A double-crosslinked hydrophobic fluid gelatin hemostatic material and its preparation method

Abstract

This invention discloses a double-crosslinked hydrophobic fluid gelatin hemostatic material and its preparation method, belonging to the field of biomedical materials technology. The material is composed of type A gelatin, transglutaminase, glycerol, and deionized water. It undergoes biological crosslinking via transglutaminase and vacuum high-temperature physical crosslinking, combined with a porous structure design, to prepare a ready-to-use shear-thinning fluid gelatin. This invention employs a biological enzyme and physical double crosslinking system, avoiding the toxic residues of chemical crosslinking agents and ensuring high biosafety. The material is directly aseptically filled in a ready-to-use fluid form, requiring no preoperative preparation or dilution; it is ready to use immediately upon opening, significantly simplifying clinical procedures. Through the biological-physical double crosslinking network design, its mechanical strength and structural stability are greatly improved. It is heat-resistant, deformation-resistant, and does not disintegrate or collapse with long-term use. It can effectively adhere to irregular wound surfaces and is suitable for wound hemostasis and repair in minimally invasive surgeries such as neurosurgery and spinal surgery.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a double-crosslinked hydrophobic fluid gelatin hemostatic material and its preparation method. Background Technology

[0002] In surgical procedures and trauma management, effective bleeding control is crucial to ensuring patient safety and surgical success. This is especially true in neurosurgery, spinal surgery, hepatobiliary surgery, and various minimally invasive laparoscopic surgeries, where the surgical field is deep and the wound surface is irregular. Traditional hemostatic methods such as compression, suturing, and electrocoagulation are often ineffective. There is an urgent clinical need for an absorbable hemostatic material that can be precisely delivered, has a rapid onset of action, and is biocompatible.

[0003] Gelatin, as a hydrolysis product of collagen, has long been regarded as an ideal hemostatic matrix material due to its inherent RGD sequence, which can promote platelet aggregation and adhesion, as well as its good biodegradability and biocompatibility. Traditional gelatin sponges have been widely used in clinical practice for decades, but their inherent block shape leads to poor conformability and difficulty in adhering to complex wounds. At the same time, its extremely high water absorption and swelling rate (usually exceeding 100%) may cause compression damage to the surrounding delicate nerve and blood vessel tissues, posing a risk of secondary injury.

[0004] To overcome the shortcomings of bulky sponges, fluid gelatin hemostatic materials have emerged. These materials typically consist of gelatin particles that are compounded to form an injectable fluid, enabling them to reach deep and irregular wounds. However, existing fluid gelatin technology still has the following significant drawbacks: 1. Inconvenience and risk of contamination: Most liquid gelatin products on the market are provided in powder form, which needs to be temporarily mixed with sterile water at the surgical site. This step not only prolongs the surgical preparation time and increases the complexity of the operation, but also easily introduces microbial contamination in emergency situations, affecting the safety and efficiency of the surgery.

[0005] 2. Insufficient hydrophobicity and high expansion: Existing gelatin materials mostly rely on physical compounding or simple cross-linking, which has limited hydrophobic properties. They easily absorb water and expand rapidly in body fluids, resulting in a dramatic increase in volume. This may not only put pressure on surrounding tissues, but also cause the material structure to disintegrate too quickly, making it impossible to maintain a stable physical barrier at the bleeding site for a sufficient period of time, thus affecting the hemostasis duration. Summary of the Invention

[0006] The purpose of this invention is to provide a double cross-linked hydrophobic fluid gelatin hemostatic material and its preparation method, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a double-crosslinked hydrophobic fluid gelatin hemostatic material, comprising the following components: The gelatin matrix is ​​type A gelatin. The cross-linking system contains transglutaminase, and its dosage is 0.1wt%-5wt% of the gelatin mass; Glycerin, used in an amount of 1 wt% of the total mass of the final fluid gelatin; Deionized water; The viscosity of the gelatin solution before crosslinking is 100-800 mPa·s, and the viscosity after crosslinking is 1000-2000 mPa·s.

[0008] As a preferred embodiment of the present invention, the degree of crosslinking is 80%-90%, and the degree of crosslinking is precisely controlled by adjusting the dosage of transglutaminase, the crosslinking temperature, and the crosslinking time.

[0009] As a preferred embodiment of the present invention, the final fluid gelatin has a moisture content of 70-85 wt%, and can be directly used for injection or coating without additional dilution.

[0010] A method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material as described in any of the above claims, comprising the following steps: Step (1): After fully dissolving type A gelatin in a constant temperature water bath at 40-65℃, prepare a homogeneous aqueous solution with a mass concentration of 4wt%-20wt%, and control its initial viscosity to be 100-800mPa·s; Step (2): Add transglutaminase to the homogeneous aqueous solution obtained in step (1), the amount of which is 0.1wt%-5wt% of the gelatin mass. At the same time, nitrogen gas is introduced and the stirring is accelerated at a speed of 100-800 r / min. The mixture is stirred and crosslinked for 10-50 min at 50-60℃ and pH 6.8-7.2. The crosslinking process is controlled by monitoring the viscosity change of the system in real time. When the viscosity reaches 1000-2000 mPa·s, the reaction is stopped, and the corresponding degree of crosslinking is 20%-50%. Step (3): Transfer the gelatin solution obtained in step (2) to a pre-cooled mold; Step (4): Place the foam obtained in step (3) in an environment of -20℃ for 60 min. After it is completely solidified, transfer it to a freeze dryer and dry it under a vacuum of -0.98 bar for 20-48 hours to obtain the freeze-dried body. Step (5): Place the freeze-dried body from step (4) in a vacuum drying oven, control the vacuum degree to -0.98 bar, the temperature to 80℃-150℃, and perform physical cross-linking for 5-24 hours; the corresponding cross-linking degree is 80%-90%. Step (6): The cross-linked body in step (5) is mechanically crushed to form porous particles; Step (7): The porous particles in step (6) are placed in a -20℃ environment and frozen for 90 min. After complete solidification, they are transferred to a freeze dryer and dried under a vacuum of -0.98 bar for 20-48 hours to obtain freeze-dried particles. The porous particles with a particle size range of 50-355 μm are collected by passing them through standard sieves of 50 mesh, 100 mesh and 150 mesh in sequence. Step (8): Add deionized water and glycerol in a preset mass ratio to the porous particles obtained in step (7), mix and dissolve to form ready-to-use shear-thinning fluid gelatin, with the moisture content controlled at 70-85 wt%, which can be directly aseptically filled.

[0011] As a preferred technical solution of the present invention, in step (2), the speed of the bubbler is 400 r / min and the bubbling time is 50 min.

[0012] As a preferred technical solution of the present invention, the freezing program in steps (4) and (7) is a gradient cooling of -20℃ for 1h → -50℃ for 2h → -80℃ for 12h, and the freeze-drying stage is first -50℃ for 24h and then -40℃ for 12h, and then heated to room temperature (20-25℃) for desorption drying for 4-6h, with a vacuum degree ≤5Pa.

[0013] As a preferred embodiment of the present invention, in step (7), a vibrating screen is used for screening at a frequency of 50 Hz and an amplitude of 1.5 mm to collect particles in the range of 50-355 μm. 10 =70μm, D 50 =180μm, D 90 =320μm.

[0014] As a preferred embodiment of the present invention, in step (8), the mass ratio of porous particles to water is 1:2.5-3.5, and the amount of glycerol added is 1 wt% of the total mass.

[0015] As a preferred technical solution of the present invention, the degree of crosslinking in step (2) is synergistically controlled by the following parameters: (a) The amount of transglutaminase used is 0.1wt%-5wt% of the gelatin mass, and the amount used is positively correlated with the degree of cross-linking; (b) The crosslinking temperature is controlled at 50-60℃, and the pH is maintained at 6.8-7.2; (c) The cross-linking time is 10-50 min. The viscosity change is monitored in real time. The reaction is stopped when the viscosity reaches 1000-2000 mPa·s.

[0016] As a preferred technical solution of the present invention, the ready-to-use fluid gelatin obtained in step (6) has a swelling rate of ≤10% and a water contact angle of ≥90°, and can be directly used for injection or wound application without preoperative preparation.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. The fluid gelatin provided by this invention is packaged directly in a ready-to-use fluid form, without the need for preoperative preparation or dilution. It can be used immediately after opening, which significantly simplifies the surgical procedure, shortens the preparation time, and completely avoids microbial contamination that may be introduced due to on-site preparation, thereby improving the convenience and safety of clinical use.

[0018] 2. The controllable cross-linking network constructed by transglutaminase and vacuum physical cross-linking, combined with the porous structure design, significantly improves the hydrophobic properties of the material and effectively reduces the swelling rate. The material maintains structural stability in body fluids and is not prone to excessive expansion, thus reducing the risk of compression on delicate tissues such as surrounding nerves and blood vessels.

[0019] 3. It adopts a physical dual cross-linking system with glutamine transaminase, which completely replaces chemical cross-linking agents such as glutaraldehyde, avoiding the introduction and residue of toxic substances. The material has good cell compatibility and meets the safety standards of high-end biomedical materials.

[0020] 4. By precisely controlling the crosslinking process through real-time viscosity monitoring, combined with gradient freeze drying and mechanical sieving processes, the high uniformity between product batches is ensured. The final fluid can be directly aseptically filled, which facilitates large-scale production, storage and transportation, and is in line with the standardization development trend of modern medical devices.

[0021] 5. The material exhibits significant shear-thinning behavior, allowing for smooth delivery via fine-diameter injection instruments and excellent conformation to various irregular wound surfaces, making it particularly suitable for minimally invasive surgical scenarios such as neurosurgery and spinal surgery. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Preparation of experimental materials Gelatin: Type A gelatin (porcine collagen) conforming to the standards of Part II of the Chinese Pharmacopoeia.

[0024] Cross-linking agent: transglutaminase (TG enzyme, activity 100U / g), purchased from Jiangsu Yiming Biotechnology Co., Ltd.

[0025] Other excipients: glycerin (pharmaceutical grade), deionized water.

[0026] Testing and Characterization Methods 1. Viscosity measurement: The initial viscosity and the viscosity after cross-linking of the gelatin solution were measured using a rotational viscometer (model: NDJ-1) at 25℃.

[0027] 2. Hydrophobicity test: Using a static contact angle meter (model: OCA20, DataPhysics, Germany), after the material is pressed into a tablet, 2μL of deionized water is dropped onto the surface, and the contact angle between the water droplet and the material surface is measured. The larger the angle, the stronger the hydrophobicity.

[0028] 3. Swelling rate determination: Accurately weigh the dried sample (Wo), immerse it in phosphate buffered saline (PBS) at pH 7.4, let it stand at 37°C for 10 minutes, then remove it, blot the surface moisture with filter paper, and weigh it (W). e ), swelling rate (%) = [(W e -W o ) / W o ×100%.

[0029] 4. Extrudability test: Load the final product fluid gelatin into a 20mL syringe, install a 22G (approximately 0.41mm in diameter) needle, and extrude it on a universal testing machine at a rate of 100mm / min. Record the maximum injection force and observe whether there is blockage or flow interruption.

[0030] 5. Verification of hemostatic effect (in vitro): Fluid gelatin was used to stop the bleeding of liver leaves of Bama miniature pigs. The hemostasis time was observed and statistically analyzed.

[0031] 6. Cell compatibility: In accordance with GB / T 16886 standard, the relative growth rate (RGR) of the material extract on L929 mouse fibroblasts was determined by CCK-8 method to evaluate cytotoxicity.

[0032] 7. Determination of crosslinking degree: The ninhydrin colorimetric method was used. 0.1 g of dried sample was accurately weighed, dissolved in 5 mL of 0.2 mol / L citrate buffer (pH 5.0), and then 1 mL of ninhydrin reagent was added. The mixture was heated in a boiling water bath for 15 min, cooled, and the absorbance was measured at 570 nm using a spectrophotometer. At the same time, uncrosslinked gelatin was used as a blank control. The free amino content in the sample was calculated according to the standard curve. Crosslinking degree (%) = [(free amino content in blank control - free amino content in sample) / free amino content in blank control] × 100%.

[0033] 8. Determination of glutaraldehyde residue: Refer to the Pharmacopoeia 2025 edition, General Chapter 3204 method for detection.

[0034] Chromatographic conditions: Octadecylsilane-bonded silica gel stationary (SG120, 4.6 mm × 250 mm, 5 μm); 70% acetonitrile solution as mobile phase; flow rate of 1.2 ml / min; detection wavelength of 360 nm; recording time of 30 minutes.

[0035] Assay: Accurately weigh an appropriate amount of glutaraldehyde reference standard, dissolve it in water, and quantitatively dilute it to a solution containing approximately 10 μg per ml. Accurately measure 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of this solution into separate test tubes, add water to each to a final volume of 1.0 ml, accurately add 1 ml of mobile phase and 0.1 ml of 2,4-dinitrophenylhydrazine solution (weigh 2.4 g of 2,4-dinitrophenylhydrazine, dissolve in 30% perchloric acid solution to a final volume of 100 ml), mix immediately on a mixer, and filter through a 0.45 μm membrane. Separately, take an appropriate amount of the test sample, centrifuge at 3000 rpm for 10 minutes, accurately measure 1 ml of the supernatant, and repeat the same procedure from the point mentioned earlier ("accurately add 1 ml of mobile phase"). Accurately inject 10 μl each of the reference solution and the test solution into the liquid chromatograph and record the chromatograms. By performing a linear regression on the peak area corresponding to the concentration of the glutaraldehyde reference solution, the linear regression equation was obtained, and the glutaraldehyde content (μg / ml) in the test solution was calculated.

[0036] Example 1: Preparation of basic ready-to-use fluid gelatin This embodiment aims to verify the feasibility of the basic process route of the present invention, and to evaluate the performance using the application environment in South China as an example.

[0037] (1) Preparation of gelatin solution: Dissolve 10g of type A gelatin in 190g of deionized water in a constant temperature water bath at 55℃ to prepare a 5wt% homogeneous aqueous solution with an initial viscosity of 150mPa·s. Let it stand to remove bubbles before use.

[0038] (2) Enzyme crosslinking and viscosity monitoring: 0.2g of transglutaminase (2wt% of gelatin mass) was added to the above solution, nitrogen gas was introduced, and the mixture was stirred at 300r / min for 30 minutes at 55℃ and pH 7.0. The viscosity of the system was monitored in real time. When the viscosity reached 1500mPa·s, the reaction was stopped. The degree of crosslinking was measured to be 40%.

[0039] (3) Pre-cooling and molding: Quickly pour the cross-linked gelatin solution into the pre-cooled stainless steel mold.

[0040] (4) Freezing and freeze-drying: The foam obtained in step (3) was placed in an environment of -20℃ for 60 min and completely solidified. Then it was transferred to a freeze dryer and dried at -60℃ and -0.98 bar vacuum for 30 hours to obtain the freeze-dried body; (5) Vacuum physical crosslinking: The freeze-dried body from step (4) was placed in a vacuum drying oven, and the vacuum degree was controlled at -0.98 bar and the temperature at 100°C for 10 hours for physical crosslinking; the corresponding crosslinking degree was 85%; (6) Crushing: The cross-linked body in step (5) is mechanically crushed to form porous particles; (7) Freezing and sieving: The porous particles in step (6) were placed in an environment of -20℃ for 60 min and completely solidified. They were then transferred to a freeze dryer and dried at -50℃ and -0.98 bar vacuum for 24 hours. The temperature was then raised to room temperature (25℃) and desorbed for 5 hours to obtain freeze-dried particles. The porous particles with a particle size range of 50-355μm were collected by passing them through standard sieves of 50 mesh, 100 mesh and 150 mesh in sequence. (8) Fluidization and filling: Add 30g of deionized water and 0.1g of glycerol to 10g of porous particles, mix and dissolve to form a ready-to-use fluid gelatin with a water content of about 75wt%, and directly perform aseptic filling to obtain a shear-thinning fluid gelatin hemostatic material.

[0041] Example 2: Preparation of high-viscosity, high-crosslinking ready-to-use fluid gelatin This embodiment aims to verify the effect of high initial viscosity and high crosslinking degree synergistically improving the hydrophobicity and structural stability of the material.

[0042] (1) Preparation of gelatin solution: Dissolve 10g of type A gelatin in 115g of deionized water in a constant temperature water bath at 55℃ to prepare an 8wt% homogeneous aqueous solution with an initial viscosity of 500mPa·s, and let it stand to remove bubbles.

[0043] (2) Enzyme crosslinking and viscosity monitoring: 0.5 g of transglutaminase (5 wt% of gelatin mass) was added to the above solution, nitrogen gas was introduced, and crosslinking was carried out at 350 r / min for 40 minutes at 58℃ and pH 7.2. The viscosity was monitored in real time and the reaction was stopped when it reached 1800 mPa·s. The degree of crosslinking was measured to be 50%.

[0044] (3) Pre-cooling and molding: Same as step (3) in Example 1; (4) Freezing and freeze drying: The gradient cooling pre-freezing program is adopted (-20℃ 1h → -50℃ 2h → -80℃ 12h), and then transferred to the freeze dryer. First, it is at -50℃ for 24h and then at -40℃ for 12h. Then, it is heated to room temperature (25℃) for desorption drying for 6 hours with a vacuum degree ≤5Pa.

[0045] (5) Vacuum physical crosslinking: The freeze-dried body from step (4) was placed in a vacuum drying oven, and the vacuum degree was controlled at -0.98 bar and the temperature at 120°C for 12 hours for physical crosslinking; the corresponding crosslinking degree was 90%. (6) Crushing: Same as step (6) in Example 1; (7) Freezing and sieving: The porous particles in step (6) were placed in an environment of -20℃ for 60 min and completely solidified. They were then transferred to a freeze dryer and dried at -50℃ and -0.98 bar vacuum for 48 hours. The temperature was then raised to room temperature (25℃) and desorbed for 8 hours to obtain freeze-dried particles. The porous particles with a particle size range of 50-355 μm were collected by passing them through standard sieves of 50 mesh, 100 mesh and 150 mesh in sequence. (8) Fluidization and filling: Add 25g of deionized water and 0.1g of glycerol to 10g of porous particles, mix and dissolve to form a ready-to-use fluid gelatin with a water content of about 71wt%, and directly perform aseptic filling to obtain a shear-thinning fluid gelatin hemostatic material.

[0046] Example 3: Preparation of low-viscosity, rapid cross-linking, ready-to-use fluid gelatin This embodiment aims to demonstrate the feasibility of rapidly crosslinking to prepare ready-to-use fluid gelatin at lower viscosity.

[0047] (1) Preparation of gelatin solution: Dissolve 10g of type A gelatin in 240g of deionized water in a constant temperature water bath at 50℃ to prepare a 4wt% homogeneous aqueous solution with an initial viscosity of 80mPa·s.

[0048] (2) Enzyme crosslinking and viscosity monitoring: Add 0.05g of transglutaminase (0.5wt%), introduce nitrogen gas, and stir at 400r / min for 15 minutes at 52℃ and pH 6.8. Stop when the viscosity reaches 1000mPa·s. The degree of crosslinking is about 20%.

[0049] Steps (3) to (8) are the same as in Example 1.

[0050] Example 4: Aseptic filling and performance verification of ready-to-use fluid gelatin This embodiment focuses on verifying the aseptic filling process and performance indicators of the final product.

[0051] Steps (1) to (7) are the same as in Example 1, to prepare porous particles.

[0052] (8) Fluidization: Add 35g of deionized water and 0.1g of glycerol to 10g of porous particles, mix evenly to form a ready-to-use fluid with a water content of about 78wt%.

[0053] (9) Aseptic filling: The fluid gelatin is filled in a clean room (Class C environment), packaged using pre-sterilized syringes or tubular bags, sealed and sterilized by ethylene oxide or irradiation to ensure the product is sterile.

[0054] (10) Performance test: After filling, the product has a water contact angle of 102°, a swelling rate of 7.5%, a maximum injection force of 15N, a hemostasis time of 95s, and an RGR of 105%, all of which meet the standards for ready-to-use medical devices.

[0055] Comparison Example 1: Traditional powders require pre-operative preparation. The steps are exactly the same as in Example 1, but no water is added in step (8), and it is packaged directly in powder form. When using it, it needs to be mixed with sterile water on site.

[0056] Comparative Example 2: Chemical cross-linking agent (glutaraldehyde cross-linking) The steps are basically the same as in Example 1, but the following modifications are made: The enzyme cross-linking in step (2) was omitted, and instead, 2.5 wt% glutaraldehyde aqueous solution was added after step (1) as a cross-linking agent. Cross-linking was carried out under the same conditions to compare and verify the superiority of biological cross-linking of glutamine transaminase compared with traditional chemical cross-linking.

[0057] Control Example 3: Single enzyme cross-linking (glutamine transaminase) The steps are basically the same as in Example 1, but the following modifications are made: The vacuum physical cross-linking in step (5) was omitted to compare and verify the superiority of the enzyme-physical double cross-linking system.

[0058] Comparison with Example 4: Single physical crosslinking (vacuum high-temperature crosslinking) The steps are basically the same as in Example 1, but the following modifications are made: The enzyme cross-linking in step (2) is omitted and replaced with step (1) followed by direct freezing and lyophilization to compare and verify the superiority of the enzyme-physical double cross-linking system.

[0059] Comparative Example 5: Johnson & Johnson's absorbable hemostatic fluid gelatin was selected. Johnson & Johnson's absorbable hemostatic fluid gelatin product was selected and compounded according to its instructions before testing.

[0060] Performance testing and data analysis The products obtained from the above embodiments and comparative examples were systematically tested, and the results are recorded in the table below: Table 1: Performance Comparison Table of Example and Control Example Products

[0061] As can be seen from the table above: 1. The ready-to-use fluid format significantly improves clinical convenience and safety. Example 1 4. All prepared products are ready-to-use fluid gelatin, which can be directly injected or applied without preoperative preparation or dilution. Test results show that the maximum injection force is between 12. Between 19 N and 22 G, the needle can pass smoothly without clogging or interruption. Compared with Control Example 1, which requires on-site preparation, and Control Example 5, which requires dilution, the ready-to-use design of this invention significantly shortens the surgical preparation time, reduces the complexity of operation, avoids the risk of microbial contamination that may be introduced due to intraoperative preparation, and improves surgical safety and efficiency. Example 4 further demonstrates the feasibility of the aseptic filling process. The product can be directly mass-produced and terminally sterilized, which meets the medical device aseptic supply specifications and has good clinical applicability and industrialization foundation.

[0062] 2. The bio-enzyme-physical dual cross-linking system ensures material safety and biocompatibility. This invention utilizes transglutaminase as a cross-linking agent in conjunction with vacuum physical cross-linking to form a dual cross-linking system. This is a purely bio-based cross-linking system, replacing chemical cross-linking agents such as glutaraldehyde, fundamentally avoiding the introduction and residue of toxic substances. Cell compatibility tests show that the relative growth rate (RGR) of L929 cells in all examples is >98%, demonstrating excellent biocompatibility. In contrast, Control Example 2, which uses glutaraldehyde cross-linking, has an RGR of only 75%, posing a significant risk of cytotoxicity. The enzyme-physical dual cross-linking process used in this invention is not only safe and controllable, but also allows for precise control of the cross-linking degree within 80% through real-time viscosity monitoring. Between 90% and 90%, repeatability and batch consistency of material properties were achieved. Compared with single crosslinking systems (Comparative Examples 3 and 4), it showed superior advantages in terms of crosslinking degree, hydrophobicity, and swelling rate.

[0063] 3. The low swelling ratio design significantly reduces the risk of tissue compression. Swelling rate is a key indicator for evaluating the safety of hemostatic materials in vivo. The high swelling property of traditional materials is one of the main risks leading to postoperative complications. This invention, through optimized cross-linking degree and porous structure synergistic regulation, strictly controls the swelling rate of the material in PBS to below 10%. Example 1 The swelling rates of Example 4 were 8.2%, 5.1%, 9.8%, and 7.5%, respectively, all of which were much lower than 85% of Example 2 and 25% of Example 5. In particular, in Example 2, after the degree of crosslinking was increased to 90%, the swelling rate further decreased to 5.1%, indicating that the dense network constructed with a high degree of crosslinking can effectively inhibit water intrusion, so that the material hardly undergoes any volume change in body fluid, maintains structural integrity, and thus fundamentally avoids secondary compression damage to delicate tissues such as surrounding nerves and blood vessels caused by material expansion.

[0064] 4. Significantly improved hydrophobic properties enhance wound adhesion and hemostasis durability. Hydrophobicity is a crucial factor affecting the adhesion and stability of materials on moist wounds. This invention significantly improves the hydrophobic properties of the material through the synergistic effect of a dense cross-linked network constructed using transglutaminase and vacuum physical cross-linking, combined with the physical structure of the porous particle surface. Water contact angle tests show that… (Example 1) The contact angles of the four examples were 102°, 118°, 95° and 102°, respectively, all greater than 90°, showing obvious hydrophobic properties. In particular, Example 2 had a contact angle as high as 118°, indicating that the high cross-linking and porous structure can effectively reduce the exposure of hydrophilic groups on the surface, enhance the material's repulsion of blood and tissue fluid, and facilitate the formation of a stable cover on the bleeding wound, thus prolonging the duration of the hemostatic barrier. In contrast, the contact angle of Control Example 5 was only 75%, and its hydrophobicity was significantly insufficient.

[0065] 5. Excellent shear-thinning behavior and fine-diameter delivery performance All products in the embodiments exhibit significant shear-thinning behavior, with viscosity decreasing as shear rate increases during injection. The injection force is moderate, allowing for smooth delivery using fine-diameter injection instruments such as 22G. This characteristic makes the material suitable for minimally invasive surgery and deep wounds. It enables the material to fully conform to irregular wound surfaces, improving the uniformity and conformity of hemostatic material coverage. It is particularly suitable for surgical scenarios with high operational precision requirements, such as neurosurgery and spinal surgery.

[0066] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A dual cross-linked hydrophobic fluid gelatin hemostatic material, characterized in that, It consists of the following components: The gelatin matrix is ​​type A gelatin. The cross-linking system contains transglutaminase, and its dosage is 0.1wt%-5wt% of the gelatin mass; Glycerin, used in an amount of 1 wt% of the total mass of the final fluid gelatin; Deionized water; The viscosity of the gelatin solution before crosslinking is 100-800 mPa·s, and the viscosity after crosslinking is 1000-2000 mPa·s.

2. The dual crosslinked hydrophobic fluid gelatin hemostatic material according to claim 1, characterized in that, The degree of crosslinking is 80%-90%, and the degree of crosslinking can be precisely controlled by adjusting the dosage of transglutaminase, crosslinking temperature, crosslinking time, and vacuum physical crosslinking temperature and time.

3. The dual crosslinked hydrophobic fluid gelatin hemostatic material of claim 1, wherein, The final fluid gelatin has a moisture content of 70-85 wt% and can be used directly for injection or coating without additional dilution.

4. A method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material as described in any one of claims 1 or 3, characterized in that, Includes the following steps: Step (1): After fully dissolving type A gelatin in a constant temperature water bath at 40-65℃, prepare a homogeneous aqueous solution with a mass concentration of 4wt%-20wt%, and control its initial viscosity to be 100-800mPa·s; Step (2): Add transglutaminase to the homogeneous aqueous solution obtained in step (1), the amount of which is 0.1wt%-5wt% of the gelatin mass. At the same time, nitrogen gas is introduced and the stirring is accelerated at a speed of 100-800 r / min. The mixture is stirred and crosslinked for 10-50 min at 50-60℃ and pH 6.8-7.

2. The crosslinking process is controlled by monitoring the viscosity change of the system in real time. When the viscosity reaches 1000-2000 mPa·s, the reaction is stopped, and the corresponding degree of crosslinking is 20%-50%. Step (3): Transfer the gelatin solution obtained in step (2) to a pre-cooled mold; Step (4): Place the foam obtained in step (3) in an environment of -20℃ for 60 min. After it is completely solidified, transfer it to a freeze dryer and dry it under a vacuum of -0.98 bar for 20-48 hours to obtain the freeze-dried body. Step (5): Place the freeze-dried body from step (4) in a vacuum drying oven, control the vacuum degree to -0.98 bar, the temperature to 80℃-150℃, and perform physical cross-linking for 5-24 hours; the corresponding cross-linking degree is 80%-90%. Step (6): The cross-linked body in step (5) is mechanically crushed to form porous particles; Step (7): The porous particles in step (6) are placed in a -20℃ environment and frozen for 90 min. After complete solidification, they are transferred to a freeze dryer and dried under a vacuum of -0.98 bar for 20-48 hours to obtain freeze-dried particles. The porous particles with a particle size range of 50-355 μm are collected by passing them through standard sieves of 50 mesh, 100 mesh and 150 mesh in sequence. Step (8): Add deionized water and glycerol in a preset mass ratio to the porous particles obtained in step (7), mix and dissolve to form ready-to-use shear-thinning fluid gelatin, with the moisture content controlled at 70-85 wt%, which can be directly aseptically filled.

5. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, In step (2), the speed of the bubbler is 400 r / min and the bubbling time is 50 min.

6. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, In steps (4) and (7), the freezing process is a gradient cooling from -20℃ for 1h to -50℃ for 2h to -80℃ for 12h. In the freeze-drying stage, the temperature is first -50℃ for 24h and then -40℃ for 12h, and then the temperature is raised to room temperature (20-25℃) for desorption drying for 4-6h, with a vacuum degree ≤5Pa.

7. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, The step (7) uses a vibrating sieve machine for sieving, with a sieving frequency of 50 Hz, an amplitude of 1.5 mm, and collecting the particles in the range of 50-355 μm, with a D 10 =70 μm, D 50 =180 μm, and D 90 =320 μm.

8. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, In step (8), the mass ratio of porous particles to water is 1:2.5-3.5, and the amount of glycerol added is 1 wt% of the total mass.

9. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, In step (2), the degree of crosslinking is synergistically controlled by the following parameters: (a) The amount of transglutaminase used is 0.1wt%-5wt% of the gelatin mass, and the amount used is positively correlated with the degree of cross-linking; (b) The crosslinking temperature is controlled at 50-60℃, and the pH is maintained at 6.8-7.2; (c) The cross-linking time is 10-50 min. The viscosity change is monitored in real time. The reaction is stopped when the viscosity reaches 1000-2000 mPa·s.

10. The method for preparing a double-crosslinked hydrophobic fluid gelatin hemostatic material according to claim 4, characterized in that, The ready-to-use fluid gelatin obtained in step (8) has a swelling rate of ≤10% and a water contact angle of ≥90°. It can be directly used for injection or wound application without preoperative preparation.

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