An absorbable gelatin hemostatic sponge for in vivo use and a method for preparing the same

Abstract

The present application relates to the technical field of medical supplies, in particular to an absorbable gelatin hemostatic sponge for in vivo use and a preparation method thereof. The hemostatic sponge is prepared by subjecting a homologous gelatin composite solution to microbial transglutaminase crosslinking, fractional freeze-drying to form pores, and then short-time activation treatment with EDC / NHS. The homologous gelatin composite solution is composed of unmodified gelatin and low-substitution degree N-carboxymethylated gelatin in a specific ratio. The method, through the synergy of homologous component compounding, internal enzymatic crosslinking and short-time coupling on the surface layer, significantly improves the blood absorption speed, wet-state support stability and in vivo hemostatic effect of the hemostatic sponge without introducing exogenous hemostatic active substances, and is particularly suitable for the rapid hemostasis of irregular wounds such as internal organ bleeding.

Description

Technical Field

[0001] This invention relates to the field of medical supplies technology, and in particular to an absorbable gelatin hemostatic sponge for internal use and its preparation method. Background Technology

[0002] Absorbable gelatin sponges, as a classic material for internal hemostasis, rely primarily on their porous structure to rapidly absorb blood and generate local pressure, as well as gelatin's ability to activate platelets and promote the aggregation of clotting factors. However, traditional gelatin sponges have a relatively limited blood absorption rate. When dealing with persistent bleeding, especially in visceral wounds with rich blood supply, hemostasis often fails or is delayed because the blood quickly washes away the material or the sponge collapses and loses support after absorbing water.

[0003] To improve hemostatic properties, existing technologies have explored various modification strategies. A common approach is to chemically modify gelatin, such as introducing more positively charged groups through amination to enhance electrostatic adsorption with negatively charged blood cells, or introducing cross-linking agents like dialdehyde starch to improve the material's wet mechanical strength. While these methods can improve individual properties, they often come at the cost of other aspects: excessive cross-linking sacrifices the material's water absorption and swelling properties and biodegradation rate, while simply increasing cationicity may accelerate platelet adhesion but has limited effect on improving the overall structural stability of the material in a blood environment, and the introduction of exogenous chemical cross-linking agents may pose biocompatibility risks.

[0004] Another strategy is to incorporate other functional components into the gelatin matrix, such as chitosan, inorganic hemostatic powders (e.g., zeolite, montmorillonite), or bioactive factors like thrombin. These composite materials can integrate multiple hemostatic mechanisms to achieve rapid hemostasis. However, the introduction of exogenous components brings new problems: interfacial compatibility between different components may affect material homogeneity; inorganic powders may remain in vivo, causing inflammation or granulomas; and biological agents such as thrombin have issues with inactivation, viral risks, and high costs, severely limiting their application.

[0005] Therefore, developing an in vivo hemostatic material that does not rely on exogenous hemostatic active substances and can simultaneously achieve rapid liquid absorption, good wet support, and efficient coagulation promotion solely through fine structural regulation of gelatin itself has become a pressing technical challenge in this field. This requires a complete redesign at the molecular level, achieving multi-dimensional synergistic enhancement of hemostatic function while retaining gelatin's good biocompatibility and absorbability. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide an absorbable gelatin hemostatic sponge for in vivo use and its preparation method, so as to solve the problems of slow hemostasis, easy collapse in wet state, and the need to introduce exogenous components or excessive cross-linking of modified materials, which leads to a decrease in biocompatibility and absorption performance in the prior art.

[0007] Based on the above objectives, the present invention provides an absorbable gelatin hemostatic sponge for in vivo use. The absorbable gelatin hemostatic sponge for in vivo use is a porous gelatin sponge obtained by cross-linking a homologous gelatin composite solution with microbial transglutaminase, pre-freezing at -20°C and then freezing at -40°C and freeze-drying to form pores, and activation treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Based on the dry basis total mass of gelatinous substances in the homologous gelatin composite solution being 100%, the gelatinous substances consist of 78%-92% unmodified gelatin and 8%-22% low-substituted N-carboxymethylated gelatin. The free amino retention rate of the low-substituted N-carboxymethylated gelatin is 84%-90%, and the free amino retention rate is calculated based on the free amino content of the unmodified gelatin being 100%.

[0008] Preferably, the homologous gelatin composite solution for enzymatic crosslinking is prepared by mass fractions of 441 parts purified water, 38.2-45.1 parts unmodified gelatin, 3.9-10.8 parts low-substituted N-carboxymethylated gelatin, and 0.08-0.13 parts lyophilized microbial transglutaminase powder.

[0009] Preferably, the low-substituted N-carboxymethylated gelatin is obtained by carboxylation with sodium chloroacetate, dialysis and freeze-drying of 10% by mass gelatin mother liquor; wherein, 0.36-0.62 parts of sodium chloroacetate are added to every 120 parts of the gelatin mother liquor, and the reaction is carried out for 100-150 min at a pH of 8.4-8.6 and a temperature of 35°C.

[0010] Preferably, the gelatin mother liquor is prepared by mass fractions of 440-460 parts purified water, 48-52 parts gelatin, 1.30-1.42 parts potassium dihydrogen phosphate and 1.68-1.80 parts anhydrous dipotassium hydrogen phosphate.

[0011] Preferably, the freeze-drying pore-forming process includes: degassing the homologous gelatin composite solution cross-linked by microbial transglutaminase and pouring it into a mold, controlling the liquid layer thickness to be 4-6 mm; placing the mold on a metal plate at -20°C for pre-freezing for 25-40 min, and then transferring it to an environment at -40°C for freezing for 3.5-5 h; after freezing, freeze-drying at -50°C and a pressure ≤30 Pa for 24 h to obtain primary gelatin sponge.

[0012] Preferably, the activation treatment includes: placing 10 parts of primary gelatin sponge in an activation solution and treating it at 4°C for 9 to 16 minutes; the activation solution comprises, by mass, 40 parts of purified water, 40 parts of anhydrous ethanol, 0.12-0.20 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.08-0.13 parts of N-hydroxysuccinimide, and the pH of the activation solution is 5.5.

[0013] Preferably, the activation treatment further includes a termination treatment and washing; the termination treatment involves transferring the activated sponge into a termination solution for 18-25 minutes; the termination solution consists of 80 parts purified water, 0.50-0.75 parts glycine, 0.216 parts potassium dihydrogen phosphate, and 0.278 parts anhydrous dipotassium hydrogen phosphate by mass.

[0014] Preferably, the absorbable gelatin hemostatic sponge used in the body is a block 10mm×10mm×5mm in shape.

[0015] Preferably, the absorbable gelatin hemostatic sponge used in vivo is obtained by sterilization with ethylene oxide; the sterilization conditions of ethylene oxide are: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, sterilization time 4h, and after sterilization, it is desorbed at 40℃ for 72h.

[0016] This invention also provides a method for preparing an absorbable gelatin hemostatic sponge for in vivo use, comprising the following steps: (1) Preparation of low-substitution-degree N-carboxymethylated gelatin; (2) Dissolve the unmodified gelatin and the low-substituted N-carboxymethylated gelatin in purified water to obtain a homologous gelatin composite solution; (3) Microbial transglutaminase was added to the homologous gelatin composite solution to carry out enzymatic cross-linking, thereby obtaining a cross-linked composite solution; (4) The cross-linked composite solution is pre-frozen at -20°C and then frozen at -40°C, and freeze-dried to obtain primary gelatin sponge; (5) The primary gelatin sponge is treated with an activating solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then terminated, washed, dried and sterilized to obtain an absorbable gelatin hemostatic sponge for in vivo use.

[0017] The beneficial effects of this invention are: (1) This invention introduces an appropriate amount of hydrophilic carboxymethyl groups into the gelatin network by homologously compounding low-substituted N-carboxymethylated gelatin with a free amino retention rate of 84%-90% with unmodified gelatin in a ratio of 78:22 to 92:8. This not only significantly improves the hydrophilicity and blood absorption rate of the material, but its moderate negative charge is also more conducive to enriching coagulation factors in the blood, thereby synergistically improving the coagulation-promoting ability without excessively altering the main properties of the gelatin.

[0018] (2) This invention innovatively employs a hierarchical cross-linking strategy of "internal enzymatic cross-linking to construct the framework and surface chemical coupling to stabilize the pore walls." The limited cross-linking by microbial transglutaminase provides the sponge with excellent wet compression recovery rate, ensuring that the shape remains consistent with the wound after water absorption; the subsequent short-term low-temperature activation treatment of EDC / NHS further stabilizes the surface of the pore walls without significantly affecting the internal pore structure, preventing excessive swelling and disintegration in the blood. The synergy of these two methods enables the material to maintain good mechanical support while rapidly absorbing blood.

[0019] (3) The synergistic effect of the above components and processes ultimately achieved excellent in vivo rapid hemostasis in animal models. The hemostatic sponge prepared by this invention can shorten the hemostasis time to as short as 58s on the standard bleeding wound surface of rat liver, with a blood loss as low as 41.8mg. This method does not introduce exogenous hemostatic powder or active factors, and is entirely based on the structural design of the gelatin material itself, which has better biosafety and clinical translation potential. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 The infrared spectra of unmodified gelatin and low-substituted N-carboxymethylated gelatin provided in Example 1 of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0023] Raw material specifications: The gelatin used is G108396 gelatin from Shanghai Aladdin Biochemical Technology Co., Ltd., grade PharmPure, conforming to USP, BP and European Pharmacopoeia standards, with a gel strength of approximately 240g Bloom and CAS number 9000-70-8; the microbial transglutaminase used is Sigma-Aldrich SAE0159 microbial transglutaminase lyophilized powder, distributed by Merck Life Sciences China Co., Ltd., with a specific activity ≥12 units / mg protein, stored at -20℃; the dialysis bags are commercially available regenerated cellulose dialysis bags with a molecular weight cutoff of 3500 Da. Example 1

[0024] Step 1: Add 50g of gelatin to 450g of purified water and let stand at 25℃ for 30min to allow the gelatin to absorb water and swell; then heat to 45℃ and stir for 60min. Add 1360mg of potassium dihydrogen phosphate and 1740mg of anhydrous dipotassium hydrogen phosphate, and continue stirring at 45℃ for 20min to obtain a gelatin mother liquor with a mass fraction of 10%. Step 2: Take 120g of the gelatin mother liquor obtained in Step 1 and stir at 35℃; separately, take 100g of purified water and add 1000mg of sodium hydroxide to prepare a 1% sodium hydroxide aqueous solution as a pH adjustment solution; adjust the pH of the gelatin mother liquor to 8.5 with the sodium hydroxide aqueous solution, then add 480mg of sodium chloroacetate and react at 35℃ for 120min; after the reaction is complete, take another 100g of purified water and add 1000mg of hydrochloric acid to prepare a 1% hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 7.5; then, stir the neutralized reaction solution... The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with 5000 g of purified water for 24 hours, with the 5000 g of purified water being replaced every 8 hours. After dialysis, the liquid in the bag was frozen at -40℃ for 8 hours, and then freeze-dried at -50℃ and a pressure ≤30 Pa for 24 hours to obtain low-substituted N-carboxymethylated gelatin powder. The free amino content was determined by the trinitrobenzenesulfonic acid method, with the free amino content of unmodified gelatin as 100%. The free amino retention rate of the obtained low-substituted N-carboxymethylated gelatin was 86.7%. Step 3: Add 42g of gelatin and 7g of the low-substituted N-carboxymethylated gelatin powder obtained in Step 2 to 441g of purified water. Stir at 45℃ for 60min to obtain a homologous gelatin composite solution with a total gelatin mass fraction of 10%. Cool the homologous gelatin composite solution to 37℃, add 100mg of microbial transglutaminase lyophilized powder, and stir at 37℃ for 40min. After the reaction is complete, raise the temperature to 70℃ and hold for 5min to stop the enzymatic reaction; then cool to 35℃. Step 4: Let the solution obtained in Step 3 stand at 35℃ for 10 minutes, then degas it under an absolute pressure of 20 kPa for 5 minutes; then pour the solution into a polytetrafluoroethylene mold, controlling the liquid layer thickness to be 5 mm. Place the mold on a metal plate at -20℃ for pre-freezing for 30 minutes, then transfer it to an environment at -40℃ for freezing for 4 hours; after freezing, freeze-dry the frozen body at -50℃ and a pressure ≤30 Pa for 24 hours to obtain primary gelatin sponge; Step 5: Take 40g purified water, 40g anhydrous ethanol, 160mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100mg N-hydroxysuccinimide, and mix them to obtain an activation solution; adjust the pH of the activation solution to 5.5 with a 1% hydrochloric acid aqueous solution and pre-cool at 4℃ for 10min; place 10g of primary gelatin sponge in the activation solution and treat at 4℃ for 12min; immediately after treatment, remove the sponge and transfer it to a termination solution for 20min; the termination solution consists of 80g purified water, 600mg glycine, 216mg potassium dihydrogen phosphate and 278mg anhydrous dipotassium hydrogen phosphate; then wash the sponge three times with purified water, each time for 10min; Step 6: Freeze the sponge washed in Step 5 at -40℃ for 6 hours, then freeze-dry it at -50℃ and pressure ≤30Pa for 24 hours to obtain a final-dry sponge; cut the final-dry sponge into 10mm×10mm×5mm blocks; sterilize with ethylene oxide after cutting under the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, and sterilization time 4 hours; after sterilization, decompose at 40℃ for 72 hours to obtain an absorbable gelatin hemostatic sponge for in vivo use. Example 2

[0025] Step 1: Add 50g of gelatin to 450g of purified water and let stand at 25℃ for 30min to allow the gelatin to absorb water and swell; then heat to 45℃ and stir for 60min. Add 1360mg of potassium dihydrogen phosphate and 1740mg of anhydrous dipotassium hydrogen phosphate, and continue stirring at 45℃ for 20min to obtain a gelatin mother liquor with a mass fraction of 10%. Step 2: Take 120g of the gelatin mother liquor obtained in Step 1 and stir at 35℃; separately, take 100g of purified water and add 1000mg of sodium hydroxide to prepare a 1% sodium hydroxide aqueous solution as a pH adjustment solution; adjust the pH of the gelatin mother liquor to 8.4 with the sodium hydroxide aqueous solution, then add 360mg of sodium chloroacetate and react at 35℃ for 100min; after the reaction is complete, take another 100g of purified water and add 1000mg of hydrochloric acid to prepare a 1% hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 7.5; then, stir the neutralized reaction solution... The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with 5000 g of purified water for 24 hours, with the 5000 g of purified water being replaced every 8 hours. After dialysis, the liquid in the bag was frozen at -40℃ for 8 hours, and then freeze-dried at -50℃ and a pressure ≤30 Pa for 24 hours to obtain low-substituted N-carboxymethylated gelatin powder. The free amino content was determined by the trinitrobenzenesulfonic acid method, with the free amino content of unmodified gelatin as 100%. The free amino retention rate of the obtained low-substituted N-carboxymethylated gelatin was 89.5%. Step 3: Add 45.1g of gelatin and 3.9g of the low-substituted N-carboxymethylated gelatin powder obtained in Step 2 to 441g of purified water. Stir at 45℃ for 60min to obtain a homologous gelatin composite solution with a total gelatin mass fraction of 10%. Cool the homologous gelatin composite solution to 37℃, add 80mg of microbial transglutaminase lyophilized powder, and stir at 37℃ for 35min. After the reaction is complete, raise the temperature to 70℃ and hold for 5min to stop the enzymatic reaction; then cool to 35℃. Step 4: Let the solution obtained in Step 3 stand at 35℃ for 10 min, then degas it under an absolute pressure of 20 kPa for 5 min; then pour the solution into a polytetrafluoroethylene mold, controlling the liquid layer thickness to be 4 mm. Place the mold on a metal plate at -20℃ for pre-freezing for 25 min, then transfer it to an environment at -40℃ for freezing for 3.5 h; after freezing, freeze-dry the frozen body at -50℃ and a pressure ≤30 Pa for 24 h to obtain primary gelatin sponge; Step 5: Take 40g purified water, 40g anhydrous ethanol, 120mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 80mg N-hydroxysuccinimide, and mix them to obtain an activation solution; adjust the pH of the activation solution to 5.5 with a 1% hydrochloric acid aqueous solution and pre-cool at 4℃ for 10min; place 10g of primary gelatin sponge in the activation solution and treat at 4℃ for 9min; immediately after treatment, remove the sponge and transfer it to a termination solution for 18min; the termination solution consists of 80g purified water, 500mg glycine, 216mg potassium dihydrogen phosphate and 278mg anhydrous dipotassium hydrogen phosphate; then wash the sponge three times with purified water, each time for 10min; Step 6: Freeze the sponge washed in Step 5 at -40℃ for 5 hours, and then freeze-dry it at -50℃ and pressure ≤30Pa for 24 hours to obtain a final-dry sponge; cut the final-dry sponge into blocks of 10mm×10mm×5mm; sterilize the blocks with ethylene oxide at the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, and sterilization time 4 hours; after sterilization, decompose the sponge at 40℃ for 72 hours to obtain an absorbable gelatin hemostatic sponge for internal use. Example 3

[0026] Step 1: Add 50g of gelatin to 450g of purified water and let stand at 25℃ for 30min to allow the gelatin to absorb water and swell; then heat to 45℃ and stir for 60min. Add 1360mg of potassium dihydrogen phosphate and 1740mg of anhydrous dipotassium hydrogen phosphate, and continue stirring at 45℃ for 20min to obtain a gelatin mother liquor with a mass fraction of 10%. Step 2: Take 120g of the gelatin mother liquor obtained in Step 1 and stir at 35℃; separately, take 100g of purified water and add 1000mg of sodium hydroxide to prepare a 1% sodium hydroxide aqueous solution as a pH adjustment solution; adjust the pH of the gelatin mother liquor to 8.6 with the sodium hydroxide aqueous solution, then add 620mg of sodium chloroacetate and react at 35℃ for 150min; after the reaction is complete, take another 100g of purified water and add 1000mg of hydrochloric acid to prepare a 1% hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 7.5; then, stir the neutralized reaction solution... The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with 5000 g of purified water for 24 hours, with the 5000 g of purified water being replaced every 8 hours. After dialysis, the liquid in the bag was frozen at -40℃ for 8 hours, and then freeze-dried at -50℃ and a pressure ≤30 Pa for 24 hours to obtain low-substituted N-carboxymethylated gelatin powder. The free amino content was determined by the trinitrobenzenesulfonic acid method, with the free amino content of unmodified gelatin as 100%. The free amino retention rate of the obtained low-substituted N-carboxymethylated gelatin was 84.4%. Step 3: Add 38.2g of gelatin and 10.8g of the low-substituted N-carboxymethylated gelatin powder obtained in Step 2 to 441g of purified water. Stir at 45℃ for 60min to obtain a homologous gelatin composite solution with a total gelatin mass fraction of 10%. Cool the homologous gelatin composite solution to 37℃, add 130mg of microbial transglutaminase lyophilized powder, and stir at 37℃ for 55min. After the reaction is complete, raise the temperature to 70℃ and hold for 5min to stop the enzymatic reaction; then cool to 35℃. Step 4: Let the solution obtained in Step 3 stand at 35℃ for 10 minutes, then degas it under an absolute pressure of 20 kPa for 5 minutes; then pour the solution into a polytetrafluoroethylene mold, controlling the liquid layer thickness to be 6 mm. Place the mold on a metal plate at -20℃ for pre-freezing for 40 minutes, then transfer it to an environment at -40℃ for freezing for 5 hours; after freezing, freeze-dry the frozen body at -50℃ and a pressure ≤30 Pa for 24 hours to obtain primary gelatin sponge; Step 5: Take 40g purified water, 40g anhydrous ethanol, 200mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 130mg N-hydroxysuccinimide, and mix them to obtain an activation solution; adjust the pH of the activation solution to 5.5 with a 1% hydrochloric acid aqueous solution and pre-cool at 4℃ for 10min; place 10g of primary gelatin sponge in the activation solution and treat at 4℃ for 16min; immediately after treatment, remove the sponge and transfer it to a termination solution for 25min; the termination solution consists of 80g purified water, 750mg glycine, 216mg potassium dihydrogen phosphate and 278mg anhydrous dipotassium hydrogen phosphate; then wash the sponge three times with purified water, each time for 10min; Step 6: Freeze the sponge washed in Step 5 at -40℃ for 7 hours, then freeze-dry it at -50℃ and pressure ≤30Pa for 24 hours to obtain a final-dry sponge; cut the final-dry sponge into 10mm×10mm×5mm blocks; sterilize with ethylene oxide after cutting under the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, and sterilization time 4 hours; after sterilization, decompose at 40℃ for 72 hours to obtain an absorbable gelatin hemostatic sponge for in vivo use. Example 4

[0027] Step 1: Add 50g of gelatin to 450g of purified water and let stand at 25℃ for 30min to allow the gelatin to absorb water and swell; then heat to 45℃ and stir for 60min. Add 1360mg of potassium dihydrogen phosphate and 1740mg of anhydrous dipotassium hydrogen phosphate, and continue stirring at 45℃ for 20min to obtain a gelatin mother liquor with a mass fraction of 10%. Step 2: Take 120g of the gelatin mother liquor obtained in Step 1 and stir at 35℃; separately, take 100g of purified water and add 1000mg of sodium hydroxide to prepare a 1% sodium hydroxide aqueous solution as a pH adjustment solution; adjust the pH of the gelatin mother liquor to 8.5 with the sodium hydroxide aqueous solution, then add 540mg of sodium chloroacetate and react at 35℃ for 135min; after the reaction is complete, take another 100g of purified water and add 1000mg of hydrochloric acid to prepare a 1% hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 7.5; then, stir the neutralized reaction solution... The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with 5000 g of purified water for 24 hours, with the 5000 g of purified water being replaced every 8 hours. After dialysis, the liquid in the bag was frozen at -40℃ for 8 hours, and then freeze-dried at -50℃ and a pressure ≤30 Pa for 24 hours to obtain low-substituted N-carboxymethylated gelatin powder. The free amino content was determined by the trinitrobenzenesulfonic acid method, with the free amino content of unmodified gelatin as 100%. The free amino retention rate of the obtained low-substituted N-carboxymethylated gelatin was 85.6%. Step 3: Add 40.2g of gelatin and 8.8g of the low-substituted N-carboxymethylated gelatin powder obtained in Step 2 to 441g of purified water. Stir at 45℃ for 60min to obtain a homologous gelatin composite solution with a total gelatin mass fraction of 10%. Cool the homologous gelatin composite solution to 37℃, add 120mg of microbial transglutaminase lyophilized powder, and stir at 37℃ for 50min. After the reaction is complete, raise the temperature to 70℃ and hold for 5min to stop the enzymatic reaction; then cool to 35℃. Step 4: Let the solution obtained in Step 3 stand at 35℃ for 10 min, then degas it under an absolute pressure of 20 kPa for 5 min; then pour the solution into a polytetrafluoroethylene mold, controlling the liquid layer thickness to be 5 mm. Place the mold on a metal plate at -20℃ for pre-freezing for 35 min, then transfer it to an environment at -40℃ for freezing for 4.5 h; after freezing, freeze-dry the frozen body at -50℃ and a pressure ≤30 Pa for 24 h to obtain primary gelatin sponge; Step 5: Take 40g purified water, 40g anhydrous ethanol, 180mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 120mg N-hydroxysuccinimide, and mix them to obtain an activation solution; adjust the pH of the activation solution to 5.5 with a 1% hydrochloric acid aqueous solution and pre-cool at 4℃ for 10min; place 10g of primary gelatin sponge in the activation solution and treat at 4℃ for 14min; immediately after treatment, remove the sponge and transfer it to a termination solution for 22min; the termination solution consists of 80g purified water, 650mg glycine, 216mg potassium dihydrogen phosphate and 278mg anhydrous dipotassium hydrogen phosphate; then wash the sponge three times with purified water, each time for 10min; Step 6: Freeze the sponge washed in Step 5 at -40℃ for 6 hours, then freeze-dry it at -50℃ and pressure ≤30Pa for 24 hours to obtain a final-dry sponge; cut the final-dry sponge into 10mm×10mm×5mm blocks; sterilize with ethylene oxide after cutting under the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, and sterilization time 4 hours; after sterilization, decompose at 40℃ for 72 hours to obtain an absorbable gelatin hemostatic sponge for in vivo use. Example 5

[0028] Step 1: Add 50g of gelatin to 450g of purified water and let stand at 25℃ for 30min to allow the gelatin to absorb water and swell; then heat to 45℃ and stir for 60min. Add 1360mg of potassium dihydrogen phosphate and 1740mg of anhydrous dipotassium hydrogen phosphate, and continue stirring at 45℃ for 20min to obtain a gelatin mother liquor with a mass fraction of 10%. Step 2: Take 120g of the gelatin mother liquor obtained in Step 1 and stir at 35℃; separately, take 100g of purified water and add 1000mg of sodium hydroxide to prepare a 1% sodium hydroxide aqueous solution as a pH adjustment solution; adjust the pH of the gelatin mother liquor to 8.5 with the sodium hydroxide aqueous solution, then add 420mg of sodium chloroacetate and react at 35℃ for 110min; after the reaction is complete, take another 100g of purified water and add 1000mg of hydrochloric acid to prepare a 1% hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 7.5; then, stir the neutralized reaction solution... The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with 5000 g of purified water for 24 hours, with the 5000 g of purified water being replaced every 8 hours. After dialysis, the liquid in the bag was frozen at -40℃ for 8 hours, and then freeze-dried at -50℃ and a pressure ≤30 Pa for 24 hours to obtain low-substituted N-carboxymethylated gelatin powder. The free amino content was determined by the trinitrobenzenesulfonic acid method, with the free amino content of unmodified gelatin as 100%. The free amino retention rate of the obtained low-substituted N-carboxymethylated gelatin was 88.5%. Step 3: Add 43.6g of gelatin and 5.4g of the low-substituted N-carboxymethylated gelatin powder obtained in Step 2 to 441g of purified water. Stir at 45℃ for 60min to obtain a homologous gelatin composite solution with a total gelatin mass fraction of 10%. Cool the homologous gelatin composite solution to 37℃, add 90mg of microbial transglutaminase lyophilized powder, and stir at 37℃ for 38min. After the reaction is complete, raise the temperature to 70℃ and hold for 5min to stop the enzymatic reaction; then cool to 35℃. Step 4: Let the solution obtained in Step 3 stand at 35℃ for 10 min, then degas it under an absolute pressure of 20 kPa for 5 min; then pour the solution into a polytetrafluoroethylene mold, controlling the liquid layer thickness to be 4.5 mm. Place the mold on a metal plate at -20℃ for pre-freezing for 28 min, then transfer it to an environment at -40℃ for freezing for 4 h; after freezing, freeze-dry the frozen body at -50℃ and a pressure ≤30 Pa for 24 h to obtain primary gelatin sponge; Step 5: Take 40g purified water, 40g anhydrous ethanol, 140mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 90mg N-hydroxysuccinimide, and mix them to obtain an activation solution; adjust the pH of the activation solution to 5.5 with a 1% hydrochloric acid aqueous solution and pre-cool at 4℃ for 10min; place 10g of primary gelatin sponge in the activation solution and treat at 4℃ for 11min; immediately after treatment, remove the sponge and transfer it to a termination solution for 19min; the termination solution consists of 80g purified water, 550mg glycine, 216mg potassium dihydrogen phosphate and 278mg anhydrous dipotassium hydrogen phosphate; then wash the sponge three times with purified water, each time for 10min; Step 6: Freeze the sponge washed in Step 5 at -40℃ for 5.5 hours, and then freeze-dry it at -50℃ and pressure ≤30Pa for 24 hours to obtain a final-dry sponge; cut the final-dry sponge into blocks of 10mm×10mm×5mm; sterilize the blocks with ethylene oxide at the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, and sterilization time 4 hours; after sterilization, decompose the sponge at 40℃ for 72 hours to obtain an absorbable gelatin hemostatic sponge for internal use.

[0029] Comparative Example 1: The difference from Example 1 is that: in step 3, 7g of the low-substituted N-carboxymethylated gelatin powder obtained in step 2 is not added, and the amount of gelatin is adjusted from 42g to 49g; the other conditions are the same as in Example 1.

[0030] Comparative Example 2: The difference from Example 1 is that the amount of sodium chloroacetate added in step 2 was adjusted from 480 mg to 960 mg, and the free amino retention rate of the resulting low-substituted N-carboxymethylated gelatin was 78.5%; the other conditions were the same as in Example 1.

[0031] Comparative Example 3: The difference from Example 1 is that the amount of gelatin used in step 3 was adjusted from 42g to 46.1g, and the amount of low-substituted N-carboxymethylated gelatin powder obtained in step 2 was adjusted from 7g to 2.9g, so that the dry basis mass ratio of unmodified gelatin to low-substituted N-carboxymethylated gelatin was 94:6; the other conditions were the same as in Example 1.

[0032] Comparative Example 4: The difference from Example 1 is that the amount of gelatin used in step 3 was adjusted from 42g to 35.3g, and the amount of low-substituted N-carboxymethylated gelatin powder obtained in step 2 was adjusted from 7g to 13.7g, so that the dry basis mass ratio of unmodified gelatin to low-substituted N-carboxymethylated gelatin was 72:28; the other conditions were the same as in Example 1.

[0033] Comparative Example 5: The difference from Example 1 is that 100mg of microbial transglutaminase lyophilized powder is not added in step 3, and the amount of gelatin is adjusted from 42g to 42.1g to keep the total amount of solid material added in step 3 consistent with that in Example 1; the other conditions are the same as in Example 1.

[0034] Comparative Example 6: The difference from Example 1 is that the amount of microbial transglutaminase lyophilized powder used in step 3 was adjusted from 100mg to 300mg, and the amount of gelatin was adjusted from 42g to 41.8g, so as to keep the total amount of solid material added in step 3 consistent with that in Example 1; the other conditions are the same as in Example 1.

[0035] Comparative Example 7: The difference from Example 1 is that the operation of placing the mold on a -20℃ metal plate for pre-freezing for 30 minutes and then transferring it to a -40℃ environment for freezing for 4 hours in step 4 is changed to directly placing the mold in a -40℃ environment for freezing for 4.5 hours; the other conditions are the same as in Example 1.

[0036] Comparative Example 8: The difference from Example 1 is that in step 5, 160 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide are not added to the activation solution, and the amount of purified water is adjusted from 40 g to 40.26 g to keep the total mass of the activation solution consistent with that of Example 1; the other conditions are the same as those in Example 1.

[0037] Comparative Example 9: The difference from Example 1 is that the treatment time of the primary gelatin sponge in the activation solution in step 5 is adjusted from 12 min to 60 min; the other conditions are the same as in Example 1.

[0038] Performance testing: Sample preparation and source: The test samples were absorbable gelatin hemostatic sponges for in vivo use prepared in Examples 1-5 and Comparative Examples 1-9. All samples were prepared according to the corresponding examples or comparative examples, uniformly cut into 10mm×10mm×5mm blocks, and uniformly sterilized with ethylene oxide under the following conditions: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, sterilization time 4h, followed by analysis at 40℃ for 72h. Before testing, the samples were placed in a clean environment at 23℃ and 50% relative humidity for 24h to equilibrate. Six parallel samples were set for each sample in each test item, and the average value was taken. The blood used for the blood-interaction test was fresh whole rabbit blood anticoagulated with sodium citrate, used within 2h after blood collection; the animals used for the liver hemostasis test were SPF-grade male SD rats weighing 250-270g.

[0039] Infrared spectroscopy: Infrared analysis was performed according to GB / T 6040-2019 "General Rules for Infrared Spectroscopic Analysis". The unmodified gelatin and the low-substitution-degree N-carboxymethylated gelatin in Example 1 were analyzed using infrared spectroscopy. The results are as follows: Figure 1 As shown.

[0040] 60s blood absorption rate: The liquid absorption test method was based on YY / T 0471.1-2004 "Test Methods for Contact Wound Dressings Part 1: Liquid Absorbency" and combined with the principles for selecting blood-interaction tests in GB / T 16886.4-2022. A balanced sponge sample was accurately weighed and recorded as m0. The sample was placed in fresh, anticoagulated rabbit whole blood at 37℃, ensuring complete immersion for 60 seconds. After removing the sample, one corner was held with tweezers and allowed to drip naturally for 30 seconds without applying external pressure. The mass was then immediately measured and recorded as m1. The 60s blood absorption rate was calculated using the formula (m1-m0) / m0, in g / g.

[0041] Wet compression recovery rate: The physical properties of the material were characterized according to GB / T 16886.19-2022. After equilibration, the sponge sample was first soaked in 37℃ phosphate buffer for 5 minutes to reach a wet state. The wet sample was then placed in the center of the flat-pressing fixture of a universal testing machine, and the compression speed was set to 5 mm / min. The sample was compressed to 50% of its original thickness, held for 30 seconds, and then unloaded at a speed of 5 mm / min. After unloading, the sample was placed back into the 37℃ phosphate buffer and allowed to stand for 60 seconds. The recovered thickness was then measured. The wet compression recovery rate was calculated as the percentage of the recovered thickness to the initial wet thickness. Each sample was tested three times consecutively, and the recovery rate after the third compression was taken as the result.

[0042] Whole blood coagulation index: Blood interaction evaluation items were selected according to GB / T 16886.4-2022. A 10mm × 10mm × 5mm sample was placed in a 37℃ constant temperature metal bath for 5 min. 200 μL of fresh sodium citrate anticoagulated rabbit whole blood was added to the sample, followed by 20 μL of 0.2 mol / L calcium chloride aqueous solution to initiate coagulation. The mixture was incubated at 37℃ for 5 min. Then, 25 mL of purified water was added, and the mixture was gently shaken at 37℃ for 10 min to allow uncoagulated red blood cells to release hemoglobin. The absorbance of the supernatant was measured at 540 nm. The blank group consisted of test tubes without a sample but containing the same volume of whole blood and calcium chloride aqueous solution. The whole blood coagulation index was calculated as a percentage of the absorbance of the sample group to that of the blank group; a lower value indicated a stronger coagulation-promoting effect.

[0043] Platelet retention rate: Blood-blood interaction evaluation items were selected according to GB / T 16886.4-2022. Fresh rabbit whole blood anticoagulated with sodium citrate was centrifuged at 200g for 10 min to obtain platelet-rich plasma. A 10mm×10mm×5mm sample was placed in a 24-well plate, and 1 mL of platelet-rich plasma was added. The platelet-rich plasma was incubated at 37℃ for 10 min. After incubation, the remaining liquid was aspirated, and the platelet-rich plasma was gently washed three times with phosphate buffer (pH 7.4), adding 2 mL each time and allowing it to stand for 20 s. The wash solution and the remaining liquid were combined, and the number of non-retained platelets was determined using a hematology analyzer. 1 mL of platelet-rich plasma not exposed to the sample was taken as the initial group. The platelet retention rate was calculated as the percentage of the difference between the initial platelet count and the number of non-retained platelets relative to the initial platelet count.

[0044] Hemostasis time and blood loss in rat liver: An animal hemostasis model was established according to the principles of GB / T 16886.4-2022 regarding the evaluation of blood-interaction. SPF-grade male SD rats, weighing 250g-270g, were anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg). After fixation, the abdominal cavity was opened, and the left lobe of the liver was exposed. A standard bleeding wound with a depth of 2mm was created on the liver surface using a 5mm diameter circular biopsy punch. Immediately afterward, free blood around the wound was aspirated with pre-weighed sterile filter paper for 5 seconds. Subsequently, a 10mm×10mm×5mm sample was placed over the wound, and a 20g weight was applied vertically through a sterile plate for 30 seconds. Timing began after the weight was removed. Every 10 seconds, the edge of the wound was gently touched with sterile filter paper. Hemostasis was considered complete when no new blood exudation was observed for 60 consecutive seconds. Hemostasis time was calculated from the removal of the weight until completion. Blood loss was calculated as the difference in total mass between the blood-collecting filter paper and the sample before and after the experiment.

[0045] The test results are shown in Table 1.

[0046] Table 1 Performance Test Results

[0047] As shown in Table 1, Comparative Example 1 used unmodified gelatin as the main body and did not introduce low-substitution N-carboxymethylated gelatin. Although it still had a certain ability to absorb blood and compress for hemostasis after enzymatic cross-linking and freeze-drying, the blood absorption ratio in 60s was only 6.4g / g, the whole blood coagulation index was 51.6%, and the liver hemostasis time and blood loss were 146s and 126.5mg, respectively. This indicates that simple gelatin sponge mainly relies on passive liquid absorption and local compression, and it is difficult to achieve both rapid enrichment of blood components and moist support on bleeding wounds. Compared with Comparative Example 1, Examples 1-5 introduced low-substituted N-carboxymethylated gelatin with a free amino retention rate of 84.4%-89.5% into the homologous gelatin system. Combined with microbial transglutaminase-limited cross-linking, pre-freezing of metal plates at -20°C followed by freezing at -40°C to form pores, and short-term low-temperature surface fixation with EDC / NHS, the 60s blood absorption ratio was increased to 9.1-12.2 g / g, the wet compression recovery rate was increased to 78.6%-86.4%, the whole blood coagulation index was reduced to 28.9%-35.1%, the platelet retention rate was increased to 54.8%-62.4%, and the liver hemostasis time was shortened to 58-80s. In Comparative Example 2, the free amino retention rate decreased to 78.5% after increasing the degree of carboxymethylation. Although it had a high blood absorption rate, the wet compression recovery rate and platelet retention rate decreased, indicating that excessive substitution is not conducive to wet pore wall stability and blood component retention. Comparative Examples 3 and 4 reduced or increased the proportion of low-substituted N-carboxymethylated gelatin, respectively, but neither achieved the comprehensive effect of Example 1. This shows that this component is not simply better the more it is, but needs to be properly compounded with unmodified gelatin. In Comparative Example 5, the wet compression recovery rate decreased to 54.8% when no microbial transglutaminase was added. In Comparative Example 6, the blood absorption ratio decreased to 7.9 g / g when the enzyme dosage was too high, indicating that insufficient or excessive internal cross-linking would weaken the fluid absorption support balance during hemostasis. In Comparative Example 7, the hemostasis time and blood loss were higher than in Example 1 after canceling graded freezing, in Comparative Example 8, the EDC / NHS activation was canceled, and in Comparative Example 9, the EDC / NHS treatment time was extended. This shows that the regular pore structure and short-term surface fixation have a positive effect on maintaining the fluid absorption channel, pore wall recovery, and wound adhesion.

[0048] from Figure 1 It can be seen that both the unmodified gelatin and the low-substituted N-carboxymethylated gelatin in Example 1 retained the typical amide characteristic absorption peaks of gelatin, with the peak at approximately 3290 cm⁻¹. -1 The area is the amide A band, approximately 2930 cm. -1 The location is where CH stretching vibrations are absorbed, approximately 1633 cm. -1 and 1540cm -1 The surrounding areas correspond to amide I and amide II bands, respectively, indicating that the gelatin backbone structure is still maintained after carboxymethylation. Compared with unmodified gelatin, low-substituted N-carboxymethylated gelatin exhibits higher molecular weight distribution at 1600 cm⁻¹. -1Enhanced carboxylate COO appeared nearby - Antisymmetric stretching absorption, and at 1412 cm -1 A more obvious COO appeared nearby - Symmetrical stretching and absorption, while 3290cm -1 The nearby broad peaks have widened slightly, reaching 1540cm. -1 The relative weakening of amide II in the vicinity indicates that some free amino groups have undergone N-carboxymethylation and a small amount of carboxymethyl groups have been introduced. Combined with the result of 86.7% retention of free amino groups in Example 1, it can be determined that this sample belongs to low-substitution degree carboxymethylation, with a moderate degree of modification. Therefore, the spectrum shows only a moderate enhancement of carboxymethyl characteristics while retaining the characteristic peaks of the gelatin skeleton, without the excessively strong carboxylate absorption commonly seen in high-substitution degree samples.

[0049] In summary, Example 1 employs a homologous compound system of unmodified gelatin and low-substituted N-carboxymethylated gelatin, and combines enzymatically limited crosslinking, graded freezing pore formation, and short-term EDC / NHS surface fixation. Without adding exogenous hemostatic powder or coagulation-active drugs, it achieves simultaneous improvement in blood absorption ratio, platelet retention, wet compression recovery, and in vivo hemostasis, demonstrating the synergistic effect between each step.

[0050] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. An absorbable gelatin hemostatic sponge for internal use, characterized in that, The absorbable gelatin hemostatic sponge for in vivo is a porous gelatin sponge obtained by cross-linking a homologous gelatin composite solution with microbial transglutaminase, pre-freezing at -20°C and then freezing at -40°C and freeze-drying to form pores, and by activation treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Based on the dry basis total mass of gelatin-like substances in the homologous gelatin composite solution being 100%, the gelatin-like substances consist of 78%-92% unmodified gelatin and 8%-22% low-substituted N-carboxymethylated gelatin. The free amino retention rate of the low-substituted N-carboxymethylated gelatin is 84%-90%, and the free amino retention rate is calculated based on the free amino content of the unmodified gelatin being 100%.

2. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The homologous gelatin complex solution for enzymatic crosslinking was prepared by mass fractions of 441 parts purified water, 38.2-45.1 parts unmodified gelatin, 3.9-10.8 parts low-substituted N-carboxymethylated gelatin, and 0.08-0.13 parts lyophilized microbial transglutaminase powder.

3. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The low-substituted N-carboxymethylated gelatin is obtained by carboxylation with sodium chloroacetate, dialysis and freeze-drying of 10% gelatin mother liquor; wherein, 0.36-0.62 parts of sodium chloroacetate are added to every 120 parts of the gelatin mother liquor, and the reaction is carried out at pH 8.4-8.6 and temperature 35°C for 100-150 min.

4. The absorbable gelatin hemostatic sponge for internal use according to claim 3, characterized in that, The gelatin mother liquor is prepared by mass fractions of 440-460 parts purified water, 48-52 parts gelatin, 1.30-1.42 parts potassium dihydrogen phosphate and 1.68-1.80 parts anhydrous dipotassium hydrogen phosphate.

5. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The freeze-drying pore-forming process includes: degassing the homologous gelatin composite solution cross-linked by microbial transglutaminase and pouring it into a mold, controlling the liquid layer thickness to be 4-6 mm; placing the mold on a metal plate at -20℃ for pre-freezing for 25-40 min, and then transferring it to an environment at -40℃ for freezing for 3.5-5 h; after freezing, freeze-drying at -50℃ and pressure ≤30 Pa for 24 h to obtain primary gelatin sponge.

6. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The activation treatment includes: treatment at 4°C for 9 to 16 minutes; the activation solution comprises, by mass, 40 parts purified water, 40 parts anhydrous ethanol, 0.12-0.20 parts 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.08-0.13 parts N-hydroxysuccinimide, and the pH of the activation solution is 5.

5.

7. The absorbable gelatin hemostatic sponge for internal use according to claim 6, characterized in that, The activation treatment is followed by a termination treatment and washing; the termination treatment involves transferring the activated sponge into a termination solution for 18-25 minutes; the termination solution consists of 80 parts purified water, 0.50-0.75 parts glycine, 0.216 parts potassium dihydrogen phosphate, and 0.278 parts anhydrous dipotassium hydrogen phosphate by mass.

8. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The absorbable gelatin hemostatic sponge used in the body is a block 10mm×10mm×5mm in size.

9. The absorbable gelatin hemostatic sponge for internal use according to claim 1, characterized in that, The absorbable gelatin hemostatic sponge used in the body was obtained by sterilization with ethylene oxide; the sterilization conditions of ethylene oxide were: temperature 45℃, relative humidity 60%, ethylene oxide concentration 600mg / L, sterilization time 4h, and after sterilization, it was desorbed at 40℃ for 72h.

10. A method for preparing an absorbable gelatin hemostatic sponge for in vivo use according to any one of claims 1-9, characterized in that, Includes the following steps: (1) Preparation of low-substitution-degree N-carboxymethylated gelatin; (2) Dissolve the unmodified gelatin and the low-substituted N-carboxymethylated gelatin in purified water to obtain a homologous gelatin composite solution; (3) Microbial transglutaminase was added to the homologous gelatin composite solution to carry out enzymatic cross-linking, thereby obtaining a cross-linked composite solution; (4) The cross-linked composite solution is pre-frozen at -20°C and then frozen at -40°C, and freeze-dried to obtain primary gelatin sponge; (5) The primary gelatin sponge is treated with an activation solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then terminated, washed, freeze-dried and sterilized to obtain an absorbable gelatin hemostatic sponge for in vivo use.

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