Hydrogel for rapid hemostasis and preparation method thereof

Abstract

The invention discloses a hydrogel for rapid hemostasis, which is characterized by comprising two monomer solutions capable of generating biological orthogonal reaction, the first monomer solution is formed by connecting high polymer with alkynyl, the second monomer solution is formed by connecting high polymer with azide group, and the hydrogel also comprises thrombin and cationic polysaccharide which are dissolved in any one of the monomer solutions. The invention also discloses a preparation method. The hydrogel has the functions of accelerating wound healing and collagen deposition, promoting angiogenesis, reducing inflammation and the like. According to the invention, the comprehensive properties of rapid hemostasis, wound barrier, infection prevention, cross-linking support and the like can be met. According to the invention, accurate regulation and control of performance can be met by adjusting the proportion and concentration of the orthogonal reaction hydrogel.

Description

Technical Field

[0001] The invention relates to a hydrogel for rapid hemostasis and a preparation method thereof, belonging to the technical field of medical materials. Background Art

[0002] In emergency medical treatment, fast, safe and effective hemostasis measures are crucial to saving lives and reducing complications. Traditional hemostasis methods, such as compression hemostasis using gauze and bandages, as well as suturing and ligating blood vessels, perform well in treating routine wounds, but are often limited in effectiveness and complicated in handling severe bleeding, especially rupture of large blood vessels caused by trauma, surgical accidents or battlefield injuries. Delays may increase the risk of life for patients, and emergency hemostasis in complex environments also carries the risk of infection.

[0003] Wound healing includes four different continuous stages: hemostasis, inflammation, proliferation, and remodeling. An ideal hemostatic material should have strong hemostatic, antibacterial, and wound healing properties. Currently, most commercial hemostatic agents have limited hemostatic and antibacterial effects and are not suitable for hemostasis of non-compressive penetrating wounds. Therefore, there is an urgent need to develop a new hemostatic material with procoagulant and antibacterial activity. Summary of the invention

[0004] The purpose of the present invention is to provide a hydrogel for rapid hemostasis, which can quickly shield wounds and reduce bleeding.

[0005] A hydrogel for rapid hemostasis includes two monomer solutions capable of undergoing bioorthogonal reactions, wherein the first monomer solution is composed of a polymer connected to an alkynyl group, and the second monomer solution is composed of a polymer connected to an azide group, and also includes thrombin and a cationic polysaccharide dissolved in any one of the monomer solutions.

[0006] Preferably, the macromolecule is a polysaccharide, a protein or a chemically synthesized polymer.

[0007] Preferably, the polymer is polylactic acid, polycaprolactone or dextran.

[0008] Preferably, the final concentration of thrombin is 0.05-5 U / mL, more preferably 0.4 U / mL, and the mass concentration of the cationic polysaccharide is 0.1%-5%, more preferably 0.4%.

[0009] The present invention also discloses a method for preparing the hydrogel, which comprises the following steps:

[0010] (1) Synthesis of the first monomer: Carbodiimide is added to a dibenzocyclooctyne acid solution, and after a period of reaction, N-hydroxysuccinimide and a polymer raw material are added, unreacted monomers are removed by dialysis, and the first monomer is obtained by freeze-drying;

[0011] Synthesis of the second monomer: add carbodiimide to the azidoacetic acid solution, react for a period of time, add N-hydroxysuccinimide and polymer raw materials, dialyze to remove unreacted monomers, and freeze-dry to obtain the second monomer;

[0012] (2) The two monomers are dissolved in water respectively, and thrombin and cationic polysaccharide are dissolved in any solution to obtain a hydrogel for rapid hemostasis.

[0013] Preferably, the synthesis of the first monomer specifically refers to weighing 83.3 mg of dibenzocyclooctyne acid and dissolving it in 20 mL of PBS buffer at pH 7.0, adding 116.4 mg of carbodiimide, reacting at 25° C. for 4 hours, then adding 86.3 mg of N-hydroxysuccinimide and 0.5 g of dextran (Dextran40), reacting at 25° C. for 24 hours, and after the reaction, dialyzing and purifying with ultrapure water. After the dialysis, freeze-drying is performed to obtain the first monomer Dex-DBCO.

[0014] Preferably, the synthesis of the second monomer specifically refers to weighing 25.3 mg of azidoacetic acid and dissolving it in 20 mL of PBS buffer at pH 7.0, adding 116.4 mg of carbodiimide, reacting at 25° C. for 4 hours, then adding 86.3 mg of N-hydroxysuccinimide and 0.5 g of dextran (Dextran40), reacting at 25° C. for 24 hours, and after the reaction, dialyzing and purifying with ultrapure water. After the dialysis, freeze-drying is performed to obtain the second monomer Dex-N3.

[0015] Preferably, in step (2), the mass concentration of the solution prepared by dissolving the two monomers in water is 0.1-5%, more preferably 1%.

[0016] When in use, spraying the two reactive monomer solutions on the wound surface can achieve the effect of rapid hemostasis.

[0017] The present invention relies on bioorthogonal reactions and utilizes two monomers to form a hydrogel that can quickly shield the wound surface. The loaded thrombin can quickly activate the coagulation system to stop bleeding. The loaded cationic polysaccharide can further shorten the coagulation time and has antibacterial and antibacterial biofilm properties. The hydrogel obtained by the present invention has the functions of promoting coagulation, quickly shielding wounds, antibacterial, accelerating wound healing and collagen deposition, promoting angiogenesis, reducing inflammation, etc., so as to meet the demand for efficient and safe hemostatic materials in the medical field, and provide strong support for saving lives and reducing complications.

[0018] The present invention can also meet the precise control of performance by adjusting the ratio and concentration of the orthogonal reaction hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a graph showing the results of measuring the amount of bleeding in a hemostatic effect verification experiment of the hydrogel for rapid hemostasis of the present invention.

[0020] Figure 2 This is a graph showing the bleeding time measurement results of an experiment to verify the hemostatic effect of the hydrogel for rapid hemostasis of the present invention. Specific implementation methods

[0022] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0023] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0024] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0025] Example 1 Synthesis of hydrogel reaction monomers

[0026] 1) Synthesis of Dex-DBCO

[0027] 83.3 mg of dibenzocyclooctyne acid (DBCO-acid) was weighed and dissolved in 20 mL of PBS buffer at pH 7.0, 116.4 mg of EDC was added, and the mixture was reacted at 25°C for 4 h, followed by 86.3 mg of N-hydroxysuccinimide (NHS) and 0.5 g of Dextran 40, and the mixture was reacted at 25°C for 24 h. After the reaction, the mixture was dialyzed and purified with ultrapure water. After the dialysis, Dex-DBCO was obtained after freeze drying.

[0028] 2) Synthesis of Dex-N3

[0029] Weigh 25.3 mg of azidoacetic acid (Azidoacetic Acid) and dissolve it in 20 mL of pH 7.0 PBS buffer, add 116.4 mg of EDC, react at 25°C for 4 hours, then add 86.3 mg of N-hydroxysuccinimide (NHS) and 0.5 g of Dextran (Dextran40), and react at 25°C for 24 hours. After the reaction, dialyze and purify with ultrapure water. After dialysis, freeze-dry to obtain Dex-N3.

[0030] Example 2: Preparation of rapid hemostatic hydrogel

[0031] Weigh 0.2g Dex-DBCO and Dex-N3 respectively and dissolve them in 20mL ultrapure water to prepare a 1% hydrogel reaction monomer solution. Dissolve thrombin and cationic polysaccharide in any reaction monomer solution to a final thrombin concentration of 0.4U / mL and a cationic polysaccharide mass concentration of 0.4%. When in use, spray the two reaction monomer solutions on the wound surface in equal amounts to obtain the rapid hemostatic hydrogel of the present invention. Cationic polysaccharides conform to the description of cationic polysaccharides in CN201911235541.1.

[0032] Example 3: Evaluation of the hemostatic effect of hydrogel

[0033] 1) SD rats were anesthetized with 40 mg / kg sodium pentobarbital solution. After the anesthesia effect was complete, the abdomen of the rats was depilated and disinfected. The abdominal epidermis was cut open at the xiphoid process with tissue scissors to open the abdominal cavity. Excess blood was wiped off with gauze, and the liver was removed from the abdominal cavity to fully expose it.

[0034] 2) Construction of liver defect model: Clean gauze and quantitative filter paper were laid under the exposed liver. A liver defect was punctured with a No. 12 disposable syringe needle (outer diameter 1.2 mm) to form a liver non-compressible bleeding model. The puncture depth was kept consistent at 5 mm. The hydrogel material was injected into the injury site immediately after liver injury.

[0035] The experiment used the blank hydrogel group as the negative control, the thrombin hydrogel group as the positive control, and the cationic polysaccharide hydrogel group as the comparative experimental group. The blank hydrogel group was prepared by weighing 0.2g Dex-DBCO and Dex-N3 respectively and dissolving them in 20mL ultrapure water to prepare a 1% hydrogel reaction monomer solution. When used, the two reaction monomer solutions were sprayed on the wound surface in equal amounts, which was the blank hydrogel group; the thrombin hydrogel group was prepared by weighing 0.2g Dex-DBCO and Dex-N3 respectively and dissolving them in 20mL ultrapure water to prepare a 1% hydrogel reaction monomer solution. Thrombin was dissolved in any reaction monomer solution to a final thrombin concentration of 0.4U / mL. When used, the two reaction monomer solutions were sprayed on the wound surface in equal amounts, which was the thrombin hydrogel group; the cationic polysaccharide hydrogel group: 0.2g Dex-DBCO and Dex-N3 were weighed and dissolved in 20mL ultrapure water to prepare a 1% hydrogel reaction monomer solution. The cationic polysaccharide was dissolved in any one of the reaction monomer solutions until the concentration of the cationic polysaccharide was 0.4%. When used, the two reaction monomer solutions were sprayed on the wound surface in equal amounts, which was the cationic polysaccharide hydrogel group. The bleeding of the defect was observed, the bleeding time was measured, and the amount of bleeding was weighed. Figure 1 and Figure 2 shown.

[0036] 3) After the experiment is completed, all animals are euthanized and disposed of safely.

[0037] The results show that the rapid hemostatic hydrogel provided by the present invention can quickly stop bleeding, greatly reduce the amount of bleeding, and shorten the bleeding time.

[0038] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A hydrogel for rapid hemostasis, characterized in that The invention comprises two monomer solutions capable of undergoing bioorthogonal reaction, wherein the first monomer solution is composed of a polymer connected with an alkynyl group, and the second monomer solution is composed of a polymer connected with an azide group, and further comprises thrombin and a cationic polysaccharide, which are dissolved in any monomer solution.

2. The hydrogel according to claim 1, characterized in that The macromolecule is polysaccharide, protein or chemically synthesized polymer.

3. The hydrogel according to claim 2, characterized in that The polymer is polylactic acid, polycaprolactone or dextran.

4. The hydrogel according to any one of claims 1 to 3, characterized in that The final concentration of the thrombin is 0.05-5 U / mL, and the mass concentration of the cationic polysaccharide is 0.1%-5%.

5. The method for preparing the hydrogel according to any one of claims 1 to 4, characterized in that: The steps include: (1) Synthesis of the first monomer: Carbodiimide is added to a dibenzocyclooctyne acid solution, and after a period of reaction, N-hydroxysuccinimide and a polymer raw material are added, unreacted monomers are removed by dialysis, and the first monomer is obtained by freeze-drying; Synthesis of the second monomer: add carbodiimide to the azidoacetic acid solution, react for a period of time, add N-hydroxysuccinimide and polymer raw materials, dialyze to remove unreacted monomers, and freeze-dry to obtain the second monomer; (2) The two monomers are dissolved in water respectively, and thrombin and cationic polysaccharide are dissolved in any solution to obtain a hydrogel for rapid hemostasis.

6. The preparation method according to claim 5, characterized in that: The synthesis of the first monomer specifically refers to weighing 83.3 mg of dibenzocyclooctyne acid and dissolving it in 20 mL of PBS buffer at pH 7.0, adding 116.4 mg of carbodiimide, reacting at 25° C. for 4 hours, then adding 86.3 mg of N-hydroxysuccinimide and 0.5 g of dextran (Dextran40), reacting at 25° C. for 24 hours, and after the reaction, dialyzing and purifying with ultrapure water. After the dialysis, freeze-drying is performed to obtain the first monomer Dex-DBCO.

7. The preparation method according to claim 5, characterized in that: The synthesis of the second monomer specifically refers to weighing 25.3 mg of azidoacetic acid and dissolving it in 20 mL of PBS buffer at pH 7.0, adding 116.4 mg of carbodiimide, reacting at 25°C for 4 hours, then adding 86.3 mg of N-hydroxysuccinimide and 0.5 g of dextran (Dextran40), reacting at 25°C for 24 hours, and after the reaction, dialyzing and purifying with ultrapure water. After the dialysis, freeze-drying is performed to obtain the second monomer Dex-N3.

8. The preparation method according to claim 5, characterized in that: In step (2), the two monomers are dissolved in water to prepare a solution with a mass concentration of 0.1-5%.

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