Self-gelatinized fluid gelatin hemostatic and preparation method thereof

Abstract

The invention discloses a self-gelation fluid gelatin hemostatic and a preparation method thereof. The self-gelation fluid gelatin hemostatic is a paste hemostatic prepared by sequentially carrying out primary cross-linking gelation, secondary thermal cross-linking and hydration on phenylboronic acid modified gelatin and a cross-linking agent containing a diol structure. And due to primary cross-linking gelatinization, the composite material has a self-gelatinized network structure based on boric acid ester bond connection. The self-gelatinized fluid gelatin hemostatic has double hemostatic mechanisms, gelatin paste can be quickly self-gelatinized and attached in situ for physical plugging after being extruded, and meanwhile, gelatin can activate and accelerate an endogenous hemostatic pathway, so that quick hemostasis is realized. After hemostasis is completed, due to the self-gelation characteristic, fluid gelatin paste which is not infiltrated by blood can be directly clamped and removed through tweezers, the step that traditional fluid gelatin needs to be flushed with normal saline and then is sucked and removed is avoided, and the operation efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to a fluid gelatin hemostat, in particular to a self-gelling fluid gelatin hemostat and a preparation method thereof. Background Art

[0002] The application of hemostatic materials in hemostasis scenarios is crucial, especially in emergency situations such as trauma, surgery, or accidental bleeding. Rapid and effective hemostasis is directly related to the patient's life safety and recovery quality. Hemostatic materials generally meet the following requirements: First, hemostatic materials must be able to quickly seal wounds, reduce blood loss, and prevent serious consequences such as hemorrhagic shock. Second, they must not only physically compress and stop bleeding, but also activate the coagulation mechanism, accelerate blood clot formation, and improve hemostasis efficiency.

[0003] Currently, gelatin sponge is a commonly used hemostatic material. Gelatin sponge can quickly absorb blood and expand, forming a physical barrier that effectively compresses the wound. It can also concentrate blood, increasing the concentration of coagulation factors and platelets around the wound, thereby accelerating the initiation of the coagulation cascade reaction. However, gelatin sponge has poor shape adaptability and poor adhesion to the wound surface. When faced with irregularly shaped wounds, deep wounds, or narrow wounds, it is often difficult to fully cover them and cannot effectively stop bleeding. Furthermore, there is a risk of excessive expansion during application, which can compress surrounding tissues.

[0004] Fluid gelatin is a fluid hemostatic agent based on cross-linked gelatin particles. Compared with solid gelatin sponge, fluid gelatin has better shape adaptability and a larger contact area with the wound surface, so it has higher hemostatic efficiency. At the same time, its expansion coefficient is also lower, which reduces unnecessary space occupation and pressure, and improves surgical efficiency and safety. The mainstream fluid gelatin products on the market include Baxter's Floseal ® , Jiangxi Bornruier Biological Co., Ltd.'s Bornflow ® These products are provided as dry gelatin powder and need to be reconstituted with saline before use. Two syringes need to be connected with Luer connectors and pushed into each other for 10 rounds. ®It is provided in the form of a hydrated paste and only needs to be pushed against each other three times before use, which significantly saves preparation time. However, the gelatin particles in traditional fluid gelatin are usually made by crushing cross-linked gelatin sponge, and the cross-linking between gelatin molecules is generally irreversible chemical cross-linking, such as formaldehyde cross-linking, glutaraldehyde cross-linking, high-temperature vacuum cross-linking, etc., which results in no interaction between the hydrated gelatin particles, making the reconstructed gelatin paste have poor mechanical strength and tissue adhesion. When the amount of bleeding is large, it is easily dispersed, so it cannot be used to stop bleeding in wounds with large bleeding volumes and high vascular pressure. In addition, the loose gelatin paste has a weak sealing effect on bleeding wounds, which limits its hemostatic effect. After hemostasis is completed, the gelatin paste that is not soaked by blood cannot be clamped as a whole and needs to be flushed and removed with saline and sucked out, which prolongs the operation time.

[0005] A Chinese invention patent with grant number CN 106983905 B discloses an injectable self-healing hemostatic material, its preparation method, and application. This invention utilizes gelatin microgel particles with positive or negative surface charges, which self-assemble into an injectable fluid gelatin through electrostatic interactions between the particles or between the particles and polymers. Because the electrostatic interactions between the microgel particles or between the particles and the polymer are physically cross-linked and reversible, the hemostatic material prepared by this inventive method exhibits excellent injectability, self-healing ability, and good mechanical strength, and can achieve rapid hemostasis even in bleeding tissues with heavy bleeding and high vascular pressure.

[0006] The Chinese invention patent with application number 202411777347.7 discloses a fast self-gelling modified gelatin-based hemostatic powder composition, its preparation method and application. The hemostatic powder is composed of phenylboronic acid modified gelatin and a long-chain polymer containing a diol structure, and can be used alone or in combination with thrombin or polyethylene glycol active derivative powder to form a composition. Under physiological conditions, the powder can quickly absorb blood, settle and self-crosslink to form a hydrogel, activate the endogenous hemostatic pathway and physically block it, and achieve rapid hemostasis. However, the hemostatic powder in this application cannot be used to prepare a paste in the same way as fluid gelatin, that is, the syringe containing the powder and the syringe containing physiological saline need to be connected with a Luer connector and then pushed together. Due to the rapid self-gelling characteristics of the hemostatic powder, blockage will occur during the first round of mutual pushing, and reconstruction cannot be completed. Therefore, the powder must be hydrated first and then filled into the syringe for use. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a self-gelling fluid gelatin hemostatic agent, which is provided in the form of a hydrated paste and can be directly extruded for use. After extrusion, the gelatin paste can quickly self-gelate, can resist the impact of blood flow and maintain in-situ adhesion and physical sealing. After hemostasis is completed, the gelatin paste that is not soaked by blood can be directly clamped and removed with tweezers, avoiding the need for traditional fluid gelatin to be rinsed with saline and then suctioned and removed, thereby improving surgical efficiency.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a self-gelling fluid gelatin hemostatic agent, which is a paste hemostatic agent prepared by first cross-linking gelatin modified with phenylboronic acid and a cross-linking agent containing a diol structure, followed by second heat cross-linking and hydration.

[0009] Optimally, the cross-linking agent is a mixture of one or more selected from tetrahydroxyethylethylenediamine, tetrahydroxypropylethylenediamine, 1,2-propylene glycol, 1,3-propylene glycol, dopamine, catechol, dopamine-containing polymers, catechol-containing polymers, polyvinyl alcohol, carboxymethyl cellulose, dextran and sugar alcohols.

[0010] Optimally, the phenylboronic acid is a mixture of one or more selected from 3-aminophenylboronic acid, 3-carboxyphenylboronic acid and 4-carboxyphenylboronic acid.

[0011] Optimally, the primary cross-linking gelation comprises the following steps: Prepare reaction solution A: Prepare a 5-20 wt / v% aqueous solution of phenylboronic acid-modified gelatin; Prepare reaction solution B: prepare a crosslinker aqueous solution with a concentration of 1-20 wt / v%; The reaction solution A and the reaction solution B are mixed at a volume ratio of 1 to 100:1 to perform cross-linking and gelation.

[0012] Optimally, the secondary thermal crosslinking is to freeze-dry the primary crosslinked gelled product and then perform high-temperature vacuum thermal crosslinking; the temperature of the high-temperature vacuum thermal crosslinking is 100-150°C, the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa.

[0013] Optimally, the hydration comprises the following steps: The secondary thermal cross-linked product is mechanically crushed and sieved to obtain powder particles with a particle size range of 50-300 mesh; The aqueous solution containing benzalkonium chloride is sprayed onto the powder particles in multiple times, stirred and rolled, and a gelatin paste is obtained when the powder particles fully absorb water to a saturated water absorption rate of 70-95%.

[0014] Another object of the present invention is to provide a method for preparing a self-gelling fluid gelatin hemostatic agent, comprising the following steps: (a) Synthesis of phenylboronic acid modified gelatin: (a1) Add gelatin powder to a phosphate buffer solution having a pH of 4 to 6, and dissolve with stirring at 40 to 60°C to prepare a gelatin solution having a concentration of 2 to 20 wt / v%; (a2) preparing a 2-20 wt / v% phenylboric acid ethanol solution, a 2-20 wt / v% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution, and a 2-10 wt / v% N-hydroxysuccinimide ethanol solution; (a3) adding the phenylboric acid ethanol solution dropwise to the gelatin solution, stirring, and sequentially adding the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution and the N-hydroxysuccinimide ethanol solution dropwise; maintaining the temperature at 25-50° C. for 24-48 hours after the addition is complete, dialyzing, and freeze-drying to obtain phenylboric acid-modified gelatin; the mass ratio of the phenylboric acid to the gelatin is 1:1-10, the mass ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gelatin is 1:1-10, and the mass ratio of the N-hydroxysuccinimide to the gelatin is 1:2-20; (b) Primary cross-linking gelation: Prepare reaction solution A: Prepare a 5-20 wt / v% aqueous solution of phenylboronic acid-modified gelatin; Prepare reaction solution B: prepare a crosslinker aqueous solution with a concentration of 1-20 wt / v%; The reaction solution A and the reaction solution B are mixed in a volume ratio of 1 to 100:1 to obtain a gel product after primary cross-linking; (c) Secondary thermal crosslinking: The gelled product is freeze-dried and then subjected to high-temperature vacuum thermal crosslinking; the high-temperature vacuum thermal crosslinking temperature is 100-150° C., the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa; (d) Hydration: The product obtained in step (c) is mechanically crushed and sieved to obtain powder particles with a particle size range of 50 to 300 meshes, and the powder particles are hydrated at a ratio of 70 to 95% of the saturated water absorption rate to obtain a gelatin paste; the gelatin paste is pre-filled into a syringe, and after irradiation sterilization, the self-gelling fluid gelatin hemostatic agent is obtained.

[0015] Optimally, in step (b), the gelled product is soaked in pure water multiple times at a volume ratio or mass ratio of 1:5 to 15, each time for 15 to 40 minutes, to remove excess cross-linking agent.

[0016] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: (1) The self-gelling fluid gelatin hemostatic agent of the present invention is provided in the form of a paste having a self-gelling network structure based on borate cross-linking. The self-gelling network is formed by cross-linking phenylboronic acid-modified gelatin and a cross-linking agent. The borate bonds are dynamically reversible and break under the action of shear force during extrusion, thereby reducing the viscosity of the paste and ensuring that it can be extruded completely and continuously, giving it a "ready-to-use" characteristic.

[0017] (2) The self-gelling fluid gelatin hemostatic agent of the present invention also contains multiple cross-linking mechanisms, which can be stored for a long time in the form of a paste, and at the same time, the gelatin paste has good mechanical strength. In the synthesis process of phenylboronic acid-modified gelatin, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) is used as a cross-linking agent, which not only allows the carboxyl / amino groups on phenylboronic acid to react with the amino / carboxyl groups on the gelatin molecular chain to complete grafting, but also allows the carboxyl / amino groups between the gelatin molecular chains to react to form amide bonds; during the gelation process of the phenylboronic acid-modified gelatin and the cross-linking agent, the two can self-cross-link to form borate ester bonds; in the subsequent thermal cross-linking process, the carboxyl groups, amino groups, and hydroxyl groups between the gelatin molecular chains can all undergo dehydration condensation to form amide bonds or ester bonds, and the carboxyl groups, amino groups, and hydroxyl groups on the gelatin molecular chain can also undergo dehydration condensation with the hydroxyl groups on the diol cross-linking agent or the hydroxyl groups on the phenylboronic acid to form ester bonds.

[0018] (3) The self-gelling fluid gelatin hemostatic agent of the present invention has a stable skeleton network thanks to the multiple cross-links formed during the thermal cross-linking process, which can effectively avoid the partial aggregation of the powder due to rapid self-gelation during the hydration process, resulting in uneven water absorption. In addition, the uniformity of the paste is ensured by multiple spraying of the solution and slow stirring and rolling operations.

[0019] (4) The self-gelling fluid gelatin hemostatic agent of the present invention has good in-situ adhesion performance and physical blocking effect. On the one hand, the viscosity of the paste can be adjusted by controlling the hydration ratio, so that it has a certain degree of adhesion. On the other hand, thanks to the dynamic reversibility of the borate bond, when the paste is extruded, the borate bond is damaged by shear force and breaks, causing some molecular chains to stretch, thereby forming chain entanglement with the tissue surface. The borate bond then recovers, fixing the entangled shape of the molecular chain, thereby achieving in-situ adhesion. At the same time, after the paste self-gels, it can effectively resist the impact of blood flow and achieve physical blocking.

[0020] (5) The self-gelling fluid gelatin hemostatic agent of the present invention has a dual hemostatic mechanism. After being extruded, the gelatin paste can quickly self-gel and adhere in situ for physical blocking. At the same time, the gelatin itself can activate and accelerate the endogenous hemostatic pathway, thereby achieving rapid hemostasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a photograph of the self-gelling fluid gelatin hemostatic agent in Example 1 (packaged in a syringe); Figure 2 This is a comparison picture of the adhesion performance test of fluid gelatin in Example 1 and Comparative Example 1; Figure 3 This is a comparison picture of the compression performance test of fluid gelatin in Example 1 and Comparative Example 1; Figure 4 These are comparison pictures of the self-gelling performance test of fluid gelatin in Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0022] The self-gelling fluid gelatin hemostatic agent of the present invention is a paste hemostatic agent prepared by sequentially subjecting phenylboronic acid modified gelatin and a cross-linking agent containing a diol structure to primary cross-linking gelation, secondary thermal cross-linking and hydration.

[0023] The cross-linking agent is preferably a mixture of one or more selected from tetrahydroxyethylethylenediamine, tetrahydroxypropylethylenediamine, 1,2-propylene glycol, 1,3-propylene glycol, dopamine, catechol, a dopamine-containing polymer, a catechol-containing polymer, polyvinyl alcohol, carboxymethyl cellulose, dextran, and sugar alcohols (sugar alcohols include xylitol, sorbitol, maltitol, etc.). The phenylboric acid is preferably a mixture of one or more selected from 3-aminophenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid.

[0024] The primary cross-linking and gelation process includes the following steps: preparing reaction solution A: preparing a phenylboronic acid-modified gelatin aqueous solution with a concentration of 5-20 wt / v%; preparing reaction solution B: preparing a cross-linking agent aqueous solution with a concentration of 1-20 wt / v%; and mixing the reaction solution A and the reaction solution B in a volume ratio of 1-100:1 to perform cross-linking and gelation.

[0025] The secondary thermal crosslinking is to freeze-dry the primary crosslinked gel product and then perform high-temperature vacuum thermal crosslinking; the temperature of the high-temperature vacuum thermal crosslinking is 100-150° C., the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa.

[0026] The hydration comprises the following steps: mechanically crushing and sieving the secondary thermally cross-linked product to obtain powder particles with a particle size range of 50 to 300 mesh; spraying an aqueous solution containing benzalkonium chloride onto the powder particles in multiple times, stirring and rolling, and obtaining a gelatin paste when the powder particles fully absorb water to a saturated water absorption rate of 70 to 95%.

[0027] The self-gelling fluid gelatin hemostat of the present invention is provided in the form of a paste with a self-gelling network structure based on borate crosslinking. The self-gelling network is formed by crosslinking phenylboronic acid-modified gelatin and a crosslinking agent. The borate bonds are dynamically reversible and break under shear forces during extrusion, thereby reducing the viscosity of the paste and ensuring complete and continuous extrusion, giving it a "ready-to-use" property. The self-gelling fluid gelatin hemostat of the present invention also contains multiple crosslinking mechanisms, enabling long-term storage in the paste form while also providing the gelatin paste with good mechanical strength. In the synthesis process of phenylboronic acid-modified gelatin, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) is used as a cross-linking agent, which not only allows the carboxyl / amino groups on phenylboronic acid to react with the amino / carboxyl groups on the gelatin molecular chain to complete grafting, but also allows the carboxyl / amino groups between gelatin molecular chains to react to form amide bonds; during the gelation process of phenylboronic acid-modified gelatin and the cross-linking agent, the two can self-cross-link to form borate ester bonds; in the subsequent thermal cross-linking process, the carboxyl groups, amino groups, and hydroxyl groups between the gelatin molecular chains can all undergo dehydration condensation to form amide bonds or ester bonds, and the carboxyl groups, amino groups, and hydroxyl groups on the gelatin molecular chains can also undergo dehydration condensation with the hydroxyl groups on the diol cross-linking agent or the hydroxyl groups on the phenylboronic acid to form ester bonds. The self-gelling fluid gelatin hemostatic agent of the present invention benefits from the multiple crosslinks formed during the thermal crosslinking process, which provides it with a stable skeleton network. This effectively avoids partial aggregation of the powder due to rapid self-gelation during the hydration process, resulting in uneven water absorption. In addition, the uniformity of the paste is ensured by repeatedly spraying the solution and slowly stirring and rolling. The self-gelling fluid gelatin hemostatic agent of the present invention has good in-situ adhesion performance. On the one hand, the viscosity of the paste can be regulated by controlling the hydration ratio, so that it has a certain degree of adhesion. On the other hand, thanks to the dynamic reversibility of the borate bond, the borate bond is broken by shear force when the paste is extruded, causing some molecular chains to stretch, thereby forming chain entanglement with the tissue surface. The borate bond then recovers, fixing the entangled form of the molecular chain, thereby achieving in-situ adhesion. At the same time, after the paste self-gelates, it can effectively resist the impact of blood flow and achieve physical blocking. The self-gelling fluid gelatin hemostatic agent of the present invention has a dual hemostatic mechanism. After being extruded, the gelatin paste can quickly self-gel and adhere in situ to perform physical blocking. At the same time, the gelatin itself can activate and accelerate the endogenous hemostatic pathway, thereby achieving rapid hemostasis.

[0028] In addition, after hemostasis is completed, thanks to its self-gelling properties, the fluid gelatin paste that is not soaked in blood can be directly removed with tweezers, avoiding the need for traditional fluid gelatin to be rinsed with saline and then removed, thereby improving surgical efficiency.

[0029] The preparation method of the self-gelling fluid gelatin hemostatic agent comprises the following steps: (a) synthesis of phenylboronic acid modified gelatin: (a1) adding gelatin powder to a phosphate buffer solution with a pH of 4 to 6, stirring and dissolving at 40 to 60°C to prepare a gelatin solution with a concentration of 2 to 20 wt / v%; (a2) preparing a phenylboronic acid ethanol solution with a concentration of 2 to 20 wt / v%, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution with a concentration of 2 to 20 wt / v%, and a 2 to 10 wt / v% N-hydroxysuccinimide ethanol solution; (a3) dropping the phenylboric acid ethanol solution into the gelatin solution, stirring, and then dropping the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution and the N-hydroxysuccinimide ethanol solution in sequence; after the addition is completed, maintaining the temperature at 25 to 50°C and continuing the reaction for 24 to 48 hours. h, dialyzing and freeze-drying to obtain phenylboronic acid-modified gelatin; the mass ratio of the phenylboric acid to the gelatin is 1:1-10, the mass ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gelatin is 1:1-10, and the mass ratio of the N-hydroxysuccinimide to the gelatin is 1:2-20; (b) Primary cross-linking gelation: preparing reaction solution A: a 5-20 wt / v% aqueous solution of phenylboronic acid-modified gelatin; preparing reaction solution B: a 1-20 wt / v% aqueous solution of a cross-linking agent; mixing the reaction solution A and the reaction solution B at a volume ratio of 1-100:1 to obtain a primary cross-linked gel product; (c) Secondary thermal crosslinking: freeze-drying the gelled product and then subjecting it to high-temperature vacuum thermal crosslinking; the temperature of the high-temperature vacuum thermal crosslinking is 100-150° C., the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa; (d) Hydration: The product obtained in step (c) is mechanically crushed and sieved to obtain powder particles with a particle size range of 50 to 300 mesh, and the powder particles are hydrated at a ratio of 70 to 95% of the saturated water absorption rate to obtain a gelatin paste; the gelatin paste is pre-filled into a syringe and sterilized by irradiation to obtain the self-gelling fluid gelatin hemostatic agent.

[0030] In step (b), the gelled product is immersed in pure water at a volume ratio or mass ratio of 1:5 to 15 for multiple times, each time for 15 to 40 minutes, to remove excess cross-linking agent.

[0031] The preferred embodiments of the present invention will be described in detail below.

[0032] The aforementioned technical contents, features, and functions of the present invention will be more clearly understood in the following detailed description of a preferred embodiment with reference to the accompanying drawings. Directional terms such as up, down, left, right, front, and back mentioned in the following embodiments are merely references to the accompanying drawings. Therefore, the directional terms used are for illustrative purposes only and are not intended to limit the present invention.

[0033] The following will describe in detail various embodiments of the present invention, with accompanying drawings as examples. In addition to these detailed descriptions, the present invention can also be widely implemented in other embodiments, and any easy replacement, modification, and equivalent changes of any of the embodiments are included in the scope of the present invention and are subject to the scope of the patent application. In the description of the specification, many specific details are provided to give readers a more complete understanding of the present invention; however, the present invention may still be implemented on the premise of omitting some or all of the specific details. In addition, well-known steps or components are not described in detail to avoid unnecessary limitations on the present invention. The same or similar components in the drawings will be represented by the same or similar symbols. It is particularly noted that the drawings are for illustration purposes only and do not represent the actual size or quantity of the components. Some details may not be fully drawn to keep the drawings concise.

[0034] Example 1 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, as follows: (a) Synthesis of phenylboronic acid modified gelatin: (a1) Prepare a 4 wt / v% gelatin solution: Add 4 g of gelatin to 100 mL of phosphate buffer (PBS) and stir at 50°C until the gelatin is completely dissolved. The phosphate buffer should have a pH of 4.5 and a concentration of 0.1 mol / L. (a2) Prepare a 10 wt / v% 3-aminophenylboronic acid solution in ethanol: Add 2 g of 3-aminophenylboronic acid to 20 mL of ethanol and stir to dissolve. Prepare a 10 wt / v% ethanol solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC): add 1 g of EDC to 10 mL of ethanol and stir to dissolve. Prepare a 4 wt / v% N-hydroxysuccinimide (NHS) ethanol solution (i.e., NHS solution): add 0.6 g NHS to 15 mL ethanol and stir to dissolve; (a3) adding all of the 3-aminophenylboronic acid solution prepared in step (a2) dropwise to the gelatin solution prepared in step (a1), stirring for 20 minutes, and then sequentially adding the EDC solution and the NHS solution (all) prepared in step (a2) dropwise, such that the mass ratio of 3-aminophenylboronic acid to gelatin is 1:2, the mass ratio of EDC to gelatin is 1:4, and the mass ratio of NHS to gelatin is approximately 1:7; After all the addition was completed, the pH value of the solution was adjusted to 5-6 with H3PO4 at a concentration of 0.85%, and the reaction was maintained at 35°C for 48 hours. After completion, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 14 kDa and freeze-dried to obtain 3-aminophenylboronic acid modified gelatin.

[0035] (b) Primary cross-linking gelation: Prepare reaction solution A: prepare a 7.5 wt / v% aqueous solution of 3-aminophenylboronic acid-modified gelatin (add 1.5 g of 3-aminophenylboronic acid-modified gelatin to 20 mL of water and stir at 50 °C to dissolve); Prepare reaction solution B: prepare an aqueous solution of polyvinyl alcohol with a concentration of 7.5 wt / v% (add 1.5 g of polyvinyl alcohol (viscosity 3.2-3.8 mPa.s) to 20 mL of water and stir at 80°C to dissolve); Take 20 mL of reaction solution A and 10 mL of reaction solution B and mix them (volume ratio 2:1) to obtain a gel product after primary cross-linking. Transfer the gel to 300 mL of pure water and soak it at room temperature for 30 min. Then change the water and repeat three times to remove excess cross-linker.

[0036] (c) Secondary thermal crosslinking: The product obtained in step (b) is freeze-dried and then subjected to high-temperature vacuum thermal cross-linking at a cross-linking temperature of 100° C., a cross-linking time of 4 hours, and a vacuum degree of ≤-0.09 MPa (within this range and without affecting the performance of the final product); (d) Hydration: The product obtained in step (c) is mechanically crushed and sieved to obtain powder particles with a particle size range of 50 to 300 meshes. The saturated water absorption rate is first determined (6.7 times), and then the powder particles are hydrated at a ratio of 90% of the saturated water absorption rate: the powder particles and an aqueous solution containing benzalkonium chloride (0.005%, volume concentration) are mixed at a mass ratio of 1:6. Specifically, 0.4 g of powder particles are taken, and 2.4 mL of benzalkonium chloride solution is sprayed onto the powder particles three times (0.8 mL each time). After each spraying, stirring and rolling, the powder particles are sprayed again after fully absorbing water until hydration is completed. The gelatin paste is then pre-filled into a syringe for irradiation sterilization. The irradiation dose is 25-40 kGy (within this range and does not affect the performance of the final product), and a self-gelling fluid gelatin hemostatic agent (such as Figure 1 shown).

[0037] Example 2 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that in step (a2), 3-carboxyphenylboronic acid is used instead of 3-aminophenylboronic acid.

[0038] Example 3 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that in step (a2), 4-carboxyphenylboronic acid is used instead of 3-aminophenylboronic acid.

[0039] Example 4 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 50 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 5:1.

[0040] Example 5 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 100 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 10:1.

[0041] Example 6 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 150 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 15:1.

[0042] Example 7 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 200 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 20:1.

[0043] Example 8 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 500 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 50:1.

[0044] Example 9 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that: in step (b), 1000 mL of reaction solution A is taken and reaction solution A and reaction solution B are mixed in a volume ratio of 100:1.

[0045] Example 10 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that high-temperature vacuum thermal cross-linking is performed according to the following group parameters.

[0046] Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Crosslinking temperature 100℃ 100℃ 120℃ 120℃ 150℃ 150℃ Cross-linking time 12 h 24 h 6 h 8 h 2 h 4 h Example 11 This embodiment provides a self-gelling fluid gelatin hemostatic agent and a preparation method thereof, which is basically the same as that in Example 1, except that the following group of cross-linking agents is used to prepare the reaction solution B.

[0047] Group 7 Group 8 Group 9 Group 10 Group 11 Group 12 Group 13 crosslinking agent Tetrahydroxypropylethylenediamine 1,3-Propanediol Dopamine Catechol Carboxymethyl cellulose Glucan Xylitol Comparative Example 1 This example provides a fluid gelatin and a preparation method thereof, wherein a 10 wt / v% gelatin solution is prepared (specifically, 10 g of gelatin powder is dissolved in 100 mL of pure water and stirred at 50°C to dissolve), the solution is fully dissolved and transferred to room temperature, and after complete gelation, it is freeze-dried and then subjected to high-temperature vacuum cross-linking (cross-linking temperature is 150°C, cross-linking time is 24 h, and vacuum degree is less than -0.09 mPa); the cross-linked product is mechanically crushed and sieved to obtain cross-linked gelatin powder particles (particle size range is 50-300 mesh). According to the test, the saturated water absorption rate of the cross-linked gelatin powder particles is 6.9 times. The powder particles are hydrated at a ratio of 90% of the saturated water absorption rate, that is, the cross-linked gelatin powder particles and an aqueous solution containing benzalkonium chloride (0.005%, volume concentration) are mixed at a mass ratio of 1:6.2. Specifically, 0.4 g of modified gelatin composite powder particles are put into a syringe, and 2.5 g of the modified gelatin composite powder particles are drawn into another syringe. mL of aqueous solution containing benzalkonium chloride (0.005%), connect two syringes with a Luer interface, and push each other 10 times to obtain a fluid gelatin paste.

[0048] Saturated water absorption test of powder particles The powder particles with a particle size range of 50-300 mesh obtained by mechanically crushing and sieving the product obtained in step (c) of Example 1 and the cross-linked gelatin powder particles in Comparative Example 1 were tested. The initial mass of the powder was weighed, recorded as m0, and the powder was soaked in a sufficient amount of physiological saline and left at room temperature. After 24 hours, excess water was removed using a 400-mesh sieve. The mass of the powder after water absorption was weighed, recorded as m1.

[0049] The saturated water absorption rate of powder particles = (m1-m0) / m0. The test was repeated three times in parallel and the average value was taken as follows: Powder particles in Example 1: Therefore, the saturated water absorption rate of the powder particles with a particle size range of 50-300 mesh obtained by mechanically crushing and sieving the product obtained in step (c) of Example 1 is 6.7 times.

[0050] Powder particles in Comparative Example 1: Therefore, the saturated water absorption rate of the cross-linked gelatin powder particles in Comparative Example 1 is 6.9 times.

[0051] Tissue adhesion test The fluid gelatin samples in Example 1 and Comparative Example 1 were tested by injecting them onto the surface of pig heart and pig liver, respectively, and keeping them in a vertical state. They were rinsed with running water and their adhesion was observed. Figure 2 The sample in Example 1 can firmly adhere to the tissue surface and can withstand the erosion of running water without falling off even in a vertical state, and the gel is not dispersed; while the traditional fluid gelatin (Comparative Example 1) has weak adhesion, is easily dispersed by water flow, and cannot maintain in-situ adhesion.

[0052] Compression performance test The fluid gelatin samples in Example 1 and Comparative Example 1 were tested by injecting them into a cylindrical mold. After demoulding, they were gently pressed with a medicine spoon to observe the state of the sample after pressing. Figure 3 The sample in Example 1 has a certain elasticity and does not collapse after pressing. This is mainly because the borate bond network constructed between the gelatin molecular chains gives it a certain mechanical strength, allowing it to withstand a certain external force; while the sample in Comparative Example 1 obviously collapses after being pressed and cannot recover, which means that there is no interaction between the gelatin particles that have only undergone high-temperature cross-linking, and they cannot resist external forces without deformation.

[0053] Self-gelation test The samples in Example 1 and Comparative Example 1 were tested by injecting them into a plastic culture dish, and then using tweezers to gather the extruded fluid gelatin to observe whether it could be re-fused.

[0054] See the results Figure 4 The sample in Example 1 can be quickly self-gelled to form a whole piece of gel after being extruded and then gathered together, which can be easily picked up by tweezers; while the sample in Comparative Example 1 cannot be picked up by tweezers as a whole piece because there is no interaction between the gelatin particles after being gathered together.

[0055] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A self-gelling fluid gelatin hemostatic agent, characterized in that: The hemostatic paste is prepared by sequentially subjecting phenylboronic acid-modified gelatin and a cross-linking agent containing a diol structure to primary cross-linking gelation, secondary thermal cross-linking and hydration.

2. The self-gelling fluid gelatin hemostatic agent according to claim 1, characterized in that: The crosslinking agent is a mixture of one or more selected from tetrahydroxyethylethylenediamine, tetrahydroxypropylethylenediamine, 1,2-propylene glycol, 1,3-propylene glycol, dopamine, catechol, dopamine-containing polymers, catechol-containing polymers, polyvinyl alcohol, carboxymethyl cellulose, dextran and sugar alcohol.

3. The self-gelling fluid gelatin hemostatic agent according to claim 1, characterized in that: The phenylboronic acid is a mixture of one or more selected from 3-aminophenylboronic acid, 3-carboxylphenylboronic acid and 4-carboxylphenylboronic acid.

4. The self-gelling fluid gelatin hemostatic agent according to claim 1, characterized in that The primary cross-linking gelation comprises the following steps: Prepare reaction solution A: Prepare a 5-20 wt / v% aqueous solution of phenylboronic acid-modified gelatin; Prepare reaction solution B: prepare a crosslinker aqueous solution with a concentration of 1-20 wt / v%; The reaction solution A and the reaction solution B are mixed at a volume ratio of 1 to 100:1 to perform cross-linking and gelation.

5. The self-gelling fluid gelatin hemostatic agent according to claim 1, characterized in that: The secondary thermal crosslinking is to freeze-dry the primary crosslinked gel product and then perform high-temperature vacuum thermal crosslinking; the temperature of the high-temperature vacuum thermal crosslinking is 100-150° C., the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa.

6. The self-gelling fluid gelatin hemostatic agent according to claim 1, characterized in that The hydration comprises the following steps: The secondary thermal cross-linked product is mechanically crushed and sieved to obtain powder particles with a particle size range of 50-300 mesh; The aqueous solution containing benzalkonium chloride is sprayed onto the powder particles in multiple times, stirred and rolled, and a gelatin paste is obtained when the powder particles fully absorb water to a saturated water absorption rate of 70-95%.

7. A method for preparing a self-gelling fluid gelatin hemostatic agent, characterized in that: The following steps are involved: (a) Synthesis of phenylboronic acid modified gelatin: (a1) Add gelatin powder to a phosphate buffer solution having a pH of 4 to 6, and dissolve with stirring at 40 to 60°C to prepare a gelatin solution having a concentration of 2 to 20 wt / v%; (a2) preparing a 2-20 wt / v% phenylboric acid ethanol solution, a 2-20 wt / v% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution, and a 2-10 wt / v% N-hydroxysuccinimide ethanol solution; (a3) adding the phenylboric acid ethanol solution dropwise to the gelatin solution, stirring, and sequentially adding the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution and the N-hydroxysuccinimide ethanol solution dropwise; maintaining the temperature at 25-50° C. for 24-48 hours after the addition is complete, dialyzing, and freeze-drying to obtain phenylboric acid-modified gelatin; the mass ratio of the phenylboric acid to the gelatin is 1:1-10, the mass ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gelatin is 1:1-10, and the mass ratio of the N-hydroxysuccinimide to the gelatin is 1:2-20; (b) Primary cross-linking gelation: Prepare reaction solution A: Prepare a 5-20 wt / v% aqueous solution of phenylboronic acid-modified gelatin; Prepare reaction solution B: prepare a crosslinker aqueous solution with a concentration of 1-20 wt / v%; The reaction solution A and the reaction solution B are mixed in a volume ratio of 1 to 100:1 to obtain a gel product after primary cross-linking; (c) Secondary thermal crosslinking: The gelled product is freeze-dried and then subjected to high-temperature vacuum thermal crosslinking; the high-temperature vacuum thermal crosslinking temperature is 100-150° C., the crosslinking time is 2-48 h, and the vacuum degree is ≤-0.09 MPa; (d) Hydration: The product obtained in step (c) is mechanically crushed and sieved to obtain powder particles with a particle size range of 50 to 300 meshes, and the powder particles are hydrated at a ratio of 70 to 95% of the saturated water absorption rate to obtain a gelatin paste; the gelatin paste is pre-filled into a syringe, and after irradiation sterilization, the self-gelling fluid gelatin hemostatic agent is obtained.

8. The method for preparing the self-gelling fluid gelatin hemostatic agent according to claim 7, characterized in that: In step (b), the gelled product is immersed in pure water at a volume ratio or mass ratio of 1:5 to 15 for multiple times, each time for 15 to 40 minutes, to remove excess cross-linking agent.

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