A chitosan-based antibacterial and hemostatic expanded sponge, its preparation method and application

Abstract

This invention discloses a chitosan-based antibacterial hemostatic expandable sponge, its preparation method, and its applications, belonging to the field of biomedical materials technology. This invention uses chitosan amino acid derivatives as the core active raw material, supplemented by a cross-linking agent, and is prepared through the synergistic effect of chemical cross-linking and physical molding. This invention prepares functionalized derivatives by branching amino acid groups onto the chitosan side, and simultaneously constructs a chemically cross-linked three-dimensional porous network using a cross-linking agent, endowing the hemostatic sponge with excellent adaptive expansion properties and wet structural stability. The resulting hemostatic sponge possesses advantages such as wet tissue adhesion, blood absorption and expansion sealing properties, broad-spectrum antibacterial activity, outstanding hemostatic effect, and good biocompatibility, making it widely adaptable to the clinical hemostatic needs of various complex wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and medical device technology, and particularly relates to a chitosan-based antibacterial and hemostatic expandable sponge, its preparation method and application. Background Technology

[0002] Uncontrolled bleeding is one of the leading causes of death in clinical surgery and emergency trauma. Currently, commonly used hemostatic materials include medical degreased gauze, gelatin sponge, collagen sponge, and fibrin glue. Among them, medical gauze can only achieve basic hemostasis through physical adsorption, with low hemostatic efficiency and extremely poor effect on patients with coagulation disorders. It is also prone to adhesion to the wound surface, causing secondary damage. Although fibrin glue has good hemostatic performance, it poses a risk of virus transmission, is expensive, and has limited sealing effect on massive bleeding, making it unsuitable for the needs of complex clinical scenarios (Nature Communications, 2025, 16(1): 4910). Although gelatin sponge and collagen sponge have good biocompatibility, they have slow hemostatic speed, poor wet mechanical strength, no inherent antibacterial activity, and are prone to postoperative infection. In addition, collagen sponge poses an immunogenicity risk and is expensive, making it difficult to promote its large-scale application (Communications Materials, 2024, 5(1): 129.).

[0003] For non-compressible bleeding such as deep irregular wounds and puncture wounds of cavities, traditional hemostatic materials are difficult to achieve rapid sealing and efficient hemostasis. At the same time, complications such as postoperative wound infection further increase the clinical treatment risk. Currently, most of the commercially available expandable hemostatic sponges in clinical use are prepared with polyvinyl alcohol (PVA) as the core raw material (such as the commercially available PVA expandable sponge, medical device registration number: Beijing Medical Device Approval No. 20152140298). However, this type of product is only applicable to the hemostasis of superficial mucosal wounds such as the nasal cavity and cannot meet the clinical treatment needs of deep irregular wounds and penetrating organ bleeding. At the same time, the product has no inherent antibacterial activity and cannot reduce the risk of wound infection, and there are significant limitations in the clinical application scenario. Chitosan is a natural cationic polysaccharide with inherent hemostatic and antibacterial activities, and extensive research has been carried out in the field of medical hemostatic materials. Currently, several patents have disclosed relevant technical solutions for chitosan-based hemostatic sponges (such as CN112778570B, a chitosan-based composite hemostatic sponge and its preparation method; CN108653797A, a medical hemostatic sponge and its preparation method). Although the above solutions have achieved certain hemostatic effects, they still focus on the hemostasis scenario of superficial mucosal wounds such as the nasal cavity and have not optimized the performance for the treatment needs of deep wounds. For the clinical application scenario of deep wounds and organ bleeding, in addition to having excellent expansion performance to achieve physical sealing of the wound, the hemostatic sponge also needs to have good wet tissue adhesion performance to avoid the material being washed off by blood flow and unable to achieve continuous and effective wound sealing. At the same time, it also needs to have stable broad-spectrum antibacterial activity to reduce the risk of postoperative wound infection and meet the treatment needs of complex clinical scenarios.

[0004] Therefore, developing a medical hemostatic material with rapid hemostasis, antibacterial, expansion sealing, wet adhesion, and excellent biocompatibility is a core technical problem亟待解决 in the fields of clinical first aid and wound repair, with important clinical value and social significance. Summary of the Invention

[0005] The present invention uses chitosan as the matrix material to provide a chitosan-based antibacterial hemostatic expandable sponge, and at the same time discloses its preparation method and application.

[0006] The core component of this hemostatic sponge is a chitosan amino acid derivative, which is chemically cross-linked and physically molded with a cross-linking agent. The hemostatic sponge, with the chitosan amino acid derivative as its core component, is a homogeneous mixture of one or two of the following: chitosan citrulline derivative, chitosan aspartic acid derivative, chitosan arginine derivative, chitosan lysine derivative, and chitosan ornithine derivative. Chitosan is a natural cationic basic polysaccharide. This invention chemically modifies it through amino acid grafting to enrich the types of functional groups in its molecular side chains and optimize its bioactivity and processability. Specifically, the positive charge inherent in the chitosan molecular chain destroys the negatively charged bacterial cell membrane through electrostatic interaction, achieving sterilization; while amino acid grafting modification improves the adsorption and aggregation of erythrocytes and platelet activation ability, resulting in superior hemostatic performance compared to unmodified chitosan. Based on this, the present invention constructs a stable three-dimensional porous network structure through a crosslinking agent, endowing the sponge with excellent expansion properties and wet resilience, and finally producing a medical hemostatic sponge that integrates broad-spectrum antibacterial, expansion and sealing, rapid hemostasis and excellent biocompatibility.

[0007] According to a first aspect of the present invention, the present invention provides a method for preparing a chitosan-based antibacterial and hemostatic expandable sponge, comprising the following steps: 1) Prepare an aqueous solution of chitosan amino acid derivatives; wherein the chitosan amino acid derivatives are one or a combination of two of the following: chitosan aspartic acid derivatives, chitosan arginine derivatives, chitosan lysine derivatives, chitosan ornithine derivatives, and chitosan citrulline derivatives. 2) A crosslinking agent is added to the aqueous solution to carry out a chemical crosslinking reaction, thereby obtaining a crosslinked system; 3) The crosslinking system is subjected to heating reaction, cooling and freeze-drying treatment in sequence to obtain the hemostatic sponge.

[0008] According to a preferred embodiment of the present invention, when the chitosan amino acid derivative is a compound composition, it is a compound composition of any one of chitosan aspartic acid derivative, chitosan arginine derivative, chitosan lysine derivative, and chitosan ornithine derivative with chitosan citrulline derivative, wherein the proportion of chitosan citrulline derivative in the total mass of chitosan amino acid derivative in the compound composition is 0~100 wt% (excluding 0 and 100%).

[0009] According to a preferred embodiment of the present invention, the chitosan amino acid derivative is prepared by dissolving chitosan in an acid solution with a mass fraction of 0.1% to 5%, then adding an amino acid activating solution, reacting at room temperature for 0.5 to 2 days, dialyzing the reaction solution at room temperature for 2 to 4 days, and then freeze-drying to obtain the chitosan amino acid derivative.

[0010] According to a preferred embodiment of the present invention, the acid solution is selected from one or more of acetic acid, formic acid, oxalic acid, phosphoric acid, hydrochloric acid, sulfuric acid, and nitric acid; the weight-average molecular weight of the chitosan is 10,000 to 2,000,000 Da.

[0011] According to a preferred embodiment of the present invention, the main raw materials for preparing chitosan amino acid derivatives are, by mass, 1-2 parts chitosan, 80-120 parts acid solution, and 1-20 parts amino acids; the molar ratio of the repeating structural units of the chitosan to the amino acids is 1:(0.1-20).

[0012] According to a preferred embodiment of the present invention, the preparation method of the amino acid activation solution is as follows: amino acids are weighed and dissolved in a 2-morpholinoethanesulfonic acid (MES) buffer solution with pH=4.0~6.5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added, and the activation reaction is carried out at room temperature for 30~60 min to obtain the amino acid activation solution.

[0013] According to a preferred embodiment of the present invention, the crosslinking agent is selected from one or more of glutaraldehyde, epichlorohydrin, genipin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether.

[0014] According to a preferred embodiment of the present invention, the mass concentration of the chitosan amino acid derivative aqueous solution is 0.5~10 wt%; the crosslinking agent is prepared as an aqueous solution for use, and the crosslinking agent aqueous solution is added to the chitosan amino acid derivative aqueous solution, wherein the mass ratio of the crosslinking agent (solute) to the chitosan amino acid derivative (solute) is 1:(50~2000).

[0015] According to a preferred embodiment of the present invention, in step 3), the crosslinking system is heated in a water bath at 20~70 ℃ for 0~10 h, then cooled to -20~-80 ℃ for freeze drying. After drying, the product is placed in an oven at 30~70 ℃ for curing for 0.5~3 days to obtain the hemostatic sponge. According to a further preferred embodiment of the present invention, the mass ratio of the crosslinking agent (solute) to the chitosan amino acid derivative (solute) is 1:(50~800).

[0016] According to a further preferred embodiment of the present invention, the mass concentration of the chitosan amino acid derivative aqueous solution is 1~6wt%.

[0017] According to a further preferred embodiment of the present invention, the crosslinking system is heated in a water bath at 45~60 ℃ for 0~6 h, and then cooled to -40~-60 ℃ for freeze drying. After drying, the product is placed in an oven at 30~60 ℃ for heat preservation and curing for 0.5~2 days to obtain the hemostatic sponge.

[0018] According to a second aspect of the present invention, a chitosan-based antibacterial and hemostatic expandable sponge prepared by the aforementioned method is provided. Preferably, the sponge has a saturated water absorption capacity of 60 to 130 times its own weight and a volume expansion rate of 700% to 1600%. Preferably, the sponge has an antibacterial rate of greater than 70% against both Staphylococcus aureus and Escherichia coli; preferably, the sponge has an in vitro coagulation index of less than 10%.

[0019] In a further preferred embodiment, the sponge has a saturated water absorption capacity of 80-130 times its own weight and a volume expansion rate of 900%-1600%. More preferably, the sponge has an antibacterial rate greater than 95% against both Staphylococcus aureus and Escherichia coli; preferably, the sponge has an in vitro coagulation index of less than 10%.

[0020] According to a third aspect of the present invention, the present invention provides the application of the chitosan-based antibacterial hemostatic expanded sponge in the preparation of hemostatic, antibacterial or tissue repair medical materials.

[0021] Compared with other methods, the beneficial technical effects of this invention are: (1) Chitosan, as a cationic polysaccharide, has advantages in antibacterial and hemostatic properties. In this invention, amino acids are modified on the side chains of chitosan, so that the derivative has more functions than single-component chitosan. On this basis, a crosslinking agent is added. The sponge obtained after a series of treatments has good water absorption, swelling and flexibility, and good biocompatibility. It also has excellent performance in hemostasis and antibacterial properties.

[0022] (2) This invention is safe and non-toxic, and has excellent flexibility and resilience. When using it, the sponge is compressed to the required size and placed into the surgical cavity. It can absorb blood and expand to seal the bleeding site. In addition, this invention can also be modified to fit the actual situation of the patient's surgical cavity to achieve rapid hemostasis. It also has antibacterial function, good moisture absorption and swelling properties, can effectively protect the wound, and has good mechanical properties, and has broad application prospects. Attached Figure Description

[0023] The accompanying drawings form part of this specification and are used to help those skilled in the art to more clearly understand the technical solutions of the present invention; the illustrative embodiments and their accompanying descriptions in the present invention are only used to explain the core concept of the present invention and do not constitute an improper limitation on the scope of protection of the present invention.

[0024] Figure 1 This is a scanning electron microscope (SEM) image of the morphology of Example 5 after it reached water absorption equilibrium; Figure 2 The water absorption rate change curves for Examples 2 and 5 within 9600s are shown. Figure 3 This is a comparison chart of the saturated water absorption of Examples 2, 5-9, and Comparative Example 2; Figure 4 This is a comparison chart of the equilibrium volume expansion rates of Examples 2 and 5-9; Figure 5 The cyclic compressive stress-strain curves for Examples 2, 5, 6, and 8 are shown. Figure 6 The wet adhesion stress-strain curves (left) and the comparison diagram of maximum adhesion force (right) for Examples 2 and 5-9 are shown. Figure 7 The in vitro degradation rate change curves are for Examples 2 and 5-9; Figure 8 The in vitro dynamic coagulation test of Examples 2, 5-9 and Comparative Examples 1-3 (left), and the comparison chart of in vitro coagulation index at 5 min (right); Figure 9 The image shows the results of the antibacterial activity test of the sample against Staphylococcus aureus. Figure 10 The image shows the results of the test for the antibacterial activity of the sample against Escherichia coli. Figure 11 This is a comparison chart of the hemolysis rate test results for the samples. The test used phosphate-buffered saline (PBS) as a negative control and deionized water as a positive control. Figure 12 The image shows the results of the cytotoxicity test of the sample on mouse embryonic fibroblast L929 cells. Figure 13 The left image shows the hemostasis time (left) and blood loss (right) of the rat liver puncture wounds in Examples 2, 8-9, and Comparative Examples 2-3. Figure 14 The graph shows the survival rate of rats 30 days after liver biopsy following treatment in Examples 2, 8-9, and Comparative Examples 2-3. Detailed Implementation

[0025] The following will provide a detailed description of several exemplary embodiments of the present invention. This detailed description should not be construed as limiting the scope of protection of the present invention, but should be regarded as a further detailed description of some technical aspects, technical features and specific implementations of the present invention.

[0026] It should be understood that the terminology used in this specification is only for describing specific embodiments and is not intended to limit the scope of protection of the present invention. Furthermore, regarding the numerical ranges described in this specification, it should be clarified that every intermediate value between the upper and lower limits of such numerical ranges has been specifically disclosed in this specification; simultaneously, all smaller numerical ranges formed between any of the aforementioned disclosed numerical values, intermediate values ​​within a numerical range, and any other disclosed numerical values, intermediate values ​​within a corresponding range, are included within the scope of protection of the present invention, and the upper and lower limits of such smaller numerical ranges can be independently included or excluded from the corresponding range.

[0027] Unless otherwise expressly stated in this specification, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Although this specification describes only preferred embodiments and raw materials, any methods and raw materials similar to or equivalent to those described herein may be used in the implementation or verification testing of this invention. All publicly available documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or raw materials associated with those documents; in the event of any conflict between the content of any incorporated document and this specification, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various improvements, adjustments, and modifications can be made to the specific embodiments described in this specification without departing from the scope and core principles of this invention. Other embodiments derived by those skilled in the art based on the content of this specification also fall within the scope of protection of this invention. This specification and its embodiments are merely illustrative and do not constitute a limitation on the scope of protection of this invention.

[0029] The terms “include,” “including,” “have,” and “contain” used in this article are all open-ended terms, meaning “includes but is not limited to.”

[0030] This invention provides a method for preparing an expandable hemostatic sponge with antibacterial function. The method uses chitosan amino acid derivatives as the main raw material, adds a crosslinking agent to its dispersion system, and prepares the sponge through conventional physical processing such as stirring, cooling, and freeze-drying.

[0031] In this embodiment of the invention, the preparation method of the chitosan amino acid derivative includes the following steps: Weigh 1-2 g of chitosan and add it to 80-120 mL of 0.5-2 wt% acetic acid solution (preferably 100 mL of 1 wt% acetic acid solution), stirring until completely dissolved to obtain a homogeneous and transparent chitosan solution; weigh 0.5-10 g of amino acids and add them to 80-120 mL of 0.1-2 mM 2-morpholinoethanesulfonic acid (MES) buffer solution (pH=4.0-6.5) (preferably 100 mL of 0.2 mM MES solution, pH=5.5), stirring until completely dissolved; then weigh 1-40 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.5-30 g of N-hydroxysuccinimide (NHS), add them to the above amino acid buffer solution, and activate the reaction at room temperature (20-25 °C) for 30-60 minutes. min; the activated amino acid mixture solution is added dropwise to the chitosan solution at a uniform rate, and the reaction is stirred at room temperature (20-25 °C) for 0.5-2 days (preferably 1 day); after the reaction is completed, the reaction solution is dialyzed at room temperature for 3-5 days (preferably 3 days) using a dialysis bag with a molecular weight cutoff of 3000-10000 Da, and then freeze-dried to obtain the chitosan amino acid derivative; wherein the molar ratio of the repeating structural unit of chitosan to the amino acid is 1:(0.1-8), and the weight-average molecular weight of chitosan is 10,000-2,000,000 Da.

[0032] In this embodiment of the invention, the preparation method of the chitosan-based antibacterial and hemostatic expandable sponge includes the following steps: Chitosan amino acid derivatives are prepared into aqueous solutions with a mass fraction of 1-10 wt% (preferably 3 wt%). The chitosan amino acid derivatives are one or a combination of two of the following: chitosan aspartic acid derivative, chitosan arginine derivative, chitosan lysine derivative, chitosan ornithine derivative, and chitosan citrulline derivative. When the chitosan amino acid derivative is a combination, it is a combination of any one of the following: chitosan aspartic acid derivative, chitosan arginine derivative, chitosan lysine derivative, and chitosan ornithine derivative, and chitosan citrulline derivative in any mass ratio.

[0033] Subsequently, a crosslinking agent (added in the form of an aqueous solution) is added to the chitosan amino acid derivative aqueous solution, wherein the mass ratio of the crosslinking agent (solute) to the chitosan amino acid derivative (solute) is 1:(50~800), and the mixture is stirred until homogeneous. The mixed solution is then heated in a water bath at 45~60 ℃ for 0~6 h (if the heating time is 0, i.e., no water bath heating is required) (preferably 50 ℃ for 4 h), and then cooled to -20~-80 ℃ (preferably -40 ℃). After freeze-drying, the solution is placed in an oven at 40~60 ℃ for 0.5~2 d for curing (preferably 50 ℃ for 1 d) to obtain the chitosan-based antibacterial hemostatic expanded sponge.

[0034] All raw materials used in the embodiments of this invention were obtained through commercial purchase.

[0035] In this embodiment of the invention, room temperature refers to 25±2 ℃.

[0036] The technical solution of the present invention will be further illustrated by the following embodiments.

[0037] Example 1 Weigh 1 g of chitosan and dissolve it in 100 mL of 1 wt% acetic acid solution, stirring until completely dissolved to obtain a chitosan solution. Weigh 2.5 g of aspartic acid and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5), then add 5.4 g of EDC and 3.2 g of NHS, and activate the reaction at room temperature for 45 min. Slowly add the above amino acid / EDC / NHS mixed solution to the chitosan solution and react at room temperature for 1 day. Dialyze the reaction solution at room temperature for 4 days and then freeze-dry to obtain the chitosan aspartic acid derivative (denoted as DC).

[0038] Weigh 1 g of chitosan and dissolve it in 100 mL of 1 wt% acetic acid solution, stirring until completely dissolved to obtain a chitosan solution. Weigh 3.3 g of citrulline and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5), then add 5.4 g of EDC and 3.2 g of NHS, and activate the reaction at room temperature for 30 min. Slowly add the above amino acid / EDC / NHS mixed solution to the chitosan solution and react at room temperature for 1 day. Dialyze the reaction solution at room temperature for 4 days and then freeze-dry to obtain the chitosan-citrulline derivative (denoted as CC).

[0039] Chitosan aspartic acid derivative and chitosan citrulline derivative were prepared into 3% aqueous solutions, respectively. The two solutions were then mixed thoroughly to achieve a chitosan citrulline derivative to chitosan aspartic acid derivative ratio of 1:4. 1,4-Butanediol diglycidyl ether was added to the mixed solution to achieve a crosslinking agent (solute) to chitosan amino acid derivative (solute) mass ratio of 1:600, and the mixture was stirred thoroughly. The mixture was heated in a 50 ℃ water bath for 4 h, then cooled to -40 ℃ for freeze-drying. The dried product was then kept in a 50 ℃ oven for 1 day to obtain the hemostatic sponge (sample name: DC / CC). 20 -0.05).

[0040] Example 2 Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a homogeneous and transparent chitosan solution. Weigh 3.2 g of arginine and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 45 min. Add the activated amino acid / EDC / NHS mixture to the chitosan solution dropwise at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan-arginine derivative (denoted as AC).

[0041] Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a chitosan solution. Weigh 3.3 g of citrulline and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 30 min. Add the activated amino acid / EDC / NHS mixture to the chitosan solution dropwise at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan-citrulline derivative (denoted as CC).

[0042] The chitosan arginine derivative and chitosan citrulline derivative obtained above were prepared into aqueous solutions with a mass fraction of 3 wt%. The two solutions were then mixed thoroughly in a specific ratio, controlling the mass ratio of chitosan citrulline derivative to chitosan arginine derivative in the mixture to be 1:4. 1,4-Butanediol diglycidyl ether was added to the mixed solution, controlling the mass ratio of crosslinking agent (solute) to chitosan amino acid derivative (solute) to be 1:600, and the mixture was stirred until homogeneous. The mixture was placed in a 50 ℃ constant temperature water bath for 4 h, then cooled to -40 ℃ for freeze-drying. After drying, the product was placed in a 50 ℃ oven for 1 day to cure, thus obtaining the expandable hemostatic sponge with antibacterial function (sample name: AC / CC). 20 -0.05).

[0043] Example 3 Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a homogeneous and transparent chitosan solution. Weigh 2.7 g of lysine and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 45 min. Add the activated lysine / EDC / NHS mixed solution dropwise to the chitosan solution at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan-lysine derivative (denoted as LC).

[0044] Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a chitosan solution. Weigh 3.3 g of citrulline and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 30 min. Add the activated citrulline / EDC / NHS mixed solution dropwise to the chitosan solution at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan-citrulline derivative.

[0045] The chitosan lysine derivative and chitosan citrulline derivative obtained above were prepared into aqueous solutions with a mass fraction of 3 wt%. The two solutions were then mixed thoroughly in a specific ratio, controlling the mass ratio of chitosan citrulline derivative to chitosan lysine derivative in the mixture to be 1:4. 1,4-Butanediol diglycidyl ether was added to the mixed solution, controlling the mass ratio of crosslinking agent (solute) to chitosan amino acid derivative (solute) to be 1:600, and the mixture was stirred until homogeneous. The mixture was placed in a 50 ℃ constant temperature water bath for 4 h, then cooled to -40 ℃ for freeze-drying. After drying, the product was placed in a 50 ℃ oven for 1 day to cure, thus obtaining the expandable hemostatic sponge with antibacterial function (sample name: LC / CC). 20 -0.05).

[0046] Example 4 Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a homogeneous and transparent chitosan solution. Weigh 2.5 g of ornithine and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 45 min. Add the activated ornithine / EDC / NHS mixed solution dropwise to the chitosan solution at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan ornithine derivative (denoted as OC).

[0047] Weigh 1 g of chitosan and add it to 100 mL of 1 wt% acetic acid solution. Stir until completely dissolved to obtain a chitosan solution. Weigh 3.3 g of citrulline and dissolve it in 100 mL of 0.2 mM MES buffer solution (pH=5.5). Then add 5.4 g of EDC and 3.2 g of NHS and activate the reaction at room temperature for 30 min. Add the activated citrulline / EDC / NHS mixed solution dropwise to the chitosan solution at a uniform rate and stir at room temperature for 1 day. After the reaction is complete, dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 Da at room temperature for 4 days. After freeze-drying, obtain the chitosan-citrulline derivative.

[0048] The chitosan ornithine derivative and chitosan citrulline derivative obtained above were prepared into aqueous solutions with a mass fraction of 3 wt%. The two solutions were then mixed thoroughly in a specific ratio, controlling the mass ratio of chitosan citrulline derivative to chitosan ornithine derivative in the mixture to be 1:4. 1,4-Butanediol diglycidyl ether was added to the mixed solution to make the mass ratio of crosslinking agent (solute) to chitosan amino acid derivative (solute) 1:600, and the mixture was stirred until homogeneous. The mixture was placed in a 40 ℃ constant temperature water bath for 4 h, then cooled to -40 ℃ for freeze-drying. After drying, the product was placed in a 50 ℃ oven for 1 day to cure, thus obtaining the expandable hemostatic sponge with antibacterial function (sample name: OC / CC). 20 -0.05).

[0049] Example 5 The only difference between this embodiment and Example 2 is that in the chitosan amino acid derivative mixed solution, the mass ratio of chitosan citrulline derivative to chitosan arginine derivative is 2:3; all other preparation steps and process parameters are completely consistent with Example 2 (sample name is denoted as AC / CC). 40 -0.05).

[0050] Example 6 The only difference between this embodiment and Example 2 is that in the chitosan amino acid derivative mixed solution, the mass ratio of chitosan citrulline derivative to chitosan arginine derivative is 3:2; all other preparation steps and process parameters are completely consistent with Example 2 (sample name is denoted as AC / CC). 60 -0.05).

[0051] Example 7 The only difference between this embodiment and Example 2 is that in the chitosan amino acid derivative mixed solution, the mass ratio of chitosan citrulline derivative to chitosan arginine derivative is 4:1; all other preparation steps and process parameters are completely consistent with Example 2 (sample name is denoted as AC / CC). 80 -0.05).

[0052] Example 8 The only difference between this embodiment and Example 2 is that a 3 wt% aqueous solution of chitosan arginine derivative was prepared using a single component, and 1,4-butanediol diglycidyl ether was added to make the mass ratio of crosslinking agent (solute) to chitosan amino acid derivative (solute) 1:600; the remaining preparation steps and process parameters are completely consistent with those of Example 2 (sample name is AC-0.05).

[0053] Example 9 The difference between this embodiment and Embodiment 2 is only that: a single-component chitosan citrulline derivative is formulated into an aqueous solution with a mass fraction of 3 wt%, and 1,4-butanediol diglycidyl ether is added, so that the mass ratio of the cross-linking agent (solute) to the chitosan amino acid derivative (solute) is 1:600; the remaining preparation steps and process parameters are exactly the same as those in Embodiment 2 (the sample name is recorded as CC-0.05).

[0054] Comparative Example 1 A commercially available medical hemostatic gauze is used as a control sample.

[0055] Comparative Example 2 A commercially available expandable polyvinyl alcohol (PVA) hemostatic sponge is used as a control sample (manufacturer: Beijing Yingjia Maidic Medical Materials Co., Ltd., medical device registration certificate number: Beijing Medical Device Registration Approval 20152140298).

[0056] Comparative Example 3 A commercially available chitosan oral hemostatic sponge is used as a control sample (manufacturer: Henan Ping'an Tree Medical Device Co., Ltd., medical device registration certificate number: Hunan Medical Device Registration 20222141564).

[0057] Comparative Example 4 A commercially available gelatin sponge is used as a control sample (manufacturer: Nanchang Hushida Medical Technology Co., Ltd., medical device registration label number: National Medical Device Registration 20233141949) The expandable hemostatic sponges obtained in each embodiment and the samples in the comparative examples are subjected to various performance standards using exactly the same test methods, and the detection methods and corresponding results are as follows: (1)Microscopic morphology test after water absorption (scanning electron microscope test) The sponge of Embodiment 5 is cut into a standard cylindrical specimen and immersed in deionized water at 25 °C until the water absorption reaches complete equilibrium; after freeze-drying treatment, the specimen is cryo-fractured, and the cross-sectional microscopic morphology is observed using a scanning electron microscope. The results are shown in Figure 1, and the interior of the sponge presents a three-dimensional porous structure.

[0058] (2)Water absorption rate and maximum water absorption test The target sponge was cut into standard cylindrical samples, and the initial dry weight of the sample was recorded as m0 and the initial volume as V0. The sample was compressed to 20% of its initial height along the height direction and fixed. Then it was immersed in deionized water at 25 ℃. The sample was taken out at preset time intervals and suspended in the air for 30s to remove unadsorbed free water from the surface. The wet weight m1 of the sample at different immersion times and the volume V1 after water absorption equilibrium were weighed and recorded. The water absorption rate and swelling rate at the corresponding time points were calculated, and the water absorption rate and maximum water absorption of the sponge were finally obtained. From the water absorption rate of the sponge (Figure 2), it can be seen that when the CC content in the sponge is less than 80%, the water absorption rate of the sponge is not affected by the CC content. Examples 2, 5, 6 and 8 can reach saturation within 5s. However, once the CC content continues to increase, the water absorption rate of the sponge will decrease significantly. The maximum water absorption capacity (Figure 3) and volume expansion rate (Figure 4) of the sponge are both positively correlated with the CC content in the sponge. It can absorb up to 130 times its own weight in water, and its volume expands to 16 times its original size.

[0059] (3) Cyclic compression performance test The target sponge was cut into standard cylindrical samples and completely immersed in deionized water at 25 °C until saturated. Cyclic compression tests were then conducted using a universal testing machine at room temperature and 50% relative humidity. The test parameters were set as follows: compression speed 10 mm / min, return speed 10 mm / min, maximum compressive strain 70%, and 20 cycles per set. The experimental results are shown in Figure 2. As the CC content in the sponge increases, the sponge structure gradually becomes more flexible, and its fatigue resistance improves. When the CC content reaches 40% (Example 5), the sponge exhibits good fatigue resistance, and its compressive stress can withstand the blood pressure of major organs in the human body (e.g., coronary artery, renal artery, and hepatic artery pressure: 8~18.5 kPa; pulmonary artery pressure: 1.1~3.3 kPa).

[0060] (4) Wet adhesion performance test The target sponge was cut into standard cubic samples (the size can be set according to actual needs) and attached to the surface of fresh pigskin that had been fully soaked in physiological saline. A constant pressure was applied using a 500 g standard weight, and the sample was left to stand for 5 minutes to allow the sponge and pigskin to fully adhere. Subsequently, a vertical tensile test was performed using a universal testing machine with a tensile speed of 100 mm / min. The maximum adhesion strength when the sponge separated from the pigskin was recorded in real time, which is the wet adhesion strength of the sponge. As can be seen from Figure 6, the adhesion ability of Example 9 and Example 8 to wet tissue is comparable. After mixing the two, it can be seen that the wet adhesion of Examples 2 and 7 did not increase significantly, while the wet adhesion of Examples 5 and 6 was significantly higher than that of Examples 2, 7, 8, and 9. Among them, Example 5 had the strongest wet adhesion force, reaching 69.2 kPa.

[0061] (5) In vitro degradation experiment After drying the sponge sample to constant weight, accurately weigh it and record its initial dry weight as m0; prepare a 4×10⁻⁶ PBS buffer solution with pH=7.4. 4 A lysozyme solution of IU / mL was used to completely immerse the sponge sample in the solution, which was then placed in a 37 ℃ constant temperature incubator to simulate the in vivo physiological environment for degradation experiments. Multiple preset time points were set. At each time point, the sponge sample was removed and repeatedly washed with deionized water to thoroughly remove residual PBS buffer solution and lysozyme from the surface. The sample was then freeze-dried until constant weight, and accurately weighed, recorded as m1. Based on the difference between m0 and m1, the degradation rate and degree of degradation of each group of sponge samples at different time points were statistically analyzed. Figure 7 As shown, with the increase of chitosan arginine content in the sponge, the degradation rate also slows down while the wet mechanical properties increase. In Example 8, the degradation rate was only 70% of the original weight after 7 days and the structure was still intact, while in Example 9, the structure completely disintegrated after 1 day.

[0062] (6) In vitro dynamic coagulation index test After drying the sponge samples to constant weight, they were cut into samples of uniform weight. 5 mL of sodium citrate-anticoagulated rabbit blood was thoroughly mixed with 50 μL of 0.1 M CaCl2 solution and allowed to stand. 100 μL of the prepared blood was added to the surface of each group of sponge samples, and the samples were incubated at 37 ℃ for 5, 30, 60, 180, and 300 s. At each preset time point, the samples were removed, 5 mL of deionized water was quickly added, and the samples were allowed to stand at 37 ℃ for another 5 min. The coagulation state of each group of samples was then recorded using a camera. After centrifugation, the supernatant was collected, and its absorbance was measured at 540 nm. Deionized water was used as a positive control, and commercially available gelatin sponge, commercially available chitosan sponge, and medical gauze were used as reference standards. The in vitro dynamic coagulation index of each group of samples at 300 s was calculated using the corresponding formula. Figure 8 As shown, the coagulation index of the sponge decreased with the increase of chitosan-citrulline derivative content, with a minimum value of 7.2%, which was significantly lower than that of comparative examples 1, 3, and 4, indicating the procoagulant function of chitosan-citrulline derivative.

[0063] (7) Antibacterial performance test ① Staphylococcus aureus antibacterial test: Take 50 μL of a concentration of 1×10 7 A CFU / mL suspension of Staphylococcus aureus was thoroughly mixed with 4 mg of sponge sample and incubated in a constant temperature shaking incubator at 37 ℃ and 100 r / min for 8 h. After incubation, each group of bacterial suspensions was serially diluted to 10⁻¹⁰ with sterile PBS buffer. 5 Take 100 μL of the diluted bacterial suspension and spread it evenly on the surface of solid agar medium. Incubate at 37 ℃ for 24 h. After visible colonies form, photograph the agar plate and count the number of colonies. In this experiment, a blank control group was prepared by co-culturing sterile PBS buffer with an equal volume and concentration of bacterial suspension; the colony count of this control group was recorded as N0. The colony count of the sponge sample group was recorded as N... s The antibacterial rate is calculated based on this.

[0064] ② Escherichia coli antibacterial test: Take 50 μL of a concentration of 1×10 7 A CFU / mL suspension of Escherichia coli was thoroughly mixed with 4 mg of sponge sample and placed in a constant temperature shaking incubator at 37 ℃ and 100 r / min for 4 h. The remaining experimental steps, operating parameters and colony counting methods were consistent with the above Staphylococcus aureus antibacterial test.

[0065] It can be seen that in this invention, the antibacterial rate of the hemostatic sponge increases with the increase of the proportion of chitosan-citrulline derivative. Figure 9 ,10 ).

[0066] (8) Hemolysis rate test After drying the sponge samples to constant weight, they were cut into samples of uniform weight and placed in 1 mL of 10% (v / v) rabbit red blood cell PBS suspension. Untreated red blood cell PBS suspension served as a negative control, and 200 μL of 0.1% (w / w) Triton X-100 solution was added to the red blood cell PBS suspension as a positive control. All groups were incubated at 37 ℃ for 1 h. After incubation, each group of samples was centrifuged at 4 ℃ and 8000 rpm for 10 min. The supernatant was collected, and its absorbance at 540 nm was measured using a multi-mode microplate reader. The hemolysis rate of each sponge group was calculated based on the absorbance value. It can be seen that the hemolysis in Examples 2 and 5-9 was not significantly different from that in PBS (Figure 11), demonstrating good blood compatibility.

[0067] (9) Cytotoxicity test The cytotoxicity of sponges against mouse embryonic fibroblast L929 cells was determined using the MTT assay. The specific procedure is as follows: ① Preparation of sample extract: Cut the sponge sample to the same mass, add it to the complete cell culture medium at a material-to-liquid ratio of 0.1 g / mL, and place it in an incubator containing 5% CO2 at 37 ℃ for 72 h. Collect the supernatant and filter it through a sterile filter membrane to obtain sample extracts of different concentrations. Store at 4 ℃ for later use. ② Cell seeding and culture: L929 cells were seeded at a rate of 6 × 10⁶ cells / year. 3 The cells were seeded at a density of cells / well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After the cells adhered to the plate, the original culture medium in the well was discarded and replaced with complete culture medium containing the sample extract. The cells were then co-cultured for another 72 h. ③ Absorbance detection: After co-culture, 20 μL of MTT solution was added to each well, and the cells were incubated at 37 ℃ for another 4 h. After incubation, the absorbance of each well at 490 nm was measured using a microplate reader. Six parallel experiments were set up for each sample concentration, with untreated normal cultured cells serving as a blank control group.

[0068] pass Figure 12 It can be seen that although the degradation rates of Examples 2 and Examples 5-9 are different, their 72-hour extracts and cell cultures did not show significant toxicity.

[0069] (10) Rat liver puncture hemostasis experiment SPF-grade SD rats weighing 230–260 g were selected for liver biopsy hemostasis experiments. Rats were anesthetized with 2.5% tribromoethanol via intraperitoneal injection and fixed on a sterile operating tray tilted at 30°. After abdominal skin preparation and disinfection, the liver was dissected and exposed. Excess tissue fluid on the liver surface was absorbed using sterile gauze, and pre-weighed sterile filter paper was placed under the liver. A penetrating perforation was created in the liver using a 0.6 cm diameter sterile punch. After 5 seconds of free bleeding to establish continuous bleeding, a sponge was compressed to the appropriate volume and inserted into the perforation. Hemostasis time was recorded in real-time using a camera (the endpoint was defined as the cessation of active bleeding from the wound). After hemostasis, the blood-absorbing filter paper was removed and accurately weighed. The amount of bleeding in each group was calculated based on the weight difference before and after filter paper removal. After hemostasis of the liver in each rat was completed, the liver was repositioned, and the abdominal wound was aseptically sutured. The rats were then returned to their cages for normal rearing, and the survival rate of the rats within 30 days post-surgery was recorded. The experimental results show that, compared with gauze, commercial gelatin sponge, and commercial chitosan sponge, the sponges prepared in Examples 5, 8, and 9 have a shorter hemostasis time and a significantly reduced bleeding volume. Figure 13 More importantly, after using the sponge hemostatic agents prepared in Examples 5, 8, and 9, the rats survived for a longer period of time. Figure 14 ).

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a chitosan-based antibacterial hemostatic expansion sponge, characterized in that, Includes the following steps: 1) Prepare an aqueous solution of chitosan amino acid derivatives; wherein the chitosan amino acid derivatives are one or a combination of two of the following: chitosan aspartic acid derivatives, chitosan arginine derivatives, chitosan lysine derivatives, chitosan ornithine derivatives, and chitosan citrulline derivatives. 2) A crosslinking agent is added to the aqueous solution to carry out a chemical crosslinking reaction, thereby obtaining a crosslinked system; 3) The crosslinking system is subjected to heating reaction, cooling and freeze-drying treatment in sequence to obtain the hemostatic sponge.

2. The method of claim 1, wherein, When the chitosan amino acid derivative is a compound composition, it is a compound composition of any one of chitosan aspartic acid derivative, chitosan arginine derivative, chitosan lysine derivative, and chitosan ornithine derivative with chitosan citrulline derivative.

3. The method of claim 1, wherein, The chitosan amino acid derivative is prepared by dissolving chitosan in an acid solution with a mass fraction of 0.1% to 5%, then adding an amino acid activating solution, reacting at room temperature for 0.5 to 4 days, and then lyophilizing the reaction solution after dialyzing at room temperature for 1 to 4 days to obtain the chitosan amino acid derivative.

4. The method of claim 3, wherein, The acid solution is selected from one or more of acetic acid, formic acid, oxalic acid, phosphoric acid, hydrochloric acid, sulfuric acid, and nitric acid; the weight-average molecular weight of the chitosan is 10,000 to 2,000,000 Da.

5. The method of claim 3, wherein, The main raw materials for preparing chitosan amino acid derivatives, by mass, are: 1-2 parts chitosan, 80-120 parts acid solution, and 1-20 parts amino acids; the molar ratio of repeating structural units of chitosan to amino acids is 1:(0.1-20).

6. The method of claim 1, wherein, The crosslinking agent is selected from one or more of glutaraldehyde, epichlorohydrin, genipin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether.

7. The method of claim 1, wherein, The mass concentration of the chitosan amino acid derivative aqueous solution is 0.1~30 wt%; the crosslinking agent is prepared as an aqueous solution and added to the chitosan amino acid derivative solution so that the mass ratio of the crosslinking agent to the chitosan amino acid derivative is 1:(40~5000).

8. The method according to claim 1, characterized in that, Step 3) The heating reaction is as follows: the crosslinking system is placed in a water bath at 20~70 ℃ and heated for 0~10 h; The cooling and freeze-drying process involves cooling the crosslinking system to -20 to -80 ℃ for freeze-drying, and then placing the product in an oven at 20 to 80 ℃ for curing for 0.5 to 4 days to obtain the hemostatic sponge.

9. A chitosan-based antibacterial and hemostatic expandable sponge, characterized in that, It is prepared by the method according to any one of claims 1 to 8.

10. The application of the chitosan-based antibacterial hemostatic expandable sponge according to claim 9 in the preparation of hemostatic, antibacterial or tissue repair medical materials.

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