Composite hemostatic gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hemostatic materials, in particular to composite hemostatic gel as well as a preparation method and application thereof. The composite hemostatic gel is prepared from the following components in percentage by mass: 0.5 to 3 percent of carboxymethyl chitosan, 0.5 to 5 percent of trehalose, 1 to 10 percent of glycerol, 0.1 to 2 percent of carbomer, 0.1 to 2 percent of recombinant blood coagulation factor III, 0.005 to 0.05 percent of benzalkonium chloride and the balance of Tris-HCl buffer solution. Based on the synergistic effect of a natural high polymer material carboxymethyl chitosan and a bioactive component recombinant blood coagulation factor III, the gel constructs a'physical adsorption-blood coagulation activation 'dual hemostasis pathway, realizes a rapid and multi-mechanism hemostasis function, has antibacterial and repair promoting characteristics, can be widely applied to various bleeding scenes such as cutting wounds and surgical wounds, and has wide application prospects. The wound healing is effectively promoted.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials technology, and in particular to a composite hemostatic gel, its preparation method, and its application. Background Technology

[0002] Traumatic bleeding is a priority issue in clinical treatment. Untimely hemostasis can easily lead to complications such as infection and delayed healing. Traditional suturing methods are limited in handling irregular wounds, small-area oozing, or emergency hemostasis due to their complexity and potential for secondary damage. To overcome these limitations, hemostatic hydrogels have become a research hotspot in the field of medical hemostatic materials due to their good biocompatibility, wound adhesion, and adjustable properties. Among them, carboxymethyl chitosan-containing hydrogels are widely used in the development of hemostatic materials because they retain the biodegradability of chitosan and have significantly improved water solubility. However, existing carboxymethyl chitosan-containing hemostatic materials still have key technical deficiencies, making it difficult to meet the actual clinical needs for efficient and stable hemostasis.

[0003] Specifically, existing technologies for preparing hemostatic materials containing carboxymethyl chitosan often use water or acidic solutions such as dilute acetic acid or hydrochloric acid as the dissolving medium for carboxymethyl chitosan. These media cannot precisely control the pH value of the solution, easily leading to uneven dissolution of carboxymethyl chitosan or affecting the stability of its molecular structure, thus adversely impacting the overall performance of the material. Furthermore, when mixing carboxymethyl chitosan with related excipients, existing technologies often employ direct mixing, which easily triggers adverse interactions between components, resulting in uneven dispersion of the excipients. This not only reduces the intended functional effectiveness of the excipients but may also pose a risk of wound irritation due to abnormal local excipient concentrations. In addition, most existing hemostatic materials containing carboxymethyl chitosan rely solely on a single physical adsorption mechanism for hemostasis, resulting in low hemostatic efficiency and failing to meet the core clinical need for rapid hemostasis. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by providing a composite hemostatic gel, its preparation method, and its application. Through the synergistic effect of the natural polymer material carboxymethyl chitosan and the bioactive component recombinant coagulation factor III, a dual hemostatic pathway of "physical adsorption-coagulation activation" is constructed, achieving rapid and multi-mechanism hemostasis. It also has antibacterial and repair-promoting properties, and can be widely applied to various bleeding scenarios such as lacerations and surgical wounds, effectively promoting wound healing.

[0005] To achieve the above objectives, the present invention provides a composite hemostatic gel, comprising the following components by weight percentage: Carboxymethyl chitosan 0.5%~3%, trehalose 0.5%~5%, glycerol 1%~10%, carbomer 0.1%~2%, recombinant coagulation factor III 0.1%~2%, benzalkonium chloride 0.005%~0.05%, with the balance being Tris-HCl buffer.

[0006] Preferably, the degree of substitution of carboxymethyl chitosan is 70% to 95%.

[0007] Preferably, the carbomer is selected from at least one of carbomer 934, carbomer 940, and carbomer 941.

[0008] Preferably, the concentration of the Tris-HCl buffer is 20 mmol / L to 50 mmol / L; the pH value of the Tris-HCl buffer is 7.0 to 7.5.

[0009] The present invention also provides a method for preparing the aforementioned composite hemostatic gel, comprising the following steps: S1. Mix Tris-HCl buffer, benzalkonium chloride solution, carboxymethyl chitosan and trehalose solution to obtain the mother liquor; S2. Mix the mother liquor, glycerol solution, recombinant coagulation factor III, carbomer solution and Tris-HCl buffer, and defoam to obtain a composite hemostatic gel.

[0010] Preferably, in S1, the benzalkonium chloride solution has a mass fraction of 5% to 15%, and the trehalose solution has a mass fraction of 20% to 30%.

[0011] Preferably, in S1, the mass fraction of carboxymethyl chitosan in the mother liquor is 3%~7%, the mass fraction of benzalkonium chloride is 0.05%~0.15%, and the mass fraction of trehalose is 2%~6%.

[0012] Preferably, in S2, the mass fraction of the glycerol solution is 45%~55%, and the mass fraction of the carbomer solution is 1%~2%.

[0013] The present invention also provides the application of the composite hemostatic gel described above, or the composite hemostatic gel prepared according to the preparation method of the composite hemostatic gel described above, in the preparation of hemostatic materials.

[0014] The beneficial effects of this invention are as follows: 1. This invention provides a composite hemostatic gel comprising, by mass percentage: 0.5%–3% carboxymethyl chitosan, 0.5%–5% trehalose, 1%–10% glycerol, 0.1%–2% carbomer, 0.1%–2% recombinant coagulation factor III, 0.005%–0.05% benzalkonium chloride, with the balance being Tris-HCl buffer. Based on the synergistic effect of the natural polymer carboxymethyl chitosan and the bioactive component recombinant coagulation factor III, this gel constructs a dual hemostatic pathway of "physical adsorption-coagulation activation," achieving rapid, multi-mechanism hemostasis. It also possesses antibacterial and wound-healing properties, making it widely applicable to various bleeding scenarios such as lacerations and surgical wounds, effectively promoting wound healing.

[0015] 2. The core component of this composite gel, carboxymethyl chitosan, is a derivative of chitosan. Chitosan, extracted from natural chitin, possesses excellent biocompatibility and biodegradability. By introducing carboxymethyl groups into the chitosan molecular chain, its water solubility is significantly improved, making it an amphoteric polyelectrolyte that can dissolve under neutral conditions. Carboxymethyl chitosan, with its positive charge, can effectively adsorb negatively charged red blood cells, rapidly sealing the wound to form a physical barrier and achieve initial hemostasis. Simultaneously, it can promote the proliferation and migration of fibroblasts, accelerating wound healing.

[0016] 3. Recombinant coagulation factor III, as the core of this system's bioactivity, plays a crucial role in the coagulation process. When blood vessels are damaged, coagulation factor III is exposed to the blood and binds to coagulation factor VII, forming a TF / FVIIa complex with the participation of calcium ions. This complex then activates downstream coagulation factors IX and X, ultimately promoting the conversion of prothrombin to thrombin. Thrombin cleaves fibrinogen to form a fibrin network, which surrounds platelets and blood cells to form a stable thrombus, thus efficiently completing the physiological hemostasis process.

[0017] 4. This invention achieves synergistic hemostasis through a dual "adsorption-activation" pathway: Carboxymethyl chitosan can rapidly form a sealing layer on the wound surface, enriching coagulation factors and providing a high-concentration microenvironment for the coagulation reaction; simultaneously, recombinant coagulation factor III directionally activates the coagulation cascade reaction, thereby significantly shortening the coagulation time. Experimental results show that the in vitro coagulation index (BCI) of this composite gel can reach as low as 18.43%, and the hemostasis time is shortened by more than 50% compared with commercially available products, demonstrating excellent procoagulant properties. Animal experiments further confirm its significantly shortened hemostasis time and obvious hemostatic advantage. In addition, cytotoxicity experiments show that the material has no significant cytotoxicity, meeting the safety requirements for biomaterials.

[0018] 5. Other functional components in the system also play important auxiliary roles: Trehalose, as a stabilizer, protects the structural integrity of recombinant coagulation factor III through hydrogen bonding, preventing its denaturation and inactivation; Glycerin acts as a moisturizer and plasticizer, improving the gel feel and preventing drying; Carbomer, as the main thickener and suspending agent of the gel matrix, provides suitable viscoelasticity and sustained-release effect; Benzalkonium chloride acts as a preservative, ensuring the microbial safety of the product during use and storage; Tris-HCl buffer maintains the system in a neutral pH environment, maximizing the activity and stability of recombinant coagulation factor III.

[0019] 6. Furthermore, carboxymethyl chitosan and carbomer form a stable three-dimensional network structure through intermolecular hydrogen bonds and molecular chain entanglement. Under neutral to alkaline conditions, the carboxyl groups of carbomer dissociate, and the chains extend and swell to form a primary gel network. Carboxymethyl chitosan forms multiple hydrogen bonds with the carboxyl / carboxylate groups of carbomer through its hydroxyl, amino, and carboxyl groups, interpenetrating and reinforcing this network, ultimately forming a continuous gel system capable of physically locking in water, thus endowing the product with excellent viscoelasticity and mechanical stability.

[0020] In summary, this composite hemostatic gel not only effectively integrates the dual hemostatic mechanisms of physical sealing and biochemical activation, but also has the advantages of reasonable composition, high stability, and good biocompatibility. It is suitable for various acute and chronic wound hemostasis needs and has good clinical application prospects. Attached Figure Description

[0021] Figure 1 These are characterization images of the composite hemostatic gel after freeze-drying in Examples 1-3 of this invention; Figure 2 These are coagulation phenomena diagrams of the present invention NJ36-1, NJ36-1 without factor, NJ33-1, NJ33-1 without factor, commercially available medical biological adhesive A, and commercially available medical biological adhesive B; Figure 3 This is a schematic diagram showing the BCI value detection results of different samples in this invention; Figure 4 This is a schematic diagram of the 12-hour cell viability values ​​of different samples in this invention; Figure 5 This is a schematic diagram of the 24-hour cell viability values ​​of different samples in this invention; Figure 6 This is a schematic diagram of the 36-hour cell viability values ​​of different samples in this invention; Figure 7 These are diagrams illustrating the hemostatic effects of different gels from this invention. Detailed Implementation

[0022] This invention provides a composite hemostatic gel, comprising the following components by weight percentage: Carboxymethyl chitosan 0.5%~3%, trehalose 0.5%~5%, glycerol 1%~10%, carbomer 0.1%~2%, recombinant coagulation factor III 0.1%~2%, benzalkonium chloride 0.005%~0.05%, with the balance being Tris-HCl buffer.

[0023] In this invention, the degree of substitution of carboxymethyl chitosan is 70% to 95%, preferably 80% to 90%.

[0024] The degree of substitution is the rate of substitution of carboxymethyl groups on the carboxymethyl chitosan unit.

[0025] In this invention, the mass percentage of trehalose is 0.5% to 5%, preferably 1% to 2%.

[0026] In this invention, the mass percentage of glycerol is 1% to 10%, preferably 1% to 5%.

[0027] In this invention, the mass percentage of recombinant coagulation factor III is 0.1% to 2%, preferably 0.5% to 1%.

[0028] In this invention, recombinant coagulation factor III is obtained by purchasing or by preparing it using conventional techniques in the art.

[0029] In this invention, the mass percentage of benzalkonium chloride is 0.005% to 0.05%, preferably 0.01% to 0.05%.

[0030] In this invention, the carbomer is selected from at least one of carbomer 934, carbomer 940, and carbomer 941.

[0031] In this invention, the concentration of the Tris-HCl buffer is 20 mmol / L to 50 mmol / L; the pH value of the Tris-HCl buffer is 7.0 to 7.5.

[0032] The concentration of Tris-HCl buffer refers to the concentration of tris(hydroxymethyl)aminomethane in the Tris-HCl buffer.

[0033] The present invention also provides a method for preparing the aforementioned composite hemostatic gel, comprising the following steps: S1. Mix Tris-HCl buffer, benzalkonium chloride solution, carboxymethyl chitosan and trehalose solution to obtain the mother liquor; S2. Mix the mother liquor, glycerol solution, recombinant coagulation factor III, carbomer solution and Tris-HCl buffer, and defoam to obtain a composite hemostatic gel.

[0034] In this invention, in step S1, benzalkonium chloride and water are mixed to obtain a benzalkonium chloride solution; the mass fraction of benzalkonium chloride in the benzalkonium chloride solution is 5%~15%.

[0035] In this invention, in step S1, trehalose and water are mixed to obtain a trehalose solution; the mass fraction of trehalose in the trehalose solution is 20%~30%.

[0036] In this invention, S1, mixing includes: sequentially adding benzalkonium chloride solution, carboxymethyl chitosan and trehalose solution to Tris-HCl buffer.

[0037] In this invention, in S1, the mass fraction of carboxymethyl chitosan in the mother liquor is 3%~7%, the mass fraction of benzalkonium chloride is 0.05%~0.15%, and the mass fraction of trehalose is 2%~6%.

[0038] In this invention, in step S2, glycerol and water are mixed to obtain a glycerol solution; the mass fraction of glycerol in the glycerol solution is 45% to 55%.

[0039] In this invention, in step S2, carbomer is added to water and allowed to swell for 10-14 hours. The carbomer is then stirred to dissolve, and an alkaline solution is added to adjust the pH to 6-7 to obtain a carbomer solution. The alkaline solution includes sodium hydroxide solution with a concentration of 0.5 mol / L to 1.5 mol / L. The mass fraction of carbomer in the carbomer solution is 1% to 2%.

[0040] In this invention, S2, mixing includes: adding glycerol solution and recombinant coagulation factor III to the mother liquor, stirring until homogeneous, adding carbomer solution, and finally adding Tris-HCl buffer solution and stirring until homogeneous.

[0041] This invention first premixes benzalkonium chloride, trehalose, and carboxymethyl chitosan in Tris-HCl buffer to form a mother liquor. This strategy ensures highly uniform dispersion of key small-molecule excipients at the molecular level, effectively avoiding problems such as insoluble precipitation that may occur when benzalkonium chloride is directly mixed with other polymers, thus enhancing the compatibility and stability of the system. Simultaneously, trehalose, in its pre-dissolved state, can more fully exert its stabilizing and protective effects. More importantly, this mother liquor provides a mild and stable buffer environment for recombinant coagulation factor III, which is sensitive to pH and the environment, allowing it to be introduced only in the final formulation stage. This significantly shortens its exposure time to potentially adverse conditions, thereby significantly protecting the bioactivity of recombinant coagulation factor III and ensuring the hemostatic efficacy of the product. Furthermore, the preparation method of this invention also improves the controllability and batch-to-batch consistency of the entire preparation process, which is beneficial for large-scale production.

[0042] The present invention also provides the application of the composite hemostatic gel described above, or the composite hemostatic gel prepared according to the preparation method of the composite hemostatic gel described above, in the preparation of hemostatic materials.

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0044] In the following embodiments of the present invention, the degree of substitution of carboxymethyl chitosan was 80%, purchased from Jiangsu Aoxin Biotechnology Co., Ltd.; trehalose was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.; glycerol was purchased from Sangon Biotech (Shanghai) Co., Ltd.; carbomer was carbomer 940, purchased from Jiangsu Ruidian Pharmaceutical Co., Ltd.; benzalkonium chloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the concentration of Tris-HCl buffer was 20 mmol / L, the pH value was 7.5, and it was purchased from Vibio (Beijing) Biotechnology Co., Ltd.; the recombinant coagulation factor III had the catalog number BX1001 and the batch number A20220801, and was provided by Shanxi Boxin Biotechnology Co., Ltd.

[0045] Example 1 This embodiment provides a composite hemostatic gel, which, by mass percentage, contains the following components: Carboxymethyl chitosan 1%, trehalose 1%, glycerol 2%, carbomer 0.5%, recombinant coagulation factor III 1%, benzalkonium chloride 0.02%, with the balance being Tris-HCl buffer.

[0046] The preparation method includes the following steps: A 10% benzalkonium chloride solution was obtained by mixing benzalkonium chloride and water; a 25% trehalose solution was obtained by mixing trehalose and water; and a 50% glycerol solution was obtained by mixing glycerol and water.

[0047] Add carbomer to 30 mL of water and allow it to swell naturally for 12 hours. After swelling, stir to dissolve the carbomer, then add 1 mol / L sodium hydroxide solution to adjust the pH to 6.5. Finally, add water to obtain a 1% carbomer solution.

[0048] Add benzalkonium chloride solution to 10 mL Tris-HCl buffer, then add carboxymethyl chitosan and stir until dissolved, then add trehalose solution, and finally adjust the volume to 20 mL with Tris-HCl buffer to obtain the mother liquor (the mass fraction of carboxymethyl chitosan in the mother liquor is 5%, the mass fraction of benzalkonium chloride is 0.1%, and the mass fraction of trehalose is 5%).

[0049] According to the mass percentage of each component in the composite hemostatic gel, take the mother liquor, add glycerol solution and recombinant coagulation factor III, stir evenly, add carbomer solution, and finally add Tris-HCl buffer, stir evenly, and after defoaming, obtain the composite hemostatic gel (labeled as NJ33-1).

[0050] Example 2 This embodiment provides a composite hemostatic gel, which, by mass percentage, contains the following components: The formula consists of 2% carboxymethyl chitosan, 1% trehalose, 5% glycerol, 0.6% carbomer, 1% recombinant coagulation factor III, 0.02% benzalkonium chloride, and the balance being Tris-HCl buffer.

[0051] The preparation method includes the following steps: A 10% benzalkonium chloride solution was obtained by mixing benzalkonium chloride and water; a 25% trehalose solution was obtained by mixing trehalose and water; and a 50% glycerol solution was obtained by mixing glycerol and water.

[0052] Add carbomer to 20 mL of water and allow it to swell naturally for 12 hours. After swelling, stir to dissolve the carbomer, then add 1 mol / L sodium hydroxide solution to adjust the pH to 6.5. Finally, add water to obtain a 2% carbomer solution.

[0053] Add benzalkonium chloride solution to 10 mL Tris-HCl buffer, then add carboxymethyl chitosan and stir until dissolved, then add trehalose solution, and finally adjust the volume to 20 mL with Tris-HCl buffer to obtain the mother liquor (the mass fraction of carboxymethyl chitosan in the mother liquor is 5%, the mass fraction of benzalkonium chloride is 0.05%, and the mass fraction of trehalose is 2.5%).

[0054] According to the mass percentage of each component in the composite hemostatic gel, take the mother liquor, add glycerol solution and recombinant coagulation factor III, stir evenly, add carbomer solution, and finally add Tris-HCl buffer, stir evenly, and after defoaming, obtain the composite hemostatic gel (labeled as NJ36-1).

[0055] Example 3 This embodiment provides a composite hemostatic gel, which, by mass percentage, contains the following components: The formula consists of 2% carboxymethyl chitosan, 1% trehalose, 5% glycerol, 0.5% carbomer, 1% recombinant coagulation factor III, 0.02% benzalkonium chloride, and the balance being Tris-HCl buffer.

[0056] The preparation method includes the following steps: A 10% benzalkonium chloride solution was obtained by mixing benzalkonium chloride and water; a 25% trehalose solution was obtained by mixing trehalose and water; and a 50% glycerol solution was obtained by mixing glycerol and water.

[0057] Add carbomer to 20 mL of water and allow it to swell naturally for 12 hours. After swelling, stir to dissolve the carbomer, then add 1 mol / L sodium hydroxide solution to adjust the pH to 6.5. Finally, add water to obtain a 2% carbomer solution.

[0058] Add benzalkonium chloride solution to 10 mL Tris-HCl buffer, then add carboxymethyl chitosan and stir until dissolved, then add trehalose solution, and finally adjust the volume to 20 mL with Tris-HCl buffer to obtain the mother liquor (the mass fraction of carboxymethyl chitosan in the mother liquor is 5%, the mass fraction of benzalkonium chloride is 0.05%, and the mass fraction of trehalose is 2.5%).

[0059] According to the mass percentage of each component in the composite hemostatic gel, take the mother liquor, add glycerol solution and recombinant coagulation factor III, stir evenly, add carbomer solution, and finally add Tris-HCl buffer, stir evenly, and after defoaming, obtain the composite hemostatic gel (labeled as NJ39).

[0060] Comparative Example 1 This comparative example provides a hemostatic gel, which differs from Example 1 in that the addition of recombinant coagulation factor III is omitted (labeled as NJ33-1 factorless).

[0061] Comparative Example 2 This comparative example provides a hemostatic gel, which differs from Example 2 in that the addition of recombinant coagulation factor III is omitted (labeled as NJ36-1 factorless).

[0062] Experimental Example 1 The viscosity of the composite hemostatic gels obtained in Examples 1-3 was tested using a Brookfield DV2T digital rotational viscometer. The test sample was placed in a beaker on a horizontal platform, and a suitable rotor was screwed into the instrument's connecting screw. The instrument's lifting knob was rotated to gradually immerse the rotor in the sample liquid until the liquid level was level with the marking line on the rotor shaft, ensuring the rotor was centered in the beaker. The instrument was then leveled again. Subsequently, the appropriate rotor and rotation speed were selected, and the measurement was performed at room temperature (25°C). The results were recorded after the test. The results showed that the viscosity values ​​of the gels prepared in Examples 1, 2, and 3 were 3770 mPa·s (test parameters: rotor RV-6, rotation speed 100 rpm), 34200 mPa·s (test parameters: rotor RV-7, rotation speed 100 rpm), and 23320 mPa·s (test parameters: rotor RV-7, rotation speed 100 rpm), respectively. The viscosity values ​​of all samples met the requirements for the gel's intended use.

[0063] Experimental Example 2 The composite hemostatic gels obtained in Examples 1-3 were freeze-dried and their macroscopic structure was observed. Characterization images of the composite hemostatic gels after freeze-drying in Examples 1-3 were obtained, as shown in the figure. Figure 1 As shown. The appearance of the samples was visually observed under room temperature and fluorescent lighting conditions. Figure 1 As can be seen, the sample of Example 1 has a uniform texture and a dense structure; the sample of Example 2 has a non-uniform texture but a denser structure; and the sample of Example 3 has a uniform texture and a relatively dense structure.

[0064] Experimental Example 3 Weigh out 100 mg each of the gel samples (NJ33-1, NJ36-1) prepared in the examples and the control samples (comparative example 1 hemostatic gel, comparative example 2 hemostatic gel, commercially available medical bio-adhesive A (Zhende medical bio-adhesive, purchased from Qingdao Boyite Biomaterials Co., Ltd., model: B, specification: 15g) and commercially available medical bio-adhesive B (Boyite Chuangyu Wound Healing Ointment Medical Bio-adhesive, purchased from Qingdao Boyite Biomaterials Co., Ltd., model: B, specification: 10g) and place them in sterile petri dishes and spread them evenly. Then, uniformly add 50 μL of fresh plasma to the surface of each gel sample, and immediately add 20 μL of 0.2 mol / L CaCl2 solution to initiate the coagulation process. Closely observe and record the coagulation phenomenon. Obtain coagulation phenomenon diagrams for NJ36-1, NJ36-1 without factor, NJ33-1, NJ33-1 without factor, commercially available medical bio-adhesive A, and commercially available medical bio-adhesive B, as shown in the figure. Figure 2 As shown. From Figure 2It can be seen that in the control group containing commercially available bio-glue A and B and factorless gel, although fibrin formation was observed, only a thin white film formed on the gel surface, with the underlying liquid remaining flowable, indicating incomplete coagulation. In contrast, the gel samples containing recombinant coagulation factor III of this invention all showed superior coagulation effects. Among them, the NJ36-1 sample showed the most significant effect, promoting complete plasma coagulation and forming a stable clot structure. In summary, under the same test conditions, the in vitro plasma coagulation effect of the products of this invention (especially NJ36-1) is significantly better than that of the factorless control group and commercially available products.

[0065] Experiment Example 4 The procoagulant activity of the gel was quantitatively evaluated using the BCI method. 100 mg of gel sample was weighed and spread evenly in a culture dish to form a thin film. The mixture was incubated at 37°C for 5 min, followed by the addition of 50 μL of fresh anticoagulated whole blood and immediately 20 μL of 0.2 mol / L CaCl2 solution. Incubation continued at 37°C for another 5 min. Then, 10 mL of deionized water was slowly added to a portion of the culture dish that was not in contact with the blood (to induce rupture of uncoagulated red blood cells and release hemoglobin through hypotonic effect). The mixture was shaken at 50 rpm and 37°C for 5 min. 1 mL of the supernatant was collected, centrifuged at 7000 rpm for 5 min, and the absorbance (OD value) of the final supernatant was measured at 540 nm. A sample containing only an equal volume of anticoagulated whole blood and CaCl2 solution served as a blank control (its OD value was denoted as A0).

[0066] The BCI value is calculated using the following formula: BCI(%) = (A1 / A0) × 100%, where A1 is the OD value of each sample group. A lower BCI value indicates less residual free hemoglobin, a faster clotting rate, and superior hemostatic performance. The BCI value test results for different samples are shown in Table 1; a schematic diagram of the BCI value test results for different samples is shown below. Figure 3 As shown.

[0067] Table 1. BCI value detection results for different samples

[0068] Table 1 and Figure 3Data shows that the BCI values ​​of all gel samples (NJ33-1, NJ36-1, and NJ39) of this invention were significantly lower than those of the control group. Among them, NJ36-1 had the lowest BCI value, at only 18.43%, exhibiting the fastest clotting rate. Compared with commercially available products (BCI value > 60%), the clotting time of the products of this invention was significantly shortened. According to the formula (BCI1 - BCI2) / BCI1 × 100%, where BCI1 is the BCI of commercially available medical bio-adhesive (A / B), and BCI2 is the BCI of NJ33-1, NJ36-1, or NJ39; the clotting ability of NJ33-1, NJ36-1, and NJ39 was increased by 67.8%, 70.6%, and 51.6% respectively compared with commercially available medical bio-adhesive A, and by 68.3%, 71.1%, and 52.2% respectively compared with commercially available medical bio-adhesive B. The quantitative results are consistent with the conclusions of the plasma coagulation observation experiment, which fully confirms that the composite hemostatic gel of the present invention achieves efficient and rapid hemostasis through the dual-pathway synergistic mechanism of "physical adsorption-coagulation activation".

[0069] Experimental Example 5 The cytotoxicity of hemostatic gels NJ33-1, NJ36-1, and NJ39 was evaluated using the CCK-8 assay. (1) Extraction: 1 g each of hemostatic gels NJ33-1, NJ36-1, and NJ39 were weighed and placed in three conical flasks. The solvent was preheated to 10% dimethyl sulfoxide solution (37°C) at a mass / volume ratio of 1:10, ensuring complete coverage of the sample. The solution was dispersed evenly by stirring with a glass rod, the flasks were sealed to prevent evaporation, and the containers were placed in a constant temperature shaker at 37°C and 150 rpm for 24 h. The extract was centrifuged and the supernatant was used for the cytotoxicity experiment (as a sample in subsequent experiments). (2) Sample dilution: The sample was diluted to 1 mg / mL using DMEM medium, and five concentration gradients of 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL were further set up. (3) Cell culture: Mouse fibroblasts (L929) in logarithmic growth phase were taken, digested with trypsin and resuspended into a single-cell suspension, and cell counts were performed. The cell density was adjusted to 1×10⁻⁶. 5Cells / mL. The cell suspension was seeded into 96-well plates, 100 μL was added to each well (each sample was tested in 3 replicates, and the average value was taken later), and the culture plate was placed in a CO2 incubator at 37°C (temperature) and 5% (CO2 concentration) for 24 h. (4) Detection: The old culture medium in the 96-well plate was aspirated, and each concentration of sample (100 μL) was added in advance. CCK-8 (10 μL) was added after 12 h, 24 h and 36 h respectively. After 4 h of reaction, the absorbance was measured at 450 nm using a Thermo Scientific microplate reader and cell viability was calculated. The formula for calculating cell viability is: Cell viability (%) = (experimental group - blank group) / (negative control group - blank group) × 100%. Experimental group: The above description is the experimental group; Negative control group: The sample described above was replaced with pure DMEM medium; Blank group: No cells were added, and the absorbance of the sample and CCK-8 reagent itself was tested at 450 nm. The final 12-hour cell viability test results for different samples are shown in Table 2; a schematic diagram of the 12-hour cell viability values ​​for different samples is shown below. Figure 4 As shown in Table 3; the 24-hour cell viability test results for different samples are shown in Table 4; a schematic diagram of the 24-hour cell viability values ​​for different samples is shown in Table 5. Figure 5 As shown in Table 4; the 36-hour cell viability test results for different samples are shown in Table 5; a schematic diagram of the 36-hour cell viability values ​​for different samples is shown in Table 6. Figure 6 As shown.

[0070] Table 2. Results of 12-hour cell viability tests for different samples.

[0071] Table 3. Results of 24-hour cell viability tests for different samples

[0072] Table 4. Results of 36-hour cell viability tests for different samples

[0073] The experimental results above show that the cell viability of the three gels was higher than 70% at different time points and concentration gradients, which meets the evaluation standards for the safety of biomaterials and indicates that the samples have no obvious cytotoxicity.

[0074] Experimental Example 6 To evaluate the hemostatic effects of different gels, a rabbit ear margin hemostasis experiment was conducted. The specific steps were as follows: A rabbit was taken, and the fur on both sides of the outer ear margin was cleaned. Starting from the distal end of the left ear, transverse incisions of approximately 1 cm were made, labeled A1 and A2 respectively. The right ear was treated similarly, with the incisions labeled B1 and B2. Then, the corresponding gels were applied to the incisions sequentially: NJ33-1 was applied to A1, NJ36-1 to A2; NJ33-1 (factor-free) was applied to B1, and NJ39 to B2. The time required from application to complete cessation of bleeding was recorded. The experimental results are shown in Table 5; the hemostatic effects of different gels are illustrated in the following figures. Figure 7 As shown.

[0075] Table 5 Experimental Results

[0076] The hemostasis time shows that NJ33-1 and NJ36-1 have better hemostatic effects, and their times are similar; NJ39 requires a longer time, but its hemostatic ability still has a certain advantage compared to factorless hemostatic gel.

[0077] Therefore, the present invention uses the above-mentioned composite hemostatic gel, which, based on the synergistic effect of natural polymer material carboxymethyl chitosan and bioactive component recombinant coagulation factor III, constructs a dual hemostatic pathway of "physical adsorption-coagulation activation", realizing rapid and multi-mechanism hemostatic function, and also has antibacterial and repair-promoting properties. It can be widely used in various bleeding scenarios such as lacerations and surgical wounds, and effectively promotes wound healing.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A composite hemostatic gel, characterized in that, By weight percentage, it contains the following components: Carboxymethyl chitosan 0.5%~3%, trehalose 0.5%~5%, glycerol 1%~10%, carbomer 0.1%~2%, recombinant coagulation factor III 0.1%~2%, benzalkonium chloride 0.005%~0.05%, with the balance being Tris-HCl buffer.

2. The composite hemostatic gel according to claim 1, characterized in that, The degree of substitution of carboxymethyl chitosan is 70%~95%.

3. The composite hemostatic gel according to claim 1, characterized in that, The carbomer is selected from at least one of carbomer 934, carbomer 940, and carbomer 941.

4. The composite hemostatic gel according to claim 1, characterized in that, The concentration of Tris-HCl buffer is 20 mmol / L to 50 mmol / L; the pH of Tris-HCl buffer is 7.0 to 7.

5.

5. The method for preparing the composite hemostatic gel according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Mix Tris-HCl buffer, benzalkonium chloride solution, carboxymethyl chitosan and trehalose solution to obtain the mother liquor; S2. Mix the mother liquor, glycerol solution, recombinant coagulation factor III, carbomer solution and Tris-HCl buffer, and defoam to obtain a composite hemostatic gel.

6. The method for preparing the composite hemostatic gel according to claim 5, characterized in that, In S1, the mass fraction of benzalkonium chloride solution is 5%~15%, and the mass fraction of trehalose solution is 20%~30%.

7. The method for preparing the composite hemostatic gel according to claim 5, characterized in that, In S1, the mass fraction of carboxymethyl chitosan in the mother liquor is 3%~7%, the mass fraction of benzalkonium chloride is 0.05%~0.15%, and the mass fraction of trehalose is 2%~6%.

8. The method for preparing the composite hemostatic gel according to claim 5, characterized in that, In S2, the mass fraction of the glycerol solution is 45%~55%, and the mass fraction of the carbomer solution is 1%~2%.

9. The application of the composite hemostatic gel according to any one of claims 1 to 4 or the composite hemostatic gel prepared by the preparation method according to any one of claims 5 to 8 in the preparation of hemostatic materials.

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