A spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, its preparation method and application

Abstract

This invention relates to a two-component cross-linked oxidized nanocellulose spray hemostatic gel, its preparation method, and its application, belonging to the field of biomedical materials. Addressing the problems of weak adhesion, insufficient mechanical properties, and biocompatibility risks in existing hemostatic materials, this invention introduces aldehyde groups using sodium periodate to prepare an adhesive component (TOCN-CHO), and simultaneously modifies the carboxyl groups of TOCN with lysine to create a cross-linking component (TOCN-Lys). After the two components are sprayed and mixed, the aldehyde groups of TOCN-CHO bind to the amino groups on the tissue surface to achieve wound sealing. Simultaneously, the amino groups of TOCN-Lys form a Schiff base cross-linking network in situ with the aldehyde groups of TOCN-CHO, enhancing mechanical strength while eliminating aldehyde toxicity. The gel retains shear-thinning properties and can be sprayed. Animal experiments show that the hemostasis time is reduced to 28 seconds. It also exhibits biodegradability and renewability, effectively solving the problem of synergistic optimization of adhesion, mechanical strength, and biocompatibility, making it suitable for emergency hemostasis and surgical scenarios.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, its preparation method, and its application. Background Technology

[0002] In the fields of trauma emergency care and surgery, developing highly efficient hemostatic materials is a key technological challenge in reducing the risk of blood loss and shortening the treatment window. Hydrogel dressings, with their three-dimensional network structure, high water content, and good biocompatibility, are considered ideal candidate materials. However, their clinical translation is hampered by two major bottlenecks: insufficient efficient and stable adhesion to wet tissue surfaces and a lack of long-term stability of mechanical properties under dynamic physiological conditions. Existing traditional hemostatic materials, such as oxidized cellulose gauze Surgicel, rely on physical compression and cannot actively adhere to irregular wounds, with hemostasis times generally exceeding 60 seconds. While commercial fibrin glue Tisseel has a certain adhesive strength, its cost is extremely high. Single-component oxidized cellulose gel, although the carboxyl groups in the cellulose structure can lower the pH of the blood, providing an acidic environment that attracts Fe ions from hemoglobin, and can also activate platelets in the blood, causing platelet aggregation and thrombus formation to control bleeding, still lacks sufficient mechanical strength due to its network structure relying solely on physical hydrogen bonds, making it easily washed away by blood flow in cases of significant bleeding.

[0003] To improve adhesion, sodium periodate oxidizes the secondary hydroxyl groups at the C2 / C3 positions to generate dialdehyde cellulose, introducing highly reactive aldehyde groups while retaining the original carboxyl groups. This allows for rapid binding with amino groups on the tissue surface via the Schiff base reaction. However, single-component aldehyde-based TOCN gels carry the risk of significant cytotoxicity due to excessive aldehyde introduction, and the mechanical properties of the purely physical cross-linked network are insufficient to withstand arterial blood pressure shocks.

[0004] Patent application CN115364273A discloses a method for preparing a sprayable nano-oxidized cellulose hemostatic gel. The method involves introducing carboxyl groups at the C6 position of cellulose using a TEMPO / NaClO / NaBr oxidation system, followed by nano-dispersion and the addition of ether and iodoform to create a sprayable material. However, due to its reliance on single-component gelation and the addition of small-molecule drugs, a stable chemical cross-linking network is not constructed. This results in insufficient mechanical strength, susceptibility to being washed away by blood flow, and adhesion performance dependent on physical action rather than chemical bonding. Therefore, its application is significantly limited in addressing irregular wounds with high bleeding volumes commonly encountered in combat trauma emergency care due to its insufficient mechanical strength, weak adhesion, and limited functionality. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, its preparation method, and its application. By constructing an aldehyde-amino two-component Schiff base cross-linking system, rapid and controllable cross-linking can be achieved at the moment of spraying. It has comprehensive advantages such as strong wound adhesion, excellent mechanical properties, good biocompatibility, convenient operation, and degradability, so as to meet the key clinical needs in emergency treatment and surgery.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes the following steps: Step 1: Disperse oxidized cellulose in water to prepare an ONC hydrogel; Step 2: Add a certain proportion of sodium periodate to the ONC hydrogel obtained in Step 1 to react and prepare OCN-CHO hydrogel. Step 3: Add lysine to the carboxyl-activated cellulose system to prepare OCN-Lys hydrogel by reacting it. Step 4: Apply the ONC-CHO hydrogel prepared in Step 2 and the ONC-Lys hydrogel prepared in Step 3 to the wound in sequence. Based on the aldehyde content in ONC-CHO hydrogel and the amino content in ONC-Lys hydrogel, the ratio of aldehyde content in ONC-CHO hydrogel to amino content in ONC-Lys hydrogel is (2-5):1. The aldehyde groups carried by ONC-CHO and the amino groups carried by ONC-Lys undergo a Schiff base reaction to achieve cross-linking and form a stable hemostatic gel.

[0007] The specific method for step 2 includes: Step 2.1: Adjust the solid content of the ONC hydrogel obtained in Step 1 to 0.5-0.7%, and add sodium periodate. The ratio of sodium periodate to solid content in ONC hydrogel is (1-4):1 by mass. Adjust the pH of the system to 2-4 with diluted glacial acetic acid or hydrochloric acid, and react in the dark at 40-50℃ for 3-6 h to obtain OCN-CHO suspension. Step 2.2: The OCN-CHO suspension obtained in Step 2.1 is repeatedly centrifuged and washed in a high-speed centrifuge until the supernatant is neutral, and the centrifuged OCN-CHO precipitate is obtained. Step 2.3: Prepare a suspension with a solid content of 0.1-1% from the centrifuged OCN-CHO precipitate obtained in Step 2.2, and then sonicate it for 5-10 minutes at a power of 1200-1500W to obtain an OCN-CHO aqueous dispersion. Step 2.4: Concentrate the OCN-CHO aqueous dispersion obtained in Step 2.3 to a solid content of 1-4% to finally prepare OCN-CHO hydrogel.

[0008] The specific method of step 3 includes: Step 3.1: Prepare an aqueous dispersion of oxidized cellulose and activate its carboxyl groups in an EDC / NHS catalytic system to obtain carboxyl-activated oxidized cellulose; Step 3.2: Add (10-15) mg / mL lysine to the carboxyl-activated oxidized cellulose obtained in step 3.1, adjust the pH of the system to 7-8 with NaHCO3, and react for 22-25 h to ensure that the amino group is fully grafted to obtain amino-grafted oxidized nanocellulose OCN-Lys. Step 3.3: Filter the amino-grafted oxidized nanocellulose OCN-Lys obtained in Step 3.2 and wash it repeatedly until neutral to obtain OCN-Lys cellulose; Step 3.4: The OCN-Lys cellulose obtained in Step 3.3 is prepared into an OCN-Lys suspension with a solid content of 0.5-1%, and then processed by a high-pressure homogenizer to obtain an OCN-Lys hydrogel with a solid content of 0.5-1%. Step 3.5: Concentrate the OCN-Lys hydrogel with a solid content of 0.5-1% obtained in step 3.4 to a solid content of 1-2% to finally prepare the OCN-Lys hydrogel.

[0009] The oxidized cellulose described herein includes functional groups containing carboxyl or hydroxyl groups after oxidation.

[0010] The present invention also provides a spray hemostatic gel layer based on two-component cross-linked oxidized nanocellulose, which is prepared by the above method.

[0011] This invention also provides an application of a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, for trauma emergency treatment, surgical hemostasis, or tissue repair.

[0012] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes the OCN-CHO hydrogel prepared in step 2, which enhances surface adhesion, and the OCN-Lys hydrogel prepared in step 3. These two hydrogels are then mixed via a Schiff base reaction between the aldehyde and amino groups in step 4. This achieves the core technical effects of rapid and strong adhesion, enhanced mechanical properties, and excellent biocompatibility. This design allows the spray to instantly form a stable covalent network at the wound site, not only resolving the contradiction between adhesion and mechanical strength in single-component materials but also effectively avoiding the problems of poor coverage with high solids content and easy washout by blood with low solids content.

[0013] 2. The aldehyde-oxidized nanocellulose in the OCN-CHO hydrogel prepared in step 2 of this invention serves as an adhesive component to achieve adhesion to tissues, with an adhesion strength reaching 40 kPa. The amino-oxidized nanocellulose in the OCN-Lys hydrogel prepared in step 3 serves as a crosslinking component, which crosslinks with the aldehyde component through the Schiff base reaction to enhance the mechanical properties of the system. In addition, it can also consume excess aldehyde groups and reduce the potential cytotoxicity of aldehyde groups.

[0014] 3. Compared with single-component OCN, this invention performs two-component modification in steps 2 and 3 on the basis of step 1, and has a better hemostatic effect through the Schiff base reaction in step 4. It can achieve effective hemostasis within 30 seconds and can be applied to larger bleeding models. At the same time, it retains the wound healing promotion properties of OCN.

[0015] 4. The preparation process of the two-component sprayable oxidizable nanocellulose hemostatic gel in steps 2 and 3 of this invention does not require special equipment, the reaction conditions are mild, and it can be industrialized.

[0016] 5. The two-component sprayable oxidative nanocellulose hemostatic gel of the present invention can quickly form a hydrogel upon contact with blood after spraying out the OCN-CHO hydrogel of step 2 and the OCN-Lys hydrogel nanofiber of step 3. No additional operation is required. It can be solidified by the Schiff base reaction of the aldehyde-amino two-component in step 4. It is simple to operate and easy to carry.

[0017] In summary, this invention achieves a highly efficient balance in adhesion strength, dynamic mechanical properties, and biosafety of the spray hemostatic gel through a two-component crosslinking design based on the Schiff base reaction of "aldehyde-based nanocellulose / amino nanocellulose". It has significant advantages such as rapid hemostasis, strong adhesion, good biocompatibility, convenient operation, and easy industrialization. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0019] Figure 1 This is a flowchart illustrating the preparation method of the two-component cross-linked oxidized nanocellulose of the present invention.

[0020] Figure 2(a) shows the contact angle test diagram of the TOCN-5 hydrogel in Experimental Analysis 1 of the present invention.

[0021] Figure 2(b) shows the contact angle test results of the TOCN-CHO-3 hydrogel in Experimental Analysis 1 of this invention.

[0022] Figure 2(c) shows the contact angle test results of the TOCN-Lys-3 hydrogel in Experimental Analysis 1 of this invention.

[0023] Figure 2(d) shows the contact angle test results of the crosslinked TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experimental Analysis 1 of this invention.

[0024] Figure 3(a) shows the stress-strain curves of TOCN-5 hydrogel, TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, and TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel after crosslinking in Experimental Analysis 2 of the present invention.

[0025] Figure 3(b) is a statistical graph of the tensile strength of TOCN-5 hydrogel, TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and the cross-linked TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experimental Analysis 2 of the present invention.

[0026] Figure 3(c) is a statistical graph of the Young's modulus of TOCN-5 hydrogel, TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and TOCN-CHO-3 hydrogel after crosslinking in Experimental Analysis 2 of the present invention.

[0027] Figure 4(a) is a SEM image of the TOCN-CHO-3 hydrogel in Experimental Analysis 3 of this invention.

[0028] Figure 4(b) is a SEM image of the TOCN-Lys-3 hydrogel in Experimental Analysis 3 of this invention.

[0029] Figure 4(c) is a SEM image of the cross-linked TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experimental Analysis 3 of the present invention.

[0030] Figure 5(a) Shear viscosity diagram of TOCN-CHO hydrogels with different aldehyde content and different solid content in Experiment 4 of this invention.

[0031] Figure 5(b) Shear viscosity diagram of TOCN-Lys hydrogels with different amino and solid contents in Experiment 4 of this invention.

[0032] Figure 6(a) is a comparison of the adhesion performance results of TOCN-CHO hydrogels with different aldehyde content and TOCN-Lys hydrogels with different amino content in Experiment 5 of this invention.

[0033] Figure 6(b) is a comparison of the adhesion performance results of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel with different solid contents in Experiment 5 of this invention.

[0034] Figure 6(c) is a comparison of the adhesion performance results of different crosslinking ratios of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experiment 5 of this invention.

[0035] Figure 6(d) is a comparison of the adhesion performance results of TOCN hydrogel, TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, 3M tissue adhesive and Hydrosorb hemostatic hydrogel in Experiment 5 of this invention.

[0036] Figure 7(a) is a comparison of the burst pressure results of TOCN-CHO hydrogels with different aldehyde content and TOCN-Lys hydrogels with different amino content in Experiment 6 of this invention.

[0037] Figure 7(b) is a comparison of the burst pressure results of different crosslinking ratios of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experiment 6 of this invention.

[0038] Figure 7(c) is a comparison of the burst pressure results of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, TOCN-CHO-3 hydrogel with a ratio of 4:1, TOCN-Lys-3 hydrogel after crosslinking, Hydrosorb hemostatic hydrogel, and TOCN hydrogel in Experiment 6 of the present invention.

[0039] Figure 8(a) shows the hemolysis experiment of TOCN-CHO hydrogel, TOCN-Lys hydrogel and TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel after double cross-linking in Experiment 7 of the present invention.

[0040] Figure 8(b) is a statistical chart showing the hemolysis rates of TOCN-CHO hydrogels with different aldehyde contents and TOCN-Lys hydrogels with different amino contents compared with commercial 3M tissue adhesive and Hydrosorb hemostatic hydrogel in Experimental Analysis 7 of this invention.

[0041] Figure 8(c) is a statistical chart of hemolysis rates of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel with different crosslinking ratios in Experimental Analysis 7 of the present invention.

[0042] Figure 9(a) is a comparison of the cell activity results of TOCN-CHO hydrogels with different aldehyde contents and TOCN-Lys hydrogels with different aldehyde contents in the experimental analysis of this invention.

[0043] Figure 9(b) is a comparison of cell activity results for different crosslinking ratios of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel in Experiment 8 of this invention.

[0044] Figure 10 This is a comparative image showing the staining results of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, and their cross-linked implantation in vivo in Experiment 9 of this invention.

[0045] Figure 11(a) shows the wound hemostasis diagram of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and their cross-linking in Experimental Analysis 10 of the present invention.

[0046] Figure 11(b) is a statistical comparison of the bleeding volume of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and their cross-linking in Experiment 10 of the present invention.

[0047] Figure 11(c) shows the hemostasis time of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and their cross-linking in Experiment 10 of the present invention. Detailed Implementation

[0048] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0049] Example 1 like Figure 1 As shown, a method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes: Step 1: Prepare oxidized cellulose nanoparticles (TOCN) using the TEMPO-NaClO-NaBr oxidation system; Step 1.1: First, place the lignocellulose wet pulp in distilled water, adjust the pH of the lignocellulose wet pulp to 2 using HCl to remove the minerals, then filter and wash with water, adjust the pH to 7 using NaOH, and finally dry at 105℃ for 1 hour to determine the cellulose content. Step 1.2: Mix TEMPO, NaBr and deionized water to prepare a solution according to the ratio of 0.016g TEMPO, 0.1g NaBr and 100mL deionized water per gram of cellulose. Then add the corresponding amount of the final product from step 1.1. Next, add NaClO to the solution at a ratio of 5mmol / g to obtain a reaction system with a NaClO content of 5mmol / g. Step 1.3: Start the reaction. Add 0.5 mol / L NaOH to the reaction system with a NaClO content of 5 mmol / g to maintain the pH of the reaction at 10. After the reaction is completed, the reaction solution is obtained. Step 1.4: Add sodium borohydride and ethanol to the reaction solution obtained in step 1.3 according to the ratio of 0.1g sodium borohydride and 1mL ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Wash and filter the reaction solution from Step 1.4, adjust the pH to neutral, and obtain oxidized nanocellulose TOCN-5.

[0050] Step 2: Prepare the oxidized nanocellulose TOCN-5 obtained in Step 1.5 into a suspension with a solid content of 1%, and disperse it in nano by a high-pressure homogenizer at 500 bar to obtain a TOCN-5 hydrogel with a solid content of 1%. Step 3: Add a certain proportion of sodium periodate to the TOCN-5 hydrogel obtained in Step 2 to prepare TOCN-CHO-3 hydrogel by reacting it. Step 3.1: Add the TOCN-5 hydrogel obtained in Step 2 to deionized water, adjust the solid content to 0.5%, and add sodium periodate. The ratio of sodium periodate to solid content in the ONC hydrogel is 4:1 by mass. Adjust the pH to 3.5 with 1% glacial acetic acid, and react at 45°C in the dark for 4 h to obtain a TOCN-CHO-3 suspension. Step 3.2: The TOCN-CHO-3 suspension prepared in step 3.1 is repeatedly centrifuged and washed in a high-speed centrifuge at 10,000 rpm for 10 min until the supernatant is neutral, and the centrifuged TOCN-CHO-3 precipitate is obtained. Step 3.3: Prepare a suspension with a solid content of 0.5% from the TOCN-CHO-3 precipitate collected in step 3.2, and sonicate it with a probe sonicator for 5 min using a 1440 W cell disruptor to obtain a TOCN-CHO-3 aqueous dispersion. Step 3.4: The TOCN-CHO-3 aqueous dispersion obtained in Step 3.3 was concentrated using a rotary evaporator at 50 °C to obtain a TOCN-CHO-3 hydrogel with a solid content of 4%.

[0051] Step 4: Add lysine to the carboxyl-activated system of the oxidized nanocellulose TOCN-5 obtained in Step 1 to prepare TOCN-Lys-1 hydrogel. Step 4.1: The oxidized nanocellulose TOCN-5 from step 1.5 was prepared with a solid content of 1%, and EDC was added and reacted for 1 h. Then NHS was added to continue activation for 1 h to obtain carboxyl-activated oxidized nanocellulose. Step 4.2: For the carboxyl-activated oxidized nanocellulose obtained in Step 4.1, add lysine at a concentration of 10 mg / mL according to the volume of water in the reaction system to allow it to react. Adjust the pH of the system to 7.8 with NaHCO3 and react for 24 h to obtain amino-grafted oxidized nanocellulose TOCN-Lys-1. Step 4.3: After the reaction is complete, filter the amino-grafted oxidized nanocellulose TOCN-Lys-1 obtained in step 4.2 through a sand core funnel and wash repeatedly until neutral to obtain TOCN-Lys-1 cellulose; Step 4.4: The TOCN-Lys-1 cellulose obtained in Step 4.3 is prepared into a TOCN-Lys-1 suspension with a solid content of 1%, and processed by a high-pressure homogenizer to obtain a TOCN-Lys-1 hydrogel with a solid content of 1%.

[0052] Example 2 like Figure 1 As shown, a method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes: Step 1: Prepare oxidized cellulose nanoparticles (TOCN) using the TEMPO-NaClO-NaBr oxidation system; Step 1.1: First, place the lignocellulose wet pulp in distilled water, adjust the pH of the lignocellulose wet pulp to 2 using HCl to remove the minerals, then filter and wash with water, adjust the pH to 7 using NaOH, and finally dry at 105℃ for 1 hour to determine the cellulose content. Step 1.2: Mix TEMPO, NaBr and deionized water to prepare a solution according to the ratio of 0.016g TEMPO, 0.1g NaBr and 100mL deionized water per gram of cellulose. Then add the corresponding amount of the final product from step 1.1. Next, add NaClO to the solution at a ratio of 5mmol / g to obtain a reaction system with a NaClO content of 5mmol / g. Step 1.3: Start the reaction. Add 0.5 mol / L NaOH to the reaction system with a NaClO content of 5 mmol / g to maintain the pH of the reaction at 10. After the reaction is completed, the reaction solution is obtained. Step 1.4: Add sodium borohydride and ethanol to the reaction solution obtained in step 1.3 according to the ratio of 0.1g sodium borohydride and 1mL ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Wash and filter the reaction solution from Step 1.4, adjust the pH to neutral, and obtain oxidized nanocellulose TOCN-5; Step 2: Prepare the oxidized nanocellulose TOCN-5 obtained in Step 1.5 into a suspension with a solid content of 1%, and disperse it in nano by a high-pressure homogenizer at 500 bar to obtain a TOCN-5 hydrogel with a solid content of 1%. Step 3: Add a certain proportion of sodium periodate to the TOCN-5 hydrogel obtained in Step 2 to prepare TOCN-CHO-2 hydrogel by reacting it. Step 3.1: Add the TOCN-5 hydrogel obtained in Step 2 to deionized water, adjust the solid content to 0.7%, and add sodium periodate. The ratio of sodium periodate to solid content in the ONC hydrogel is 3:1 by mass. Adjust the pH to 4 with 1% glacial acetic acid and react at 50°C in the dark for 3 h to obtain TOCN-CHO-2 suspension. Step 3.2: The TOCN-CHO-2 suspension prepared in step 3.1 is repeatedly centrifuged and washed in a high-speed centrifuge at 12000 rpm for 10 min until the supernatant is neutral, and the centrifuged TOCN-CHO-2 precipitate is obtained. Step 3.3: Prepare a suspension with 1% solid content from the TOCN-CHO-2 precipitate collected in Step 3.2, and use a 1440 W cell disruptor for probe sonication for 10 min to obtain a TOCN-CHO-2 aqueous dispersion. Step 3.4: The TOCN-CHO-2 aqueous dispersion obtained in Step 3.3 was concentrated using a rotary evaporator at 52°C to obtain a TOCN-CHO-2 hydrogel with a solid content of 3%.

[0053] Step 4: Add lysine to the carboxyl-activated system of the oxidized nanocellulose TOCN-5 obtained in Step 1 to prepare TOCN-Lys-1 hydrogel. Step 4.1: The oxidized nanocellulose TOCN-5 from step 1.5 was prepared with a solid content of 1%, and EDC was added and reacted for 2 h. Then NHS was added and activated for another 2 h to obtain carboxyl-activated oxidized nanocellulose. Step 4.2: For the carboxyl-activated oxidized nanocellulose obtained in Step 4.1, add lysine at a concentration of 12 mg / mL according to the volume of water in the reaction system to allow it to react. Adjust the pH of the system to 8 with NaHCO3 and react for 24 h to obtain amino-grafted oxidized nanocellulose TOCN-Lys-1. Step 4.3: After the reaction is complete, filter the amino-grafted oxidized nanocellulose TOCN-Lys-1 obtained in step 4.2 through a sand core funnel and wash repeatedly until neutral to obtain TOCN-Lys-1 cellulose; Step 4.4: The TOCN-Lys-1 cellulose obtained in Step 4.3 is prepared into a TOCN-Lys-1 suspension with a solid content of 0.5%, and processed by a high-pressure homogenizer to obtain a TOCN-Lys-1 hydrogel with a solid content of 0.5%. Step 4.5: Concentrate the TOCN-Lys-1 hydrogel with a solid content of 0.5% obtained in Step 4.4 to a solid content of 1% to finally prepare the TOCN-Lys-1 hydrogel.

[0054] Example 3 like Figure 1 As shown, a method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes: Step 1: Prepare oxidized cellulose nanoparticles (TOCN) using the TEMPO-NaClO-NaBr oxidation system; Step 1.1: First, place the lignocellulose wet pulp in distilled water, adjust the pH of the lignocellulose wet pulp to 2 using HCl to remove the minerals, then filter and wash with water, adjust the pH to 7 using NaOH, and finally dry at 105℃ for 1 hour to determine the cellulose content. Step 1.2: Mix TEMPO, NaBr and deionized water to prepare a solution according to the ratio of 0.016g TEMPO, 0.1g NaBr and 100mL deionized water per gram of cellulose. Then add the corresponding amount of the final product from step 1.1. Next, add NaClO to the solution at a ratio of 5mmol / g to obtain a reaction system with a NaClO content of 5mmol / g. Step 1.3: Start the reaction. Add 0.5 mol / L NaOH to the reaction system with a NaClO content of 5 mmol / g to maintain the pH of the reaction at 10. After the reaction is completed, the reaction solution is obtained. Step 1.4: Add sodium borohydride and ethanol to the reaction solution obtained in step 1.3 according to the ratio of 0.1g sodium borohydride and 1mL ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Wash and filter the reaction solution from Step 1.4, adjust the pH to neutral, and obtain oxidized nanocellulose TOCN-5.

[0055] Step 2: Prepare the oxidized nanocellulose TOCN-5 obtained in Step 1.5 into a suspension with a solid content of 1%, and disperse it in nano by a high-pressure homogenizer at 500 bar to obtain a TOCN-5 hydrogel with a solid content of 1%. Step 3: Add a certain proportion of sodium periodate to the TOCN-5 hydrogel obtained in Step 2 to prepare TOCN-CHO-1 hydrogel by reacting it. Step 3.1: Add the TOCN-5 hydrogel obtained in Step 2 to deionized water, adjust the solid content to 0.6%, and add sodium periodate. The ratio of sodium periodate to solid content in the ONC hydrogel is 1:1 by mass. Adjust the pH to 2 with 1% glacial acetic acid, and react at 40°C in the dark for 6 hours to obtain a TOCN-CHO-1 suspension. Step 3.2: The TOCN-CHO-1 suspension prepared in step 3.1 is repeatedly centrifuged and washed in a high-speed centrifuge at 12000 rpm for 2 min until the supernatant is neutral, and the centrifuged TOCN-CHO-1 precipitate is obtained. Step 3.3: Prepare a suspension with a solid content of 0.1% from the TOCN-CHO-1 precipitate collected in step 3.2, and use a 1440 W cell disruptor for probe sonication for 6 min to obtain a TOCN-CHO-1 aqueous dispersion. Step 3.4: The TOCN-CHO-1 aqueous dispersion obtained in Step 3.3 was concentrated using a rotary evaporator at 40 °C to obtain a TOCN-CHO-1 hydrogel with a solid content of 2%.

[0056] Step 4: Add lysine to the carboxyl-activated system of the oxidized nanocellulose TOCN-5 obtained in Step 1 to prepare TOCN-Lys-1 hydrogel. Step 4.1: The oxidized nanocellulose TOCN-5 from step 1.5 was prepared with a solid content of 1%, and EDC was added and reacted for 1.5 h. Then NHS was added to continue activation for 1.5 h to obtain carboxyl-activated oxidized nanocellulose. Step 4.2: For the carboxyl-activated oxidized nanocellulose obtained in Step 4.1, add lysine at a concentration of 15 mg / mL according to the volume of water in the reaction system to allow it to react. Adjust the pH of the system to 7 with NaHCO3 and react for 23 h to obtain amino-grafted oxidized nanocellulose TOCN-Lys-1. Step 4.3: After the reaction is complete, filter the amino-grafted oxidized nanocellulose TOCN-Lys-1 obtained in step 4.2 through a sand core funnel and wash repeatedly until neutral to obtain TOCN-Lys-1 cellulose; Step 4.4: The TOCN-Lys-1 cellulose obtained in Step 4.3 was prepared into a TOCN-Lys-1 suspension with a solid content of 0.8%, and then processed by a high-pressure homogenizer to obtain a TOCN-Lys-1 hydrogel with a solid content of 0.8%. Step 4.5: Concentrate the TOCN-Lys-1 hydrogel with a solid content of 0.8% obtained in Step 4.4 to a solid content of 1.7% to finally prepare the TOCN-Lys-1 hydrogel.

[0057] Example 4 like Figure 1 As shown, a method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes: Step 1: Prepare oxidized cellulose nanoparticles (TOCN) using the TEMPO-NaClO-NaBr oxidation system; Step 1.1: First, place the lignocellulose wet pulp in distilled water, adjust the pH of the lignocellulose wet pulp to 2 using HCl to remove the minerals, then filter and wash with water, adjust the pH to 7 using NaOH, and finally dry at 105℃ for 1 hour to determine the cellulose content. Step 1.2: Mix TEMPO, NaBr and deionized water to prepare a solution at a ratio of 0.016g TEMPO, 0.1g NaBr and 100mL deionized water per gram of cellulose. Then add the corresponding amount of the final product from step 1.1. Next, add NaClO to the solution at a ratio of 10mmol / g to obtain a reaction system with a NaClO content of 10mmol / g. Step 1.3: Start the reaction. Add 0.5 mol / L NaOH to the reaction system with NaClO content of 10 mmol / g to maintain the pH of the reaction at 10. After the reaction is completed, the reaction solution is obtained. Step 1.4: Add sodium borohydride and ethanol to the reaction solution obtained in step 1.3 according to the ratio of 0.1g sodium borohydride and 1mL ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Wash and filter the reaction solution from Step 1.4, adjust the pH to neutral, and obtain oxidized nanocellulose TOCN-10.

[0058] Step 2: Prepare a suspension of oxidized nanocellulose TOCN-10 obtained in Step 1.5 with a solid content of 1%, and disperse it in nano using a high-pressure homogenizer at 500 bar to obtain a TOCN-10 hydrogel with a solid content of 1%. Step 3: Add a certain proportion of sodium periodate to the TOCN-10 hydrogel obtained in Step 2 to prepare TOCN-CHO-1 hydrogel by reacting it. Step 3.1: Add the TOCN-10 hydrogel obtained in Step 2 to deionized water, adjust the solid content to 0.55%, and add sodium periodate. The ratio of sodium periodate to solid content in the ONC hydrogel is 1:1 by mass. Adjust the pH to 3.2 with 1% glacial acetic acid, and react at 43°C in the dark for 3.5 h to obtain a TOCN-CHO-1 suspension. Step 3.2: The TOCN-CHO-1 suspension prepared in step 3.1 is repeatedly centrifuged and washed at 10,000 rpm for 5 min in a high-speed centrifuge until the supernatant is neutral, and the centrifuged TOCN-CHO-1 precipitate is obtained. Step 3.3: Prepare a suspension with a solid content of 0.5% from the TOCN-CHO-1 precipitate collected in step 3.2, and use a 1440 W cell disruptor for probe sonication for 8 min to obtain a TOCN-CHO-1 aqueous dispersion. Step 3.4: The TOCN-CHO-1 aqueous dispersion obtained in Step 3.3 was concentrated using a rotary evaporator at 50 °C to obtain a TOCN-CHO-1 hydrogel with a solid content of 1%.

[0059] Step 4: Add lysine to the carboxyl-activated system of the oxidized nanocellulose TOCN-10 obtained in Step 1 to prepare TOCN-Lys-2 hydrogel. Step 4.1: The oxidized nanocellulose TOCN-10 from step 1.5 was prepared with a solid content of 1%, and EDC was added and reacted for 1 h. Then NHS was added to continue activation for 1 h to obtain carboxyl-activated oxidized nanocellulose. Step 4.2: For the carboxyl-activated oxidized nanocellulose obtained in Step 4.1, add lysine at a concentration of 10 mg / mL according to the volume of water in the reaction system to allow it to react. Adjust the pH of the system to 7.8 with NaHCO3 and react for 25 h to obtain amino-grafted oxidized nanocellulose TOCN-Lys-2. Step 4.3: After the reaction is complete, filter the amino-grafted oxidized nanocellulose TOCN-Lys-2 obtained in step 4.2 through a sand core funnel and wash it repeatedly until neutral to obtain TOCN-Lys-2 cellulose; Step 4.4: The TOCN-Lys-2 cellulose obtained in Step 4.3 is prepared into a TOCN-Lys-2 suspension with a solid content of 1%, and processed by a high-pressure homogenizer to obtain a TOCN-Lys-2 hydrogel with a solid content of 1%. Step 4.5: Concentrate the TOCN-Lys-2 hydrogel with 1% solid content obtained in Step 4.4 to 2% solid content to finally prepare the TOCN-Lys-2 hydrogel.

[0060] Example 5 like Figure 1 As shown, a method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose includes: Step 1: Prepare oxidized cellulose nanoparticles (TOCN) using the TEMPO-NaClO-NaBr oxidation system; Step 1.1: First, place the lignocellulose wet pulp in distilled water, adjust the pH of the lignocellulose wet pulp to 2 using HCl to remove the minerals, then filter and wash with water, adjust the pH to 7 using NaOH, and finally dry at 105℃ for 1 hour to determine the cellulose content. Step 1.2: Prepare a solution by mixing TEMPO, NaBr and deionized water at a ratio of 0.016g TEMPO, 0.1g NaBr and 100mL deionized water per gram of cellulose. Then add the corresponding amount of the final product from step 1.1. Next, add NaClO to the solution at a ratio of 12.5mmol / g to obtain a reaction system with a NaClO content of 12.5mmol / g. Step 1.3: Start the reaction. Add 0.5 mol / L NaOH to the reaction system with NaClO content of 12.5 mmol / g to maintain the pH of the reaction at 10. After the reaction is completed, the reaction solution is obtained. Step 1.4: Add sodium borohydride and ethanol to the reaction solution obtained in step 1.3 according to the ratio of 0.1g sodium borohydride and 1mL ethanol per gram of cellulose, and let it react for 3 hours; Step 1.5: Wash and filter the reaction solution from Step 1.4, adjust the pH to neutral, and obtain oxidized nanocellulose TOCN-12.5.

[0061] Step 2: Prepare the oxidized nanocellulose TOCN-12.5 obtained in Step 1.5 into a suspension with a solid content of 1%, and disperse it in nano using a high-pressure homogenizer at 500 bar to obtain a TOCN-12.5 hydrogel with a solid content of 1%. Step 3: Add a certain proportion of sodium periodate to the TOCN-12.5 hydrogel obtained in Step 2 to prepare TOCN-CHO-2 hydrogel by reacting it. Step 3.1: Add the TOCN-12.5 hydrogel obtained in Step 2 to deionized water, adjust the solid content to 0.6%, and add sodium periodate. The ratio of sodium periodate to solid content in the ONC hydrogel is 3:1 by mass. Adjust the pH to 3.3 with 1% glacial acetic acid and react at 44℃ in the dark for 3.5 h to obtain TOCN-CHO-2 suspension. Step 3.2: The TOCN-CHO-2 suspension prepared in step 3.1 is repeatedly centrifuged and washed in a high-speed centrifuge at 8000 rpm for 15 min until the supernatant is neutral, and the centrifuged TOCN-CHO-2 precipitate is obtained. Step 3.3: Prepare a suspension with a solid content of 0.9% from the TOCN-CHO-2 precipitate collected in Step 3.2, and sonicate it with a probe sonicator for 9 min using a 1440 W cell disruptor to obtain a TOCN-CHO-2 aqueous dispersion. Step 3.4: The TOCN-CHO-2 aqueous dispersion obtained in Step 3.3 was concentrated using a rotary evaporator at 48 °C to obtain a TOCN-CHO-2 hydrogel with a solid content of 2%.

[0062] Step 4: Add lysine to the carboxyl-activated system of the oxidized nanocellulose TOCN-12.5 obtained in Step 1 to prepare TOCN-Lys-3 hydrogel. Step 4.1: The oxidized nanocellulose TOCN-12.5 from step 1.5 was prepared with a solid content of 1%, and EDC was added and reacted for 1 hour. Then NHS was added to continue activation for 1 hour to obtain carboxyl-activated oxidized nanocellulose. Step 4.2: For the carboxyl-activated oxidized nanocellulose obtained in Step 4.1, add lysine at a concentration of 10 mg / mL according to the volume of water in the reaction system to allow it to react. Adjust the pH of the system to 7.5 with NaHCO3 and react for 22 h to obtain amino-grafted oxidized nanocellulose TOCN-Lys-3. Step 4.3: After the reaction is complete, filter the amino-grafted oxidized nanocellulose TOCN-Lys-3 obtained in step 4.2 through a sand core funnel and wash repeatedly until neutral to obtain TOCN-Lys-3 cellulose; Step 4.4: The TOCN-Lys-3 cellulose obtained in Step 4.3 was prepared into a TOCN-Lys-3 suspension with a solid content of 0.9%, and then processed by a high-pressure homogenizer to obtain a TOCN-Lys-3 hydrogel with a solid content of 0.9%. Step 4.5: Concentrate the TOCN-Lys-3 hydrogel with a solid content of 0.9% obtained in Step 4.4 to a solid content of 2% to finally prepare the TOCN-Lys-3 hydrogel.

[0063] Experimental Analysis In order to retain the shear-thinning sprayable properties of TOCN, this invention successfully improves its mechanical properties by introducing chemical cross-linking and other methods, while minimizing the introduction of polymer materials.

[0064] Sodium periodate can selectively oxidize the secondary hydroxyl groups at C2 and C3 of the cellulose molecular chain to generate dialdehyde cellulose, thereby introducing carboxyl groups while retaining the oxidized cellulose carboxyl groups.

[0065] Modifying the carboxyl group of oxidized cellulose with lysine can introduce amino groups into the cellulose molecular chain.

[0066] Aldehyde-oxidized nanocellulose acts as an adhesive component to achieve tissue adhesion, while amino-oxidized nanocellulose acts as a cross-linking component, cross-linking with the aldehyde component through a Schiff base reaction to enhance the mechanical properties of the system. Furthermore, it consumes excess aldehyde groups, reducing the potential cytotoxicity associated with aldehydes. The two components form a three-dimensional covalent network after spray mixing. Animal experiments show that the two-component gel reduces hemostasis time to 28 seconds and maintains approximately 100% cell viability, significantly superior to traditional single-component systems.

[0067] In this embodiment of the invention, the components with sodium periodate to cellulose mass ratios of 1:1, 3:1 and 4:1 are named TOCN-CHO-1, TOCN-CHO-2 and TOCN-CHO-3, respectively.

[0068] Oxidized cellulose nanoparticles prepared by adding NaClO at proportions of 5, 10, and 12.5 mmol / g to the TEMPO-NaClO-NaBr oxidation system were then aminated to obtain amino-oxidized cellulose nanoparticles, which were named TOCN-Lys-1, TOCN-Lys-2, and TOCN-Lys-3, respectively, for subsequent experiments.

[0069] Experimental Analysis 1: Hydrophilicity / Hydrophilicity Analysis The surface tension of the TOCN-5 hydrogel obtained in step 2 of Example 1, the TOCN-CHO-3 hydrogel obtained in step 3 of Example 1, the TOCN-Lys-3 hydrogel obtained in step 4 of Example 5, and the crosslinked hydrogels of the TOCN-CHO-3 hydrogel obtained in step 3 of Example 1 and the TOCN-Lys-3 hydrogel obtained in step 4 of Example 5 were tested using a contact angle meter (KRUSS, America). The tests included the following: The four hydrogel materials were dried into thin films to measure their water contact angles. As shown in Figure 2(a), the contact angle of TOCN-5 is 48.93°. With the introduction of aldehyde groups, as shown in Figure 2(b), the hydrophilicity of TOCN-CHO hydrogel is improved, and the contact angle increases to 37.60°. This leads to a significant increase in the viscosity of TOCN-CHO hydrogel at the same solid content. Due to the introduction of long carbon chains and the formation of amide bonds, as shown in Figure 2(c), TOCN-Lys hydrogel is improved to be more hydrophobic, and the contact angle becomes 86.84°. As shown in Figure 2(d), the hydrophilicity and hydrophobicity of the crosslinked TOCN-CHO-3 hydrogel obtained in step 3 of Example 1 and the TOCN-Lys-3 hydrogel obtained in step 4 of Example 5 are shown, with a contact angle of 58.41°, which is between the two.

[0070] Experimental Analysis 2: Mechanical Property Analysis Mechanical properties were tested on the TOCN-5 hydrogel obtained in step 2 of Example 1, the TOCN-CHO-3 hydrogel obtained in step 3 of Example 1, and the TOCN-Lys-3 hydrogel obtained in step 4 of Example 5, as well as their 1:1 crosslinked components. The results included the following: The four hydrogel materials were tested, and the hydrogel material closest to the average value was selected as its stress-strain curve, as shown in Figure 3(a). The tensile strength and Young's modulus were calculated and statistically analyzed, as shown in Figure 3(b). Compared with TOCN hydrogel, the test results showed that TOCN-CHO-3 hydrogel decreased to 100 MPa due to changes in fiber length and fiber skeleton; while TOCN-Lys-3 hydrogel increased its tensile strength to about 170 MPa due to the introduction of amino and amide bonds, which provided more hydrogen bonds. The construction of the cross-linked double network structure further increased its tensile strength to about 195 MPa, and this structure also significantly increased the elongation.

[0071] As shown in Figure 3(c), Young's modulus is only related to the chemical composition of the material and is not related to its tissue changes. Therefore, the difference between the above four hydrogel materials is very small.

[0072] Experimental Analysis 3: Microscopic Morphology Analysis The microstructure of the TOCN-CHO-3 hydrogel obtained in step 3 of Example 1 and the TOCN-Lys-3 hydrogel obtained in step 4 of Example 5, as well as their crosslinked components, was observed using a scanning electron microscope (Nova Nano SEM 230), including the following: The hydrogels all exhibit this porous network structure, as shown in Figure 4(a). The pore size of TOCN-CHO-3 hydrogel is approximately 20-30 μm, as shown in Figure 4(b), while the pore size of TOCN-Lys-3 hydrogel is approximately 40-60 μm, as shown in Figure 4(c). The pore size after cross-linking is approximately 10-20 μm. The dense porous structure formed after cross-linking is partly due to the physical network formed by hydrogen bonding between oxidized cellulose nanoparticles, and partly due to the chemical network formed by the cross-linking of aldehyde and amino groups. The presence of this dual-network structure leads to a smaller pore size.

[0073] Experimental Analysis 4: Rheological Properties Shear viscosity tests were performed on TOCN-CHO hydrogels with different solid contents and different aldehyde contents obtained in Examples 1, 2, and 3, namely TOCN-CHO-3 hydrogel obtained in Example 1, TOCN-CHO-2 hydrogel obtained in Example 2, and TOCN-CHO-1 hydrogel obtained in Example 3, and TOCN-Lys hydrogels with different amino contents obtained in Examples 1, 4, and 5, namely TOCN-Lys-1 hydrogel obtained in Example 1, TOCN-Lys-2 hydrogel obtained in Example 4, and TOCN-Lys-3 hydrogel obtained in Example 5, including the following: As the solid content increases, the viscosity of both materials increases while maintaining the shear-thinning property. As shown in Figure 5(a), the viscosity of TOCN-CHO hydrogel increases rapidly compared to before the reaction, which indicates the increase in intermolecular forces of TOCN-CHO hydrogel. At the same time, the viscosity of TOCN-CHO hydrogel also increases with the increase of aldehyde content.

[0074] As shown in Figure 5(b), the introduction of lysine leads to the consumption of carboxyl groups on the TOCN hydrogel, resulting in a weakening of hydrogen bonds. Consequently, the viscosity of the TOCN-Lys hydrogel decreases compared to before the reaction. Therefore, as the amino content increases, the viscosity of the TOCN-Lys hydrogel decreases.

[0075] Experimental Analysis 5: Adhesion Performance Study Excellent adhesion properties are an important condition for the practical application of hemostatic hydrogels. Their adhesion properties are characterized by a shear adhesion test, including the following: As shown in Figure 6(a), the adhesion properties of TOCN-CHO hydrogels with different aldehyde content (i.e., TOCN-CHO-3 hydrogel obtained in Example 1, TOCN-CHO-2 hydrogel obtained in Example 2, and TOCN-CHO-1 hydrogel obtained in Example 3) and TOCN-Lys hydrogels with different amino content (i.e., TOCN-Lys-1 hydrogel obtained in Example 1, TOCN-Lys-2 hydrogel obtained in Example 4, and TOCN-Lys-3 hydrogel obtained in Example 5) obtained in Examples 1, 4, and 5 were tested under a 2% solid content condition. With increasing aldehyde content, the adhesion properties significantly improved, as aldehydes can react with amino groups on proteins on the tissue surface through a Schiff base reaction, thereby achieving adhesion. With increasing amino content, the adhesion properties improved slightly, as amino groups can generate electrostatic interactions with tissues, thus enhancing adhesion.

[0076] The effects of solid content on the TOCN-CHO-3 and TOCN-Lys-3 hydrogels, which exhibited the best adhesion performance, were further investigated, as shown in Figure 6(b). The adhesion performance of both hydrogels improved with increasing solid content.

[0077] To further investigate the effect of aldehyde content on adhesion properties, crosslinking 2% TOCN-CHO-3 hydrogel and 4% TOCN-Lys-3 hydrogel was performed to study their adhesion properties. Aldehyde and amino groups were crosslinked at ratios of 1:1, 2:1, 3:1, 4:1, and 5:1 for subsequent studies, and their adhesion properties are shown in Figure 6(c). With increasing ratios, i.e., increasing aldehyde content in the system, the adhesion properties gradually improved, reaching 8 kPa at a ratio of 5:1.

[0078] Finally, the hydrogels with the strongest adhesion properties, TOCN-CHO-3 and TOCN-Lys-3, were compared with TOCN-5 hydrogel, commercial 3M tissue adhesive, and commercial Hydrosorb hemostatic hydrogel. The test results are shown in Figure 6(d). Compared with 3M tissue adhesive (whose main component is cyanoacrylate) and Hydrosorb hemostatic hydrogel (whose main component is polyethylene), the adhesion properties of the hydrogel of this invention are between the two. Therefore, the adhesion properties of the two-component oxidized nanocellulose hydrogel of this invention are suitable for medical hemostasis and other applications.

[0079] Experimental Analysis 6: Burst Pressure Test Simulating real wound conditions through burst pressure testing includes the following: As the content of aldehyde or amino groups increases, the burst pressure also increases, as shown in Figure 7(a). This increase in burst pressure largely comes from the increase in its adhesion. Specifically, the burst pressure value of TOCN-CHO-3 hydrogel under the condition of 2.5% solid content is 250 mmHg, and the burst pressure value of TOCN-Lys-3 hydrogel reaches 180 mmHg, which is higher than the human arterial blood pressure of 120 mmHg. This indicates that it has the ability to stop bleeding in a massive hemorrhage model.

[0080] Further burst pressure tests were conducted on hydrogels with different crosslinking ratios, as shown in Figure 7(b). With increasing aldehyde and amino group ratios, the burst pressure initially decreased and then increased, reaching a maximum of 330 mmHg at a 4:1 ratio, after which it decreased again. Low aldehyde content resulted in poor adhesion, leading to low burst pressure, while high aldehyde content resulted in low crosslinking density, leading to poor mechanical properties. At the 4:1 ratio, the combined effect of both factors resulted in the maximum burst pressure.

[0081] Finally, the present invention was compared with commercially available Hydrosorb hemostatic hydrogel and unmodified TOCN hydrogel, as shown in Figure 7(c). It was found that the burst pressure of both TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel and cross-linked hydrogel was much better than that of Hydrosorb hemostatic hydrogel and unmodified TOCN hydrogel.

[0082] Experimental Analysis 7: Blood Compatibility Blood compatibility tests were performed on TOCN-CHO hydrogels with various aldehyde contents obtained in Examples 1, 2, and 3 (i.e., TOCN-CHO-3 hydrogel obtained in Example 1, TOCN-CHO-2 hydrogel obtained in Example 2, and TOCN-CHO-1 hydrogel obtained in Example 3), and TOCN-Lys hydrogels with various amino contents obtained in Examples 1, 4, and 5 (i.e., TOCN-Lys-1 hydrogel obtained in Example 1, TOCN-Lys-2 hydrogel obtained in Example 4, and TOCN-Lys-3 hydrogel obtained in Example 5), as well as oxidized nanocellulose gels crosslinked with aldehyde and amino groups in ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. The tests included the following: Figure 8(a) shows the hemolysis experiment of the above hydrogel material, commercial 3M tissue adhesive, and Hydrosorb hemostatic hydrogel as control groups. As shown in Figure 8(b), except for the commercial 3M tissue adhesive, the hemolysis rate of the other samples is lower than the standard of 5%, indicating that the oxidized nanocellulose hydrogel has good blood compatibility.

[0083] Figure 8(c) shows the hemolysis statistics of different crosslinking ratios of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel. The hemolysis rate is still lower than the standard of 5%, indicating that the blood compatibility of the two-component oxidized nanocellulose hydrogel is good.

[0084] Experimental Analysis 8: In vitro biocompatibility In vitro biocompatibility studies were conducted using a CCK-8 cell proliferation assay with mouse fibroblasts, including the following: As shown in Figure 9(a), TOCN-CHO hydrogels obtained in Examples 1, 2, and 3 (i.e., TOCN-CHO-3 hydrogel obtained in Example 1, TOCN-CHO-2 hydrogel obtained in Example 2, and TOCN-CHO-1 hydrogel obtained in Example 3), and TOCN-Lys hydrogels obtained in Examples 1, 4, and 5 (i.e., TOCN-Lys-1 hydrogel obtained in Example 1, TOCN-Lys-2 hydrogel obtained in Example 4, and TOCN-Lys-3 hydrogel obtained in Example 5) were tested. The crosslinked components were TOCN-CHO-3 hydrogel obtained in Example 1 and TOCN-Lys-3 hydrogel obtained in Example 5. The cell activity of TOCN-CHO hydrogel after 5 days of culture was about 80%, and the cell activity of TOCN-Lys hydrogel after 5 days of culture was about 110%. This indicates that TOCN-Lys hydrogel may have a promoting effect on cell proliferation. Meanwhile, the cell activity of 3M tissue adhesive and Hydrosorb hemostatic hydrogel after 5 days of culture was only about 50%.

[0085] As shown in Figure 9(b), the cell viability of the cross-linked hydrogel was about 100% after 5 days of culture. This indicates that the cell viability of the TOCN-CHO-3 hydrogel, which was not very good, was good after being cross-linked with the proliferation-promoting TOCN-Lys-3 hydrogel. This proves that the excess aldehyde group tissue was consumed and the toxicity reached a good level of biocompatibility.

[0086] Experimental Analysis 9: In vivo biocompatibility TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, and hydrogels with different crosslinking ratios of TOCN-CHO-3 hydrogel and TOCN-Lys-3 hydrogel were implanted in vivo for in vivo biocompatibility studies, including the following: The TOCN-CHO-3 hydrogel obtained in Example 1, the TOCN-Lys-3 hydrogel obtained in Example 5, and cross-linked gels in ratios of 1:1, 3:1, and 5:1 were implanted into the body. Seven days later, sections were prepared and HE staining was performed to observe the condition of the implantation site. Figure 10 As shown, no pathological changes were found in the heart, liver, spleen, lungs, and kidneys, indicating that the two-component oxidized nanocellulose hydrogel of the present invention has good biocompatibility in vivo.

[0087] Experimental Analysis 10: Study on Hemostatic Performance The hemostatic properties of TOCN-CHO-3 hydrogel, TOCN-Lys-3 hydrogel, and the two-component cross-linked gel were tested, including the following: The liver of rats was exposed by incising the abdomen, and the material was sprayed onto the bleeding site. The blood loss weight and hemostasis time within 1.5 minutes were recorded. Untreated wounds served as controls. Each sample was repeated four times (n = 4). The bleeding patterns of the rats are shown in Figure 11(a), and the bleeding volume and hemostasis time are statistically analyzed in Figures 11(b) and 11(c). Under no treatment, the bleeding volume of rats reached approximately 960 mg within 90 seconds. With 2% solid content TOCN hydrogel, the bleeding volume was approximately 460 mg; with 2% TOCN-CHO-3 hydrogel, the bleeding volume was only approximately 200 mg; and with 4% TOCN-Lys-3 hydrogel, the bleeding volume was approximately 270 mg. This difference in bleeding volume mainly stemmed from the adhesive properties. The bleeding amount was only 120 mg under the cross-linking treatment of the two materials. When treated with TOCN-CHO-3 hydrogel spray, the bleeding site was adhered and sealed. The TOCN-Lys-3 hydrogel was then sprayed again, causing the gel to squeeze the bleeding site, similar to the method of physical compression hemostasis.

[0088] In addition, the amount of bleeding treated with Hydrosorb hemostatic hydrogel was 290 mg. Since bleeding in rats did not stop without any treatment, the hemostasis time was not statistically analyzed. The hemostasis time for 2% TOCN hydrogel was approximately 180 s, for 2% TOCN-CHO-3 hydrogel it was approximately 90 s, and for 4% it was approximately 120 s. The TOCN-Lys-3 hydrogel, when sprayed, tended to concentrate in one area of ​​the wound, resulting in poor dispersion and blood overflow from both sides, thus prolonging the hemostasis time. With cross-linking treatment, rapid hemostasis was achieved, with a hemostasis time of only about 28 s, demonstrating good hemostatic effect. Furthermore, the amount of bleeding treated with Hydrosorb hemostatic hydrogel resulted in a hemostasis time of approximately 145 s.

[0089] The femoral artery of rats was cut, and a hemostatic material was sprayed on it. Arterial bleeding was observed. When treating femoral artery bleeding with TOCN-CHO-3 hydrogel, it was found that after spraying the material, large areas of bleeding would wash it away. When treated with TOCN-Lys-3 hydrogel, blood would overflow from the sides of the gel. The commercially available Hydrosorb hemostatic hydrogel also exhibited the same bleeding phenomenon. Only under dual-spray conditions was femoral artery hemostasis achieved.

[0090] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, characterized in that, Includes the following steps: Step 1: Disperse oxidized cellulose in water to prepare an ONC hydrogel; Step 2: Add a certain proportion of sodium periodate to the ONC hydrogel obtained in Step 1 to react and prepare OCN-CHO hydrogel. Step 3: Add lysine to the carboxyl-activated cellulose system to prepare OCN-Lys hydrogel by reacting it. Step 4: Apply the ONC-CHO hydrogel prepared in Step 2 and the ONC-Lys hydrogel prepared in Step 3 to the wound in sequence. Based on the aldehyde content in ONC-CHO hydrogel and the amino content in ONC-Lys hydrogel, the ratio of aldehyde content in ONC-CHO hydrogel to amino content in ONC-Lys hydrogel is (2-5):

1. The aldehyde groups carried by ONC-CHO and the amino groups carried by ONC-Lys undergo a Schiff base reaction to achieve cross-linking and form a stable hemostatic gel.

2. The method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose according to claim 1, characterized in that, The specific method for step 2 includes: Step 2.1: Adjust the solid content of the ONC hydrogel obtained in Step 1 to 0.5-0.7%, and add sodium periodate. The ratio of sodium periodate to solid content in ONC hydrogel is (1-4):1 by mass. Adjust the pH of the system to 2-4 with diluted glacial acetic acid or hydrochloric acid, and react in the dark at 40-50℃ for 3-6 h to obtain OCN-CHO suspension. Step 2.2: The OCN-CHO suspension obtained in Step 2.1 is repeatedly centrifuged and washed in a high-speed centrifuge until the supernatant is neutral, and the centrifuged OCN-CHO precipitate is obtained. Step 2.3: Prepare a suspension with a solid content of 0.1-1% from the centrifuged OCN-CHO precipitate obtained in Step 2.2, and then sonicate it for 5-10 minutes at a power of 1200-1500W to obtain an OCN-CHO aqueous dispersion. Step 2.4: Concentrate the OCN-CHO aqueous dispersion obtained in Step 2.3 to a solid content of 1-4% to finally prepare OCN-CHO hydrogel.

3. The method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose according to claim 1, characterized in that, The specific method of step 3 includes: Step 3.1: Prepare an aqueous dispersion of oxidized cellulose and activate its carboxyl groups in an EDC / NHS catalytic system to obtain carboxyl-activated oxidized cellulose; Step 3.2: Add (10-15) mg / mL lysine to the carboxyl-activated oxidized cellulose obtained in step 3.1, adjust the pH of the system to 7-8 with NaHCO3, and react for 22-25 h to ensure that the amino group is fully grafted to obtain amino-grafted oxidized nanocellulose OCN-Lys. Step 3.3: Filter the amino-grafted oxidized nanocellulose OCN-Lys obtained in Step 3.2 and wash it repeatedly until neutral to obtain OCN-Lys cellulose; Step 3.4: The OCN-Lys cellulose obtained in Step 3.3 is prepared into an OCN-Lys suspension with a solid content of 0.5-1%, and then processed by a high-pressure homogenizer to obtain an OCN-Lys hydrogel with a solid content of 0.5-1%. Step 3.5: Concentrate the OCN-Lys hydrogel with a solid content of 0.5-1% obtained in step 3.4 to a solid content of 1-2% to finally prepare the OCN-Lys hydrogel.

4. The method for preparing a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose according to claim 1, characterized in that, The oxidized cellulose described herein includes functional groups containing carboxyl or hydroxyl groups after oxidation.

5. A spray hemostatic gel layer based on two-component cross-linked oxidized nanocellulose, prepared by any one of claims 1 to 4.

6. An application of a spray hemostatic gel based on two-component cross-linked oxidized nanocellulose, characterized in that, Used for trauma first aid, surgical hemostasis, or tissue repair.

Images