aspects.

[0007] In one aspect, the present application introduces an anti-freezing complex, which comprises nanocellulose, proline and glycerol; the addition of glycerol can replace part of the water solvent as a co-solvent, and its hydroxyl group can combine with water molecules to make more water exist in the form of bound water, thereby significantly reducing the freezing point and inhibiting the formation of ice crystals; the proline molecule has an amphiphilic structure of hydrophobic ring-hydrophilic carboxyl group, which can be adsorbed on the surface of ice crystals through hydrophobic interaction at low temperature, thereby preventing the further growth of ice crystals and reducing the physical damage of ice crystals to the gel network; the surface of nanocellulose is rich in hydroxyl groups, which can combine with free water in the hydrogel through hydrogen bonds to convert it into bound water that is not easy to freeze, thereby reducing the formation of ice crystals at low temperature. The three components play an anti-freezing role through different mechanisms, realizing the control of the freezing behavior of the hydrogel in extremely cold environments at multiple levels, such as preventing the generation of ice crystals, reducing the freezing point, and preventing the further growth of ice crystals.

[0008] On the other hand, the conventional anti-freezing component glycerol has a weak inhibitory effect on the formation of ice crystals when added in a small amount, and when added in a large amount, its small molecules will penetrate into the polymer network, causing the mechanical properties and adhesion properties of the entire gel network to decrease. Therefore, while ensuring the anti-freezing performance, the present application also ensures that the mechanical properties are not affected through the following measures: (1) the present application reduces the amount of glycerol added and adds nanocellulose and proline to realize anti-freezing through multiple actions and combinations, thereby reducing the decrease in mechanical properties caused by the excessive addition of glycerol; (2) the other anti-freezing components nanocellulose and proline added by the present application can not only play an anti-freezing role, but also enhance the mechanical properties; the hydroxyl groups on the surface of CNC form dense hydrogen bonds with the amino groups and hydroxyl groups on the molecular chains of GelMA, thereby enhancing the rigidity of the GelMA three-dimensional network and anchoring the GelMA segments through hydrogen bonds to reduce the network collapse caused by the contraction of molecular chains at low temperature; the pyrrolidine ring of proline can be embedded in the gap between the segments to prevent excessive contraction and crystallization of the segments at low temperature through hydrophobic interaction. That is, the addition of nanocellulose and proline to some extent resists the excessive plasticization of the entire polymer network caused by the addition of glycerol. (3) the present application adds an EA complex to the system; the EA complex is a functional complex formed by the combination of EGCG and APBA through a dynamic covalent boronic ester bond; the dynamic reversibility of the boronic ester bond enables the complex to dissociate and recombine when the environment changes, thereby resisting the changes in the polymer network in extremely cold environments and ensuring good mechanical properties of the complex when in use.

[0009] In summary, the present application realizes the dual functions of anti-freezing and mechanical properties by designing the components of the anti-freezing complex and the polymerization process and network composition of the entire GelMA hydrogel, thereby ensuring its application in high-altitude extremely cold environments.

[0010] Preferably, in step S1, the molar ratio of epigallocatechin gallate and 3-acrylamidobenzenesulfonic acid in the EA complex is 1:1.5-2.5.

[0011] Preferably, in step S2, the anti-freezing complex is prepared by uniformly dispersing nanocellulose in deionized water, and then adding proline and glycerol to obtain the anti-freezing complex; the weight ratio of nanocellulose, proline, glycerol and deionized water in the anti-freezing complex is 1:2-2.5:2-3:3-5.

[0012] The nanocellulose, preferably nanocrystalline cellulose, is dispersed in deionized water, and then proline and glycerol are added to obtain an anti-freezing complex with good dispersing performance. Preferably, in this step, the amount of deionized water is 3-4 times the amount of nanocellulose by weight to ensure sufficient dispersion of nanocellulose.

[0013] Preferably, in step S2, the amount of the anti-freezing complex is 15%-20% of the amount of the methacrylated gelatin by weight.

[0014] Preferably, the ratio of the amount of methacrylated gelatin to the amount of acrylic acid (w / v) is 1 mg / (30-40 μL).

[0015] Preferably, the amount of N-succinimidyl acrylate is 1.8-2.2 times the amount of methacrylated gelatin by weight.

[0016] Preferably, the initiator is ammonium persulfate, and the promoter is tetramethyl ethylenediamine.

[0017] Preferably, in step S3, the volume of each component is 5-10 parts of the gel precursor solution, 1-2 parts of acrylic acid, 1-2 parts of the EA complex solution, 0.7-1.5 parts of the ammonium persulfate solution, and 0.12-0.15 parts of tetramethyl ethylenediamine. The mass / volume concentration of the EA complex solution is 15-20 % w / v. The mass / volume concentration of the ammonium persulfate solution is 50 % w / v.

[0018] Preferably, in step S3, the EA complex, the gel precursor solution, acrylic acid, the initiator and the promoter are mixed and heated at 60-70°C for 1-3 hours to prepare the anti-freezing hemostatic gel material.

[0019] The application also provides an anti-freezing hemostatic gel material prepared by the above preparation method.

[0020] The present application has at least the following advantages and beneficial effects: (1) The present application realizes the multi-level anti-freezing effect by designing the composition of the anti-freezing compound, and the various anti-freezing components interact with each other to maintain good mechanical properties while anti-freezing, thereby ensuring the use of the gel product in the high-altitude extremely cold environment.

[0021] (2) The present application realizes the destruction and recombination of the polymer network in the extremely cold environment by adding the EA compound with dynamic cross-linking bonds, thereby realizing the structural stability and mechanical property stability of the entire gel material.

[0022] (3) The proline and EGCG in the EA compound of the present application both have antioxidant properties, and the antioxidant functions of the two are superimposed, which can reduce the oxidative damage of wound cells caused by low temperature and hypoxia, and the proline stabilizes the phenolic hydroxyl structure of EGCG through hydrogen bonds, enhances its antioxidant durability, so that the prepared hydrogel has good antibacterial and anti-inflammatory properties.

[0023] (4) The APBA, GelMA, AAc, AAC-NHs, EGCG, proline, CNC and glycerol in the present application participate in the cross-linking reaction together and exist in the cross-linked network, and various components interact with each other, such as the formation of borate ester bonds between free APBA and the ortho-dihydroxy group of GelMA to enhance the hydrogel network; the negatively charged phenolic hydroxyl group of EGCG can be electrostatically attracted to the amino group of GelMA to further stabilize the structure; the polyphenolic hydroxyl group in EGCG can form hydrogen bonds with the carboxyl group of AAc to increase the cross-linking density and improve the toughness of the hydrogel, etc., thereby realizing the mechanical property stability of the entire gel product in the extremely cold environment. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a schematic diagram of the preparation process and injectability of the GelMA-EA hydrogel of Example 1, Figure 1 (a) is the gelation process of the GelMA-EA hydrogel, Figure 1 (b) is a hydrogel injectable demonstration diagram.

[0025] Figure 2 Figure 2 is a rheological property evaluation diagram of the GelMA-EA hydrogel in Example 1, Figure 2 (a) is the frequency scanning result, Figure 2 (b) is the flow curve.

[0026] Figure 3 Figure 3 is a state diagram of the samples of Example 1-Example 5 in Example 2 after being frozen at-15℃; Figure 4 Figure 4 is a state diagram of the samples of Comparative Example 1-Comparative Example 5 in Example 2 after being frozen at-15℃; Figure 5 State diagram of the sample of Example 1-Example 5 in Experimental Example 2 after freezing at -40℃; Figure 6 State diagram of the sample of Comparative Example 1-Comparative Example 5 in Experimental Example 2 after freezing at -40℃; Figure 7 Mechanical performance diagram of the sample of Example 1 in Experimental Example 3 after freezing at -40℃; Figure 8 Hemostatic effect of the mouse model in Experimental Example 4, Figure 8 (a) is a liver incision model; Figure 8 (b) is a common carotid artery injury model; Figure 8 (c) and Figure 8 (d) is an abdominal aortic injury model.

[0027] Figure 9 Hemostatic effect of the pig model in Experimental Example 5, Figure 9 (a) is a liver incision model; Figure 9 (b) and Figure 9 (c) is a common carotid artery injury model. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the present application will be further described below in combination with specific examples. If no specific conditions are indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If no manufacturer is indicated for the reagents or materials used, they are all conventional products that can be purchased on the market. All the features disclosed in the specification can be combined in any manner, except for the features or / and steps that are mutually exclusive.

[0029] The following examples can make the professional technical personnel more fully understand the present application, but do not limit the present application in any way.

[0030] Example 1 The present embodiment provides a preparation method of an anti-freezing hemostatic gel material, comprising the following steps: S1 Preparation of EA complex; weigh 92 mg of epigallocatechin gallate (EGCG) in 500 μL of DMSO and completely dissolve; another 76 mg of 3-aminophenylboronic acid (APBA) is completely dissolved in 500 μL of DMSO. Mix the prepared EGCG solution and APBA solution 1:1, stand for 15 min to obtain the EA complex solution.

[0031] S2 Preparation of gel precursor solution: Take 15 mg of methacrylated gelatin (GelMA), add 3 mg of antifreeze complex, ultrasonic dispersion for 0.5 h in 500 μL of acrylic acid (AAc) and 3 mL of deionized water (RO), heat in a 50°C water bath for 30 min to completely dissolve, then add 30 mg of acrylic acid N-succinimidyl ester (AAc-NHS).

[0032] The weight ratio of nanocellulose, proline, glycerol and deionized water in the antifreeze complex satisfies: 1:2:2:4. The preparation method of the antifreeze complex is: take 1 mg of nanocellulose and ultrasonic dispersion in 4 mL of deionized water for 1 h; then add 2 mg of proline and 2 mg of glycerol and continue to mix for 0.5 h to obtain the antifreeze complex.

[0033] S3 Preparation of antifreeze hemostatic gel material: take 500 μL of gel precursor solution, 100 μL of AAc, 150 μL of EA complex solution, 70 μL of ammonium persulfate solution APS (mother liquor 50% w / v), 18.75 μL of tetramethyl ethylenediamine (TEME) to prepare a hydrogel gelation system, heat at 60°C for 2 h to obtain the GelMA-EA hydrogel of the embodiment. Appendix Figure 1 The preparation of the GelMA-EA hydrogel of the embodiment is demonstrated, wherein (a) is the gelation process of the GelMA-EA hydrogel, and (b) is a demonstration diagram of the injectability of the hydrogel.

[0034] Example 2 The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in step S2, the addition amount of the antifreeze complex is 2.25 mg.

[0035] Example 3 The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in step S2, the weight ratio of nanocellulose, proline, glycerol and deionized water in the antifreeze complex satisfies: 1:2.5:3:3.

[0036] Example 4 The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in step S3, the addition amount of the EA complex solution is 200 μL.

[0037] Example 5 The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in step S3, the addition amount of the EA complex solution is 120 μL.

[0038] Comparative Example 1 This comparative example provides a method for preparing an anti-freeze hemostatic gel material, which is different from Example 1 in that no anti-freeze complex is added in step S2.

[0039] Comparative Example 2 This comparative example provides a method for preparing an anti-freeze hemostatic gel material, which is different from Example 1 in that the anti-freeze complex in step S2 does not contain nanocellulose.

[0040] Comparative Example 3 This comparative example provides a method for preparing an anti-freeze hemostatic gel material, which is different from Example 1 in that the anti-freeze complex in step S2 does not contain proline.

[0041] Comparative Example 4 This comparative example provides a method for preparing an anti-freeze hemostatic gel material, which is different from Example 1 in that the anti-freeze complex in step S2 does not contain nanocellulose and proline.

[0042] Comparative Example 5 This comparative example provides a method for preparing an anti-freeze hemostatic gel material, which is different from Example 1 in that the amount of EA complex solution added in step S3 is 30 μL.

[0043] Experimental Example 1 Rheological evaluation of GelMA-EA hydrogel prepared in Example 1 Rheology is a basic evaluation for evaluating the sol-gel conversion of hydrogel. The rheological properties of the product prepared in Example 1 were evaluated, and a multifunctional rotary rheometer was used to characterize the rheology of the hydrogel. The test temperature was 25 °C, the GelMA-EA hydrogel was placed on the parallel plate, the gap between the parallel plates was adjusted to 1 mm, and the storage modulus (G') and loss modulus (G") were investigated. The frequency scanning test was used to detect the GelMA-EA hydrogel at 25 °C, and the angular frequency was set to 100 rad s – 1 to 0.01 rad s – 1 The viscoelastic modulus of the hydrogel at different frequencies was determined in the strain of 1 % mode. The flow scanning test was used to detect the shear thinning property of the GelMA-EA hydrogel, and the shear rate range was set to 1 s - ¹to 100 s - ¹and the rheological properties of the hydrogel were determined at 25 °C in the logarithmic scanning mode.

[0044] The results are shown in Figure 2 Figures Figure 2 (a) frequency scanning results, the storage modulus (G') of the GelMA-EA hydrogel at 10 2 ~ 10 2The value consistently exceeds the loss modulus (G') within the rad / s frequency range, confirming elastic-dominated solid-like behavior. The G' value, ranging from 0.01 to 100 MPa, indicates significant elasticity in the hydrogel, likely supported by a highly cross-linked network structure or strong intermolecular forces (such as hydrogen bonds and hydrophobic interactions). Meanwhile, Figure 2 The flow curve results in (b) indicate that its apparent viscosity (η) ranges from 1000 Pa·s (γ˙ = 1 s⁻¹). 1 The concentration decreased significantly to 0.1 Pa·s (γ˙ = 1000 s). 1 The material exhibits typical shear-thinning behavior. This characteristic indicates that the material's fluidity is enhanced under high shear conditions (such as through an injection needle), while it can maintain its gel structure under static conditions (low shear rate), meeting the design requirements for injectable hydrogels.

[0045] Experiment Example 2 Antifreeze properties of hydrogels from Examples 1-5 and Comparative Examples 1-5 1. Place two samples of each hydrogel group in a freezer at -15°C for 24 hours. Then, remove the samples from the freezer and observe their condition.

[0046] The results are attached. Figure 3 and attached Figure 4 As shown, Figure 3 The samples in Examples 1 through 5 are labeled A, E, and E respectively. Figure 4 The samples in the middle (e) correspond to comparative examples 1 through 5, respectively. (See attached...) Figure 3 and attached Figure 4 It can be seen that the samples of Examples 1-5 and Comparative Examples 1-5 did not undergo significant changes in state after being frozen at -15℃ for 24 hours, and all maintained good antifreeze properties.

[0047] 2. Place one sample of each hydrogel group in a -40°C freezer for 24 hours. Then, remove the samples from the freezer and observe their condition.

[0048] The results are attached. Figure 5 and attached Figure 6 As shown, Figure 5 The samples in Examples 1 through 5 are labeled A, E, and E respectively. Figure 6 The samples in the middle (e) correspond to comparative examples 1 through 5, respectively. (See attached...) Figure 5It can be seen that the samples of Example 1-Example 5 are still not frozen at an extremely low temperature of-40℃, and have good anti-freezing effect; while the samples of Comparative Example 1-Comparative Example 4 all present obvious freezing state, proving that the anti-freezing effect is poor without introducing the anti-freezing complex or a single anti-freezing component; although Comparative Example 5 introduces the anti-freezing complex, the content of phenolic hydroxyl groups in the whole hydrogel is low due to the introduction of a small amount of EA complex with dynamic cross-linking bond, and the sample is still frozen at an extremely low temperature of-40℃, resulting in poor mechanical properties.

[0049] Experimental Example 3 Anti-freezing performance of GelMA-EA hydrogel prepared in Example 1 The hydrogel of Example 1 was placed in a low-temperature refrigerator at-40℃ for 24 h, and then the sample was taken out from the refrigerator to test the tensile effect and tissue adhesion.

[0050] Results are shown in the following table: Figure 7 Figure 7 It can be seen that the GelMA-EA hydrogel of Example 1 still has good flexibility at-40℃, can withstand torsion, stretching( Figure 7 a-c) or compression( Figure 7 d-f) and other deformations, and has high elasticity, and can quickly recover after deformation. This shows that the interaction between various groups in the GelMA-EA hydrogel network increases the mechanical properties, and the introduction of the EA complex with dynamic cross-linking bond further enhances the anti-freezing performance and maintains good mechanical properties under frozen conditions.

[0051] Experimental Example 4 Hemostatic effect of GelMA-EA hydrogel prepared in Example 1 in different vascular injury models of rats ​Test method: Liver hemostasis: The hemostatic effect of GelMA-EA hydrogel was evaluated using a rat (male, 250 ~ 300 g, 7 ~ 8 weeks) liver hemorrhage model. The rat was anesthetized, the rat liver was exposed through an abdominal incision, and pre-weighed filter paper was placed under the liver. After cutting a piece of liver with a 6 mm circular incision, the hydrogel hemostatic material was covered on the wound to stop bleeding, and the hemostatic time was recorded. Carotid artery hemostasis: The rat was anesthetized by isoflurane inhalation, a longitudinal incision was made on the front of the neck to expose and isolate the right common carotid artery. A 1 mL syringe needle was used to pierce the artery wall to create a penetrating injury model. After pulling out the needle, blood gushed out. After 5 seconds of free bleeding, the blood around the wound was gently absorbed with sterile gauze. About 100 μL of GelMA-EA hydrogel was quickly injected to cover the bleeding point, and whether hemostasis was observed was timed. Abdominal aorta: After the rat was deeply anesthetized, an incision was made along the midline of the abdomen, the abdominal cavity was opened, and the abdominal aorta behind the peritoneum was exposed. The proximal and distal ends of the segment of the blood vessel were temporarily blocked with a blood vessel clamp. Then, a puncture needle was used to make a break in the anterior wall of the artery. After removing the blood vessel clamp, the wound was cleaned with gauze after 5 seconds of free bleeding, about 100-150 μL of gel solution was precisely coated on the break to block it, and hand pressure was applied for a period of time (such as 1-2 minutes) and then slowly released, and whether hemostasis was observed.

[0052] The experimental results are shown in the accompanying Figure 8 figures, which show that in the liver incision model ( Figure 8 a), the hydrogel achieves effective hemostasis within 40 seconds after injection, demonstrating its rapid sealing ability for low pressure tissues. The hemostatic time of the common carotid artery injury model ( Figure 8 b) is 50 seconds, indicating that the material has certain adaptability to medium blood flow pressure environment. It is worth noting that the hemostatic time of the abdominal aorta ( Figure 8 c-d) is 40 seconds, which may be related to the small diameter of the blood vessels and the hemodynamic characteristics of the rat model. This result suggests that GelMA-EA hydrogel exhibits excellent immediate sealing performance in rodents.

[0053] Experimental Example 5 Hemostatic effect of GelMA-EA hydrogel prepared in Example 1 in domestic pigs Test method: healthy domestic pigs were selected for the experiment, weighing 40-50 kg. Intravenous propofol was used for anesthesia. The animals were placed in a supine position, and the abdominal surgical area was prepared for skin disinfection. The liver was exposed through an abdominal incision. A 15 mm circular incision was used to remove a piece of liver, creating a liver bleeding model. After 2-3 seconds of natural bleeding, the surface blood was absorbed with sterile gauze. About 100 μL of GelMA-EA hydrogel was quickly injected to cover the bleeding point, and the hemostasis time was observed. The animal position was the same as above, and a longitudinal incision was made in the medial margin of the sternocleidomastoid muscle on one side of the neck. The common carotid artery was carefully separated. First, the proximal and distal blood flow was temporarily blocked with artery clamps. Then, a transverse incision was made on the anterior wall of the isolated artery segment using ophthalmic scissors, and the proximal and distal artery clamps were removed in turn to restore blood flow. After 5 seconds of observation, the GelMA-EA hydrogel hemostatic material to be tested was immediately applied to the bleeding wound surface.

[0054] Figure 9 The injectable GelMA-EA hydrogel showed hemostatic effect in the liver injury and different blood vessel injury models of domestic pigs. Among them, the liver wound ( Figure 9 a) achieved effective hemostasis within 46 seconds after applying the hydrogel, indicating the material's rapid sealing ability for low pressure tissues. In the common carotid artery injury model ( Figure 9 b-c), the hydrogel showed adaptability to the medium blood flow pressure environment, with a hemostatic time of 29 seconds, and its rapid cross-linking property may have facilitated the immediate sealing of the blood vessel break. This result suggests that the GelMA-EA hydrogel exhibits excellent immediate sealing performance in rats and domestic pigs.

[0055] In summary, the anti-freeze hemostatic hydrogel material of the present application has excellent anti-freeze performance, mechanical properties and immediate hemostatic performance, and has good application effect in the high-altitude extremely cold environment.

[0056] The present application is not limited to the above-mentioned embodiments, and any person should know that structural changes made under the inspiration of the present application fall within the protection scope of the present application. Any technical solution with the same or similar technical solutions as the present application falls within the protection scope of the present application.

Claims

1. A method of preparing an anti-freeze hemostatic gel material, characterized by, It comprises the following steps: S1: preparing an EA complex; the EA complex is prepared by the reaction of epigallocatechin gallate and 3-acrylamide phenylboronic acid; S2: preparing a gel precursor solution; heating methacrylated gelatin and acrylic acid in deionized water until completely dissolved; adding an anti-freezing complex and mixing uniformly; then adding acrylic acid N-succinimidyl ester to obtain the gel precursor solution; The anti-freezing complex is prepared by mixing nanocellulose, proline and glycerol; S3: preparing an anti-freezing hemostatic gel material; mixing and reacting the EA complex, the gel precursor solution, acrylic acid, an initiator and a promoter to prepare the anti-freezing hemostatic gel material.

2. The method of claim 1, wherein the anti-freeze hemostatic gel material is prepared by the steps of: In step S1, the molar ratio of epigallocatechin gallate to 3-acrylamide phenylboronic acid in the EA complex satisfies 1:1.5-2.

5.

3. The method of claim 1, wherein the anti-freeze hemostatic gel material is prepared by the steps of: In step S2, the anti-freezing complex is prepared by uniformly dispersing nanocellulose in deionized water; then adding proline and glycerol and continuing to mix to obtain the anti-freezing complex; in the anti-freezing complex, the weight ratio of nanocellulose, proline, glycerol and deionized water satisfies 1:2-2.5:2-3:3-5.

4. The method for preparing the antifreeze hemostatic gel material according to claim 3, characterized in that, In step S2, the amount of the anti-freezing complex is 15%-20% of the methacrylated gelatin by weight.

5. The method of claim 4, wherein the anti-freeze hemostatic gel material is prepared by, The amount ratio (w / v) of the methacrylated gelatin to the acrylic acid satisfies 1 mg / (30-40 μL).

6. The method of claim 4, wherein the anti-freeze hemostatic gel material is prepared by the steps of: The amount of the acrylic acid N-succinimidyl ester is 1.8-2.2 times of the methacrylated gelatin by weight.

7. The method of claim 1-6, wherein the anti-freeze hemostatic gel material is prepared by the steps of: The initiator is ammonium persulfate and the promoter is tetramethyl ethylenediamine.

8. The method of claim 7, wherein the anti-freeze hemostatic gel material is prepared by, In step S3, the volume amount of each component satisfies: gel precursor solution 5-10 parts; acrylic acid 1-2 parts; EA complex solution 1.0-2.0 parts; ammonium persulfate solution 0.7-1.5 parts; tetramethyl ethylenediamine 0.12-0.15 parts. The mass / volume concentration of the EA complex solution is 15-20% w / v. The mass / volume concentration of the ammonium persulfate solution is 50% w / v. In step S3, after mixing the EA complex, the gel precursor solution, acrylic acid, an initiator and a promoter, heating at 60-70°C for 1-3 h to prepare the anti-freezing hemostatic gel material.

10. An anti-freezing hemostatic gel material prepared by the preparation method of the anti-freezing hemostatic gel material according to any one of claims 1-9.

9. The method of claim 1, wherein the anti-freeze hemostatic gel material is prepared by the steps of: ​ ​

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