An antifreeze hemostatic gel material and a method of making the same

Abstract

The application relates to the field of biological materials, and provides an anti-freezing hemostatic gel material and a preparation method thereof, which comprises the following steps: S1, preparing an EA complex; the EA complex is prepared by reacting epigallocatechin gallate and 3-acrylamide phenylboronic acid; S2, preparing a gel precursor solution: taking methylacrylated gelatin and acrylic acid in deionized water and heating to completely dissolve; adding an anti-freezing complex and uniformly mixing; subsequently, 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 an accelerator to prepare the anti-freezing hemostatic gel material. The anti-freezing hemostatic gel material has good adhesion and high mechanical strength, and can well adapt to the use requirements in 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 wounded 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 wounded in the harsh plateau environment.

[0004] 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 in hemostasis, 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 material, which is made into hemostatic sponge, hemostatic powder, medical dressing, injectable gel, etc. and applied to the field of hemostasis. Due to its unique physical and chemical properties, it can quickly absorb blood and swell, thereby achieving hemostatic function.

[0005] However, water gel hemostatic material, due to its main component being a high-molecular material rich in water, in a low-temperature environment, the water molecules inside will crystallize to form 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.

[0006] 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 reduces the strength of the intermolecular association in the hydrogel, resulting in poor mechanical properties of the gel, which limits its application in practice.

[0007] 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

[0008] 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 adapt to the use requirements in plateau environment.

[0009] The present application is realized by the following technical solutions:

[0010] A preparation method of an antifreeze hemostatic gel material, comprising the following steps:

[0011] S1, preparing an EA complex; the EA complex is prepared by reacting epigallocatechin gallate (EGCG) and 3-acrylamidophenylboronic acid (APBA);

[0012] S2, preparing a gel precursor solution: methylacrylated gelatin (GelMA) and acrylic acid (AAc) are heated to complete dissolution in deionized water; an antifreeze complex is added and mixed uniformly; then N-succinimidyl acrylate (AAc-NHS) is added, to obtain the gel precursor solution;

[0013] The antifreeze complex is prepared by mixing nanocellulose, proline and glycerol;

[0014] S3, preparing an antifreeze hemostatic gel material: the EA complex, the gel precursor solution, acrylic acid, an initiator and a promoter are mixed and reacted to prepare the antifreeze hemostatic gel material.

[0015] In order to adapt to the conditions of extreme cold and low pressure hypoxia in plateau environment, the application mainly realizes the application of hemostatic gel material in this environment from two aspects.

[0016] 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.

[0017] 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 the anti-freezing effect 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 that the system has good mechanical properties when in use.

[0018] 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.

[0019] Preferably, in step S1, the molar ratio of epigallocatechin gallate and 3-acrylamidobenzenesulfonic acid in the EA complex is 1:1.5-2.5.

[0020] 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.

[0021] 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.

[0022] Preferably, in step S2, the amount of the anti-freezing complex is 15%-20% of the amount of the methacrylated gelatin by weight.

[0023] Preferably, the ratio of the amount of methacrylated gelatin to the amount of acrylic acid (w / v) is 1 mg / (30-40 μL).

[0024] Preferably, the amount of N-succinimidyl acrylate is 1.8-2.2 times the amount of methacrylated gelatin by weight.

[0025] Preferably, the initiator is ammonium persulfate, and the promoter is tetramethyl ethylenediamine.

[0026] 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.

[0027] The mass / volume concentration of the EA complex solution is 15-20% w / v.

[0028] The mass / volume concentration of the ammonium persulfate solution is 50% w / v.

[0029] 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.

[0030] The application also provides an anti-freezing hemostatic gel material prepared by the above preparation method.

[0031] The present application has at least the following advantages and beneficial effects:

[0032] (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, and ultimately ensure the use of the gel product in the high-altitude extremely cold environment.

[0033] (2) The present application realizes the destruction and reorganization of the polymer network in the extremely cold environment by adding the EA compound with dynamic cross-linking bonds, and realizes the structural stability and mechanical property stability of the entire gel material.

[0034] (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 at the same time, 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.

[0035] (4) The APBA, GelMA, AAc, AAC-NHs, EGCG, proline, CNC, glycerol of 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 and the carboxyl group of AAc can form hydrogen bonds to increase the cross-linking density and improve the toughness of the hydrogel, etc., ultimately realizing the mechanical property stability of the entire gel product in the extremely cold environment. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is the preparation process and injectability diagram 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.

[0037] Figure 2 is the rheological property evaluation diagram of the GelMA-EA hydrogel in Experimental Example 1, Figure 2 (a) is the frequency scanning result, Figure 2 (b) is the flow curve.

[0038] Figure 3 is the state diagram of the samples of Example 1-Example 5 in Experimental Example 2 after being frozen at-15℃;

[0039] Figure 4State diagram of the sample of Comparative Example 1-Comparative Example 5 in Experimental Example 2 after being frozen at -15℃;

[0040] Figure 5 State diagram of the sample of Experimental Example 1-Experimental Example 5 in Experimental Example 2 after being frozen at -40℃;

[0041] Figure 6 State diagram of the sample of Comparative Example 1-Comparative Example 5 in Experimental Example 2 after being frozen at -40℃;

[0042] Figure 7 Mechanical performance diagram of the sample of Experimental Example 1 in Experimental Example 3 after being frozen at -40℃;

[0043] 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.

[0044] 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

[0045] 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. In the examples, if no specific conditions are indicated, the conventional conditions or the conditions recommended by the manufacturers are used. If no manufacturer is indicated for the reagents or materials used, they are all conventional products that can be purchased in the market. All the features disclosed in the present specification can be combined in any manner, except for the features or / and steps that are mutually exclusive.

[0046] The following examples can make the present skilled person more fully understand the present application, but do not limit the present application in any way.

[0047] Example 1

[0048] The present embodiment provides a preparation method of an anti-freezing hemostatic gel material, comprising the following steps:

[0049] S1 Preparation of EA complex; 92 mg of epigallocatechin gallate (EGCG) was weighed and completely dissolved in 500 μL of DMSO; 76 mg of 3-aminophenylboronic acid (APBA) was weighed and completely dissolved in 500 μL of DMSO. The prepared EGCG solution and APBA solution were mixed at a ratio of 1:1, and the EA complex solution was obtained after standing for 15 min.

[0050] S2 Preparation of gel precursor solution: 15 mg of methacrylated gelatin (GelMA) was added to 3 mg of antifreeze complex and ultrasonically dispersed for 0.5 h in 500 μL of acrylic acid (AAc) and 3 mL of deionized water (RO), and then 30 mg of acrylic acid N-succinimidyl ester (AAc-NHS) was added after heating in a water bath at 50°C for 30 min until completely dissolved.

[0051] In the antifreeze complex, the weight ratio of nanocellulose, proline, glycerol and deionized water satisfies: 1:2:2:4. The preparation method of the antifreeze complex is as follows: 1 mg of nanocellulose is ultrasonically dispersed in 4 mL of deionized water for 1 h; then 2 mg of proline and 2 mg of glycerol are added and mixed for 0.5 h to obtain the antifreeze complex.

[0052] S3 Preparation of antifreeze hemostatic gel material: 500 μL of gel precursor solution, 100 μL of AAc, 150 μL of EA complex solution, 70 μL of ammonium persulfate solution APS (stock solution 50% w / v), and 18.75 μL of tetramethyl ethylenediamine (TEME) were prepared to form a hydrogel gelation system, and the GelMA-EA hydrogel of the embodiment was obtained after heating at 60°C for 2 h. Figure 1 The preparation of the GelMA-EA hydrogel of the embodiment is shown, wherein (a) is the gelation process of the GelMA-EA hydrogel, and (b) is a demonstration diagram of the injectability of the hydrogel.

[0053] Example 2

[0054] The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in the step S2, the addition amount of the antifreeze complex is 2.25 mg.

[0055] Example 3

[0056] The embodiment provides a preparation method of an antifreeze hemostatic gel material, which is different from the embodiment 1 in that in the step S2, the weight ratio of nanocellulose, proline, glycerol and deionized water in the antifreeze complex satisfies: 1:2.5:3:3.

[0057] Example 4

[0058] The embodiment provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that in step S3, the adding amount of the EA complex solution is 200 μL.

[0059] Example 5

[0060] The embodiment provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that in step S3, the adding amount of the EA complex solution is 120 μL.

[0061] Comparative example 1

[0062] The comparative example provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that no anti-freezing complex is added in step S2.

[0063] Comparative example 2

[0064] The comparative example provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that no nanocellulose is contained in the anti-freezing complex in step S2.

[0065] Comparative example 3

[0066] The comparative example provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that no proline is contained in the anti-freezing complex in step S2.

[0067] Comparative example 4

[0068] The comparative example provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that no nanocellulose and proline are contained in the anti-freezing complex in step S2.

[0069] Comparative example 5

[0070] The comparative example provides a preparation method of an anti-freezing hemostatic gel material, which is different from the embodiment 1 in that the adding amount of the EA complex solution is 30 μL in step S3.

[0071] Experimental example 1

[0072] Rheological evaluation of the GelMA-EA hydrogel prepared in the embodiment 1

[0073] Rheology is a basic evaluation of sol-gel conversion of hydrogel. The rheological properties of the product prepared in Example 1 were evaluated, and the multifunctional rotary rheometer was used to characterize the rheology of the hydrogel. The test temperature was 25℃, 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 change of the viscoelastic modulus of GelMA-EA hydrogel at different frequencies at 25℃, and the angular frequency was set to 100 rad s – 1 to 0.01 rad s – 1 The change of 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 GelMA-EA hydrogel, and the shear rate range was set to 1 s - to 100 s - and the rheological properties of the hydrogel were determined at 25℃ in the logarithmic scanning mode.

[0074] The results are shown in Figure 2 , see Figure 2 (a) Frequency scanning results, the storage modulus (G') of GelMA-EA hydrogel is always higher than the loss modulus (G") in the frequency range of 10 2 ~ 10 2 rad / s, which confirms the elastic dominant solid-like behavior. The G' value is in the range of 0.01 ~ 100 MPa, indicating that the hydrogel has significant elasticity, which may be supported by a highly cross-linked network structure or strong intermolecular forces (such as hydrogen bonds, hydrophobic interaction). At the same time, Figure 2 (b) Flow curve results show that the apparent viscosity (η) of the hydrogel decreases significantly from 1000 Pa·s (γ˙=1 s 1 ) to 0.1 Pa·s (γ˙=1000 s 1 ), showing a typical shear thinning behavior. This property indicates that the material has enhanced flowability under high shear conditions (such as through a syringe needle), while maintaining the gel structure in a static state (low shear rate), which meets the design requirements of injectable hydrogel.

[0075] Experimental Example 2

[0076] Anti-freezing performance of hydrogels of Example 1-Example 5 and Comparative Example 1-Comparative Example 5

[0077] 1. Each group of hydrogel samples was placed in a low-temperature refrigerator at -15℃ for 24h. Then, the state of the sample was observed after the sample was taken out of the refrigerator.

[0078] The results are shown in Figure 3and the accompanying drawings Figure 4 as shown in the accompanying drawings, Figure 3 In Table A, A-E respectively correspond to the samples of Example 1-Example 5. Figure 4 In Table a, a-e respectively correspond to the samples of Comparative Example 1-Comparative Example 5. The accompanying drawings show Figure 3 and the accompanying drawings Figure 4 It can be seen that the samples of Example 1-5 and Comparative Example 1-5 are frozen at a low temperature of-15℃ for 24h, and the state does not change significantly, and can maintain good anti-freezing performance.

[0079] 2. Each group of hydrogel samples is placed in a low-temperature refrigerator at-40℃ for 24h, and then the state of the samples is observed after the samples are taken out of the refrigerator.

[0080] The results are shown in the accompanying drawings Figure 5 and the accompanying drawings Figure 6 as shown in the accompanying drawings, Figure 5 In Table A, A-E respectively correspond to the samples of Example 1-Example 5. Figure 6 In Table a, a-e respectively correspond to the samples of Comparative Example 1-Comparative Example 5. The accompanying drawings show Figure 5 It can be seen that the samples of Example 1-Example 5 are frozen at an extremely low temperature of-40℃, and still do not freeze, maintaining a relatively good anti-freezing effect; the samples of Comparative Example 1-Comparative Example 4 all show a significant freezing state, proving that the anti-freezing effect is poor without the introduction of an anti-freezing compound or a single anti-freezing component; although Comparative Example 5 introduces an anti-freezing compound, because it introduces a small amount of EA compound with a dynamic crosslinking bond, the content of phenolic hydroxyl groups in the entire hydrogel is low, and it still freezes a small amount at an extremely low temperature of-40℃, resulting in poor mechanical properties.

[0081] Experimental Example 3

[0082] Anti-freezing performance of GelMA-EA hydrogel prepared in Example 1

[0083] The hydrogel of Example 1 is placed in a low-temperature refrigerator at-40℃ for 24h, and then the tensile effect and tissue adhesion of the sample are tested after the sample is taken out of the refrigerator.

[0084] The results are shown in the accompanying drawings Figure 7 as shown in the accompanying drawings, Figure 7 It can be concluded that the GelMA-EA hydrogel of Example 1 still has good flexibility at-40℃, and can withstand twisting, stretching Figure 7 a-c) or compression Figure 7d-f) isovolumic, and also has high elasticity, which can quickly recover after deformation. This shows that the interaction between various groups in the GelMA-EA hydrogel network increases its mechanical properties, and the introduction of the EA compound with dynamic cross-linking bonds further enhances its freeze resistance, maintaining good mechanical properties under frozen conditions.

[0085] Experimental Example 4

[0086] Hemostatic effect of the GelMA-EA hydrogel prepared in Example 1 in different blood vessel injury models of rats

[0087] Test method: liver hemostasis: the hemostatic effect of the GelMA-EA hydrogel was evaluated using a rat (male, 250 ~ 300 g, 7 ~ 8 weeks) liver bleeding model. The rat was anesthetized, the rat liver was exposed through an abdominal incision, and a 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 arterial 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 and covered on the bleeding point, and the hemostasis was observed by timing. Abdominal aorta: after the rat was deeply anesthetized, an abdominal 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 vascular clamp. Then, a puncture needle was used to make a break in the anterior wall of the artery. The vascular clamp was removed, and after 5 seconds of free bleeding, the wound around was cleaned with gauze, about 100-150 μL of gel solution was accurately 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.

[0088] 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, which reflects 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 the environment with medium blood flow pressure. 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. The results suggest that the GelMA-EA hydrogel exhibits excellent immediate sealing performance in rodents.

[0089] Experimental Example 5

[0090] Hemostatic effect of GelMA-EA hydrogel prepared in Example 1 in a domestic pig in vivo

[0091] Test method: Healthy domestic pigs were selected for the experiment, weighing 40-50 kg. Intravenous propofol was used for anesthesia. The animal was 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 round 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 was observed. The animal was placed in the same position, and a longitudinal incision was made on 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 free 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.

[0092] Figure 9 The injectable GelMA-EA hydrogel was demonstrated to have hemostatic effect in a domestic pig liver injury and different blood vessel injury models. 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 characteristics may have facilitated the immediate sealing of the blood vessel breach. This result suggests that the GelMA-EA hydrogel exhibits excellent immediate sealing performance in rats and domestic pigs.

[0093] 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 high-altitude extremely cold environments.

[0094] 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, any technical solutions with the same or similar to the present application, fall within the protection scope of the present application.

Claims

1. A method for preparing an antifreeze hemostatic gel material, characterized in that, Includes the following steps: S1 Preparation of EA complex: The EA complex is prepared by reacting epigallocatechin gallate and 3-acrylamide phenylboronic acid. S2 Preparation of gel precursor solution: Methacrylamide gelatin and acrylic acid are heated in deionized water until completely dissolved; antifreeze complex is added and mixed evenly; then N-succinimide acrylate is added to obtain the gel precursor solution; The antifreeze complex is prepared by mixing nanocellulose, proline, and glycerol; the preparation method of the antifreeze complex is as follows: nanocellulose is uniformly dispersed in deionized water; then proline and glycerol are added and mixed further to obtain the antifreeze complex; in the antifreeze complex, the weight ratio of nanocellulose, proline, glycerol and deionized water satisfies: 1:2~2.5:2~3:3~5; S3 Preparation of antifreeze hemostatic gel material: The EA complex, the gel precursor solution, acrylic acid, initiator and accelerator are mixed and reacted to prepare the antifreeze hemostatic gel material; The volumetric amounts of each component should meet the following requirements: 5-10 parts of gel precursor solution; 1-2 parts of acrylic acid. EA complex solution, 1.0~2.0 parts; The mass / volume concentration of the EA complex solution is 15~20% w / v.

2. The method for preparing the antifreeze hemostatic gel material according to claim 1, characterized in that, In step S1, the molar ratio of epigallocatechin gallate and 3-acrylamide phenylboronic acid in the EA complex satisfies 1:1.5~2.

5.

3. The method for preparing the antifreeze hemostatic gel material according to claim 1, characterized in that, In step S2, the amount of the antifreeze complex added is 15% to 20% by weight of the methacrylamide gelatin.

4. The method for preparing the antifreeze hemostatic gel material according to claim 3, characterized in that, The ratio of the methacrylamide gelatin to the acrylic acid is 1 mg / (30~40 μL).

5. The method for preparing the antifreeze hemostatic gel material according to claim 3, characterized in that, The amount of N-succinimide acrylate used is 1.8 to 2.2 times the amount of methacrylamide gelatin by weight.

6. The method for preparing the antifreeze hemostatic gel material according to any one of claims 1 to 5, characterized in that, The initiator is ammonium persulfate and the accelerator is tetramethylethylenediamine.

7. The method for preparing the antifreeze hemostatic gel material according to claim 6, characterized in that, In step S3, the volume of each component is as follows: 5-10 parts of gel precursor solution; 1-2 parts of acrylic acid; EA complex solution 1.0~2.0 parts; ammonium persulfate solution 0.7~1.5 parts; 0.12-0.15 parts of tetramethylethylenediamine; The mass / volume concentration of the ammonium persulfate solution is 50% w / v.

8. The method for preparing the antifreeze hemostatic gel material according to claim 1, characterized in that, In step S3, the EA complex, the gel precursor solution, acrylic acid, initiator and accelerator are mixed and heated at 60℃~70℃ for 1~3h to prepare the antifreeze hemostatic gel material.

9. An antifreeze hemostatic gel material, prepared by the preparation method of the antifreeze hemostatic gel material according to any one of claims 1 to 8.

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