Multilayer nanostructured hemostatic powder and method of making same

Abstract

The application discloses a kind of multilayer nanostructure hemostatic powder and preparation method thereof, belong to the field of biomedical hemostatic material.To solve the problem of insufficient mechanical strength of existing silk fibroin-polyphenol hemostatic material, the silk fibroin solution is mixed with regenerated chitin nanofiber solution in proportion, and tea polyphenol solution is added dropwise to induce co-assembly and gelation, and the powder is prepared by washing, freeze-drying and grinding.The powder can quickly absorb water and self-gel after contacting with blood, and form a stable three-dimensional network hydrogel within a few seconds.The regenerated chitin nanofiber guides the formation of hierarchical nanostructure of silk fibroin through supramolecular interaction, significantly enhances the mechanical strength and adhesion, while tea polyphenol provides antibacterial, antioxidant and procoagulant functions.The hemostatic powder of the application has the functions of rapid hemostasis, strong adhesion in wet state, resistance to flushing and promotion of wound healing, and is suitable for various acute bleeding scenarios.

Description

Technical Field

[0001] This invention belongs to the field of hemostatic powder, specifically involving a multi-layered nanostructured hemostatic powder and its preparation method. When the hemostatic powder SCT comes into contact with blood or other body fluids, it will rapidly absorb water and undergo a self-gelling reaction within seconds. The silk fibroin SF, regenerated chitin nanofibers RChNF and tea polyphenols TPs in the powder interact and synergistically construct a stable gel network, realizing a rapid transformation from powder to gel. Background Technology

[0002] Currently, there is an urgent clinical need to develop highly effective adhesive hemostatic sealants for acute bleeding caused by aortic or visceral rupture. Inspired by mussel foot proteins, incorporating chemicals containing catecholamine structures (such as natural plant polyphenols) into adhesive systems has become a potential solution. Natural plant polyphenols, such as tannic acid (TA) and tea polyphenols (TPs), contain multiple pyrogallol and catechol groups in their molecules, which can interact with polar groups on polymer chains, thereby constructing catechol-containing complexes through simple physical blending.

[0003] Among them, silk fibroin SF-TA hydrogel has been proven to be an effective tissue adhesion hemostatic agent with good biocompatibility and extensibility. Benefiting from the dynamic non-covalent interactions and three-dimensional network structure between its components, SF-TA gel also exhibits excellent self-healing capabilities and can be formulated into self-gelling powders, making it easier to address irregular or poorly visualized bleeding wounds. The catechol groups in polyphenols can also interact with proteins in the blood, inducing fibrinogen aggregation at the bleeding site, thereby promoting the hemostatic cascade reaction, which is crucial for thrombosis. Furthermore, this type of SF-polyphenol mixed hydrogel also demonstrates excellent antibacterial properties and hemostatic ability in promoting wound healing.

[0004] However, existing SF-polyphenol composite gels have a significant drawback: their mechanical strength is generally insufficient. This limits the application of the material in wounds that are susceptible to external abrasion or high blood pressure (such as those at the heart or arterial bleeding sites).

[0005] To address the issue of mechanical strength, researchers have attempted to introduce reinforcing phases, such as chitin nanofibers (CNF). However, directly introducing CNF introduces new problems: the abundant polar groups (such as hydroxyl groups) on the CNF surface can competitively bind to SF molecules with polyphenols, which may disrupt the original SF-polyphenol network structure and consequently reduce the material's adhesion.

[0006] Therefore, there is an urgent need in this field for a new type of hemostatic material that can balance adhesion and mechanical strength, and effectively enhance the mechanical properties of the material without sacrificing its original performance.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention aims to develop a multifunctional hemostatic powder to solve the problems of insufficient mechanical strength, difficulty in balancing adhesion and mechanical properties, and single function in the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for preparing a multi-layered nanostructured hemostatic powder includes the following steps:

[0011] 1) Extraction and dissolution of silk fibroin (SF): Silkworm cocoons were cut into pieces and placed in a 9.0-9.5M lithium bromide solution prepared with deionized water to dissolve; the resulting mixed solution was dialyzed through a dialysis bag; using reverse osmosis, 10wt% polyethylene glycol was added to concentrate the dialyzed SF solution to obtain a clear SF solution.

[0012] 2) Preparation of regenerated chitin nanofibers (RChNF): The purified chitin powder was placed in sodium hydroxide solution to obtain a mixture, and then allowed to stand. The mixture was then repeatedly frozen and thawed 2-4 times. Impurities were removed by high-speed centrifugation. The supernatant was allowed to stand, then heated, and dialyzed with deionized water through a dialysis bag until all salts were removed to obtain an RChNF aqueous solution.

[0013] 3) Preparation of hemostatic powder:

[0014] The prepared RChNF aqueous solution and SF solution were mixed in proportion, and water was used as a diluent. The mixture was slowly added dropwise to a 10-20 wt% TPs solution under stirring to initiate a gelation reaction. The gel that settled at the bottom was collected and washed with deionized water to remove excess TPs. The washed gel was freeze-dried to remove moisture. The dried gel was ground into powder to obtain SCT, a multi-layered nanostructured hemostatic powder with self-gelling properties.

[0015] Using the above technical solution, the TPs solution concentration is ≥10wt% to induce the precipitation of SF / RChNF complex.

[0016] Preferably, in step 1), the dissolution conditions are 55-65℃ for 0.5-1.5h; the polyethylene glycol is PEG-20000, and the concentration of the SF solution is 10-15w / v.

[0017] Preferably, the molecular weight cutoff of the dialysis bag in step 1) is 8000-14000.

[0018] Preferably, in step 2), the concentration of the sodium hydroxide solution is 0.3-0.5 g / mL; and the mass ratio of chitosan powder to sodium hydroxide solution is 1:6 to 1:10.

[0019] Preferably, the sodium hydroxide solution is prepared by dissolving 11g of NaOH in 19g of deionized water; the mass ratio of chitosan powder to sodium hydroxide solution is 2:15.

[0020] Preferably, in step 2), the operation of the repeated freeze-thaw unit is as follows: after the mixture is left to stand at room temperature for 8 hours, it is frozen at -40°C for 5 hours; after complete thawing, 70g of deionized water is added, and it is frozen again at -40°C for 5 hours, then thawed at room temperature and stirred vigorously. One unit is one cycle.

[0021] Preferably, in step 2), the obtained supernatant is allowed to stand at 25°C for 24 hours, and then heated at 60°C for 2 hours; the concentration of the obtained RChNF aqueous solution is 2-4 wt%.

[0022] Preferably, in step 3), the prepared RChNF aqueous solution and SF solution are mixed in a ratio of 1:3 to 1:11.

[0023] Furthermore, the preferred mass ratio of SF to RChNF is 5:1 to 10:1. Too high a RChNF ratio may weaken the self-gelling ability, while too low a ratio may not effectively increase the mechanical strength of the gel.

[0024] Preferably, the freezing conditions in step 3) are -40°C to -50°C; the particle size of the freeze-dried powder is 1-5µm.

[0025] This invention also discloses a method for using a multi-layered nanostructured hemostatic powder. The hemostatic steps are as follows: at the bleeding wound, the SCT powder is directly and evenly covered on the surface of the bleeding tissue; after the powder comes into contact with blood, it rapidly expands and gels, closely adhering to the contour of the wound to form a physical barrier to prevent blood from flowing out; at the same time, the TPs components interact with proteins and cells in the blood, accelerating the coagulation process and achieving rapid hemostasis.

[0026] Preferably, the time for the powder to rapidly expand and gel upon contact with blood is no more than 10 seconds.

[0027] The above technical solution involves the following process: when SCT powder comes into contact with blood or other bodily fluids, it rapidly absorbs water and undergoes a self-gelling reaction within seconds, forming a hydrogel with a three-dimensional network structure. During this process, SF, RChNF, and TPs in the powder interact and synergistically construct a stable gel network, achieving a rapid transformation from powder to gel.

[0028] The beneficial effects of this invention are:

[0029] 1) RChNF-guided hierarchical assembly of SF: Utilizing the positive charge on the RChNF surface and the electrostatic interaction and hydrogen bonding between it and SF molecules, SF is induced to transform from a micelle structure to a fiber network structure, forming a composite material with a multi-level nanostructure. This enhances mechanical properties while avoiding competitive binding with TPs, thus maintaining the adhesion and functionality of the material.

[0030] 2) Multifunctional integration of TPs: The introduction of TPs not only provides the material with catechol groups to enhance its adhesion to the tissue surface, but also utilizes its own antioxidant and antibacterial properties to endow the hemostatic powder with multiple functions such as rapid coagulation, antibacterial protection and wound healing promotion.

[0031] 3) Rapid self-gelling property: By optimizing the ratio of SF, RChNF and TPs and the material preparation process, the hemostatic powder can quickly undergo a gelation reaction after contact with blood to form a hydrogel with high adhesion and mechanical strength, realizing the rapid transformation from powder to an effective hemostatic barrier, and adapting to the hemostatic needs of irregular wounds.

[0032] In summary, this invention provides a multi-layered nanostructured hemostatic powder that can form a firm adhesion with bleeding tissue on moist tissue surfaces or irregular wound sites, quickly stop bleeding, and resist damage from external forces such as blood flushing, tissue friction, and arterial blood pressure. Attached Figure Description

[0033] Figure 1 Schematic diagrams of the mechanical strength and adhesive strength of composite materials ((a) shows two materials bonded together with hemostatic powder; (b) shows the mechanical strength of the composite material; (c) shows the adhesive strength).

[0034] Figure 2 Statistical charts of hemostasis efficiency for tail amputation and liver cutting wounds ((a) Schematic diagram of rat tail vein bleeding model; (b) Representative images of tail bleeding sites and hemostasis after treatment with gauze and SCT powder respectively; (c, d) Quantitative analysis results of tail bleeding volume in different groups (SCT powder group, gauze group and blank control group); (e) Schematic diagram of rat liver bleeding model; (f) Representative images of liver bleeding sites and hemostasis effects after treatment with gauze and SCT powder respectively; (g, h) Quantitative analysis results of liver bleeding volume in different groups).

[0035] Figure 3 Schematic diagram of powder blood uptake ratio and in vitro coagulation efficiency ((a) blood uptake rate of powder samples in Comparative Example 1 and Examples 2-4; (b) photographs of blood clots formed after adding calcified whole blood to different samples).

[0036] Figure 4Schematic diagram of the in vitro antibacterial properties of powders ((a) evaluation of antibacterial activity of SF / RChNF, S2 and TPs; (b) and (c) statistical analysis results of antibacterial activity of the corresponding samples). Detailed Implementation

[0037] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0038] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0039] The experimental method is as follows:

[0040] 1. Blood absorption rate measurement

[0041] Powders (S1-S4) with an initial mass of m0 were immersed in anticoagulated blood at 37°C for 10 seconds. Unabsorbed blood was then removed, and the mass of the powder after absorption was recorded as m. The blood uptake ratio (BUR) is calculated using the following formula:

[0042]

[0043] 2. Coagulation test

[0044] Add 40 μL of 1 mol / L calcium chloride (CaCl2) solution to 3600 μL of citric acidified blood and vortex mix for 10 s. Then, add 50 mg of different samples to 100 μL of blood sample. At preset time points, gently wash the blood in each well with PBS to completely remove any unclotted blood. Clotting time is defined as the time it takes for a homogeneous and stable blood clot to form in the well after washing.

[0045] 3. In vitro antibacterial test

[0046] The in vitro antibacterial activity of the materials was evaluated using the plate spread method. MRSA and E. coli were cultured in standard Luria-Bertani (LB) medium. 50 mg of lyophilized SF / CNF, S2, and TPs powders were added to solutions containing 1 mL of MRSA and E. coli (bacterial concentration approximately 5 × 10⁻⁶). 6In well plates containing CFU / mL, a microbial suspension without any samples was used as a control group. After incubation at 37°C for 2 h, each sample was diluted 100-fold, and 20 μL was plated onto solid LB medium. After incubation at 37°C for 12 h, the colony count N on the plates was counted. Antibacterial activity was calculated using the following formula:

[0047]

[0048] Wherein, NC represents the number of colonies on the control group plate, and NS represents the number of colonies on the sample group plate.

[0049] Example 1:

[0050] (a) Extraction and dissolution of raw materials:

[0051] Degummed silkworm cocoons were selected as raw material, chopped, and placed in a 9.3M lithium bromide solution for dissolution at 60°C for 1 hour. The resulting solution was dialyzed against deionized water using a dialysis bag with a molecular weight cutoff of 8000-14000 to remove residual salts. The dialyzed SF solution was then concentrated using reverse osmosis by adding 10wt% polyethylene glycol (PEG-20000) solution, ultimately yielding a clear SF solution with a concentration of 12w / v.

[0052] (II) Preparation of regenerated chitin nanofibers RChNF:

[0053] Preparation of regenerated chitin nanofibers: 11g of sodium hydroxide was dissolved in 19g of deionized water, followed by the addition of 4g of purified chitin powder. The mixture was allowed to stand at room temperature for 8 hours, then frozen at -40℃ for 5 hours. After complete thawing, 70g of deionized water was added, and the mixture was frozen again at -40℃ for 5 hours, followed by thawing at room temperature with vigorous stirring. This freeze-thaw process was repeated three times, and the mixture was centrifuged at 8000 rpm for 10 minutes to remove impurities. The resulting solution was allowed to stand at 25℃ for 24 hours, then heated at 60℃ for 2 hours. Finally, the solution was dialyzed against the deionized water using a dialysis bag until all salts were removed, yielding a 3wt% RChNF aqueous solution.

[0054] (III) Preparation of hemostatic powder

[0055] Solution mixing: The prepared 3wt% CNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 0.5:5, with water as a diluent.

[0056] Induced gelation and precipitation collection: The above mixed solution was slowly added dropwise to a 10wt% TPs solution under stirring to initiate the gelation reaction. The gel that settled at the bottom was collected and washed with deionized water to remove excess TPs. The washed gel was freeze-dried at -40°C to remove moisture. The dried gel was ground into powder to obtain a multifunctional hemostatic powder (SCT powder) with self-gelling behavior.

[0057] Hemostatic application steps: Apply SCT powder evenly to the bleeding wound surface. When the SCT powder comes into contact with blood or other bodily fluids, it rapidly absorbs water and undergoes a self-gelling reaction within seconds, forming a hydrogel with a three-dimensional network structure. The powder then quickly expands and gels, closely adhering to the wound contour and forming a physical barrier to prevent blood leakage. During this process, SF, RChNF, and TPs in the powder interact to synergistically construct a stable gel network, achieving a rapid transformation from powder to gel. Simultaneously, the TPs component interacts with proteins and cells in the blood, accelerating the coagulation process and achieving rapid hemostasis.

[0058] Example 2:

[0059] (a) Extraction and dissolution of raw materials:

[0060] Degummed silkworm cocoons were selected as raw materials, chopped, and placed in a 9.0M lithium bromide solution for dissolution at 60℃ for 1 hour. The resulting solution was dialyzed against deionized water using a dialysis bag with a molecular weight cutoff of 8000-14000 to remove residual salts. The dialyzed SF solution was then concentrated using reverse osmosis by adding 10wt% polyethylene glycol (PEG-20000) solution, ultimately yielding a clear SF solution with a concentration of 12w / v.

[0061] (II) Preparation of regenerated chitin nanofibers (RChNF):

[0062] Preparation of regenerated chitin nanofibers: 10g of sodium hydroxide was dissolved in 20g of deionized water, followed by the addition of 3g of purified chitin powder. The mixture was allowed to stand at room temperature for 8 hours, then frozen at -40℃ for 5 hours. After complete thawing, 70g of deionized water was added, and the mixture was frozen again at -40℃ for 5 hours, followed by thawing at room temperature with vigorous stirring. This freeze-thaw process was repeated three times, and the mixture was centrifuged at 8000 rpm for 10 minutes to remove impurities. The resulting solution was allowed to stand at 25℃ for 24 hours, then heated at 60℃ for 2 hours. Finally, the deionized water was dialyzed through a dialysis bag until all salts were removed, yielding a 3wt% RChNF aqueous solution.

[0063] (III) Preparation of hemostatic powder

[0064] Solution mixing: The prepared 3wt% CNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 0.5:5.5, with water as a diluent.

[0065] Induced gelation and sedimentation collection: The above mixed solution was slowly added dropwise to a 15wt% TPs solution under stirring to initiate the gelation reaction. The gel that settled at the bottom was collected and washed with deionized water to remove excess TPs. The washed gel was freeze-dried at -40°C to remove moisture. The dried gel was ground into powder to obtain a multifunctional hemostatic powder (SCT powder) with self-gelling behavior.

[0066] Hemostatic application steps: Apply SCT powder evenly to the bleeding wound surface. When the SCT powder comes into contact with blood or other bodily fluids, it rapidly absorbs water and undergoes a self-gelling reaction within seconds, forming a hydrogel with a three-dimensional network structure. The powder then quickly expands and gels, closely adhering to the wound contour and forming a physical barrier to prevent blood leakage. During this process, SF, RChNF, and TPs in the powder interact to synergistically construct a stable gel network, achieving a rapid transformation from powder to gel. Simultaneously, the TPs component interacts with proteins and cells in the blood, accelerating the coagulation process and achieving rapid hemostasis.

[0067] Example 3:

[0068] (a) Extraction and dissolution of raw materials:

[0069] Degummed silkworm cocoons were selected as raw material, chopped, and placed in a 9.3M lithium bromide solution for dissolution at 60°C for 1 hour. The resulting solution was dialyzed against deionized water using a dialysis bag with a molecular weight cutoff of 8000-14000 to remove residual salts. The dialyzed SF solution was then concentrated using reverse osmosis by adding 10wt% polyethylene glycol (PEG-20000) solution, ultimately yielding a clear SF solution with a concentration of 12w / v.

[0070] (II) Preparation of regenerated chitin nanofibers (RChNF):

[0071] Preparation of regenerated chitin nanofibers: 12g of sodium hydroxide was dissolved in 18g of deionized water, followed by the addition of 5g of purified chitin powder. The mixture was allowed to stand at room temperature for 8 hours, then frozen at -40℃ for 5 hours. After complete thawing, 70g of deionized water was added, and the mixture was frozen again at -40℃ for 5 hours, followed by thawing at room temperature with vigorous stirring. This freeze-thaw process was repeated three times, and the mixture was centrifuged at 8000 rpm for 10 minutes to remove impurities. The resulting solution was allowed to stand at 25℃ for 24 hours, then heated at 60℃ for 2 hours. Finally, the deionized water was dialyzed through a dialysis bag until all salts were removed, yielding a 3wt% RChNF aqueous solution.

[0072] (III) Preparation of hemostatic powder

[0073] Solution mixing: The prepared 3wt% CNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 1:5, with water as a diluent.

[0074] Induced gelation and precipitation collection: The above mixed solution was slowly added dropwise to a 20wt% TPs solution under stirring to initiate the gelation reaction. The gel that settled at the bottom was collected and washed with deionized water to remove excess TPs. The washed gel was freeze-dried at -40°C to remove moisture. The dried gel was ground into powder to obtain a multifunctional hemostatic powder (SCT powder) with self-gelling behavior.

[0075] Hemostatic application steps: Apply SCT powder evenly to the bleeding wound surface. When the SCT powder comes into contact with blood or other bodily fluids, it rapidly absorbs water and undergoes a self-gelling reaction within seconds, forming a hydrogel with a three-dimensional network structure. The powder then quickly expands and gels, closely adhering to the wound contour and forming a physical barrier to prevent blood leakage. During this process, SF, RChNF, and TPs in the powder interact to synergistically construct a stable gel network, achieving a rapid transformation from powder to gel. Simultaneously, the TPs component interacts with proteins and cells in the blood, accelerating the coagulation process and achieving rapid hemostasis.

[0076] Example 4:

[0077] (a) Extraction and dissolution of raw materials:

[0078] Degummed silkworm cocoons were selected as raw material, chopped, and placed in a 9.3M lithium bromide solution for dissolution at 60°C for 1 hour. The resulting solution was dialyzed against deionized water using a dialysis bag with a molecular weight cutoff of 8000-14000 to remove residual salts. The dialyzed SF solution was then concentrated using reverse osmosis by adding 10wt% polyethylene glycol (PEG-20000) solution, ultimately yielding a clear SF solution with a concentration of 12w / v.

[0079] (II) Preparation of regenerated chitin nanofibers (RChNF):

[0080] Preparation of regenerated chitin nanofibers: 11g of sodium hydroxide was dissolved in 19g of deionized water, followed by the addition of 4g of purified chitin powder. The mixture was allowed to stand at room temperature for 8 hours, then frozen at -40℃ for 5 hours. After complete thawing, 70g of deionized water was added, and the mixture was frozen again at -40℃ for 5 hours, followed by thawing at room temperature with vigorous stirring. This freeze-thaw process was repeated three times, and the mixture was centrifuged at 8000 rpm for 10 minutes to remove impurities. The resulting solution was allowed to stand at 25℃ for 24 hours, then heated at 60℃ for 2 hours. Finally, the solution was dialyzed against the deionized water using a dialysis bag until all salts were removed, yielding a 3wt% RChNF aqueous solution.

[0081] (III) Preparation of hemostatic powder

[0082] Solution mixing: The prepared 3wt% CNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 1.5:4.5, with water as a diluent.

[0083] Induced gelation and precipitation collection: The above mixed solution was slowly added dropwise to a 10wt% TPs solution under stirring to initiate the gelation reaction. The gel that settled at the bottom was collected and washed with deionized water to remove excess TPs. The washed gel was freeze-dried at -40°C to remove moisture. The dried gel was ground into powder to obtain a multifunctional hemostatic powder (SCT powder) with self-gelling behavior.

[0084] The hemostasis procedure is the same as in Example 1.

[0085] Example 5:

[0086] In step 1), the dissolution conditions are 55°C for 1.5 hours; the polyethylene glycol is PEG-20000; the concentration of the SF solution is 10 w / v; the concentration of the RChNF aqueous solution is 2 wt%; and the hemostasis steps are the same as in Example 2.

[0087] Example 6:

[0088] In step 1), the dissolution conditions are 65°C for 0.5 h; the polyethylene glycol is PEG-20000, the concentration of the SF solution is 15 w / v, the concentration of the RChNF aqueous solution is 4 wt%, and the hemostasis steps are the same as in Example 3.

[0089] Comparative Example 1:

[0090] The prepared 3wt% RChNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 0:6, with water as a diluent.

[0091] Comparative Example 2:

[0092] The prepared 3wt% RChNF aqueous solution and 12wt% SF solution were mixed at a mass ratio of 3.0:0, with water as a diluent.

[0093] Comparative Example 3:

[0094] Only RChNF aqueous solution and SF were added, without TPs, otherwise it was the same as in Example 2.

[0095] The gelation time of Examples 1-4 was compared with that of Comparative Examples 1-2, and the results are shown in the table below (S1-4 represent Examples 1-4, and S2' represents the sample of the powder prepared in Example 2 after regeneration and gelation):

[0096] sample SF (wt%) RChNF(wt %) gelation time (s) Adhesion strength (MPa) Adhesion strength of the regenerated gel (MPa) S1 5.0 0.5 8.9 0.384 0.463 S2 5.5 0.5 9.5 0.357 0.441 S3 5.0 1.0 9.0 0.378 0.407 S4 4.5 1.5 9.0 0.256 0.182 Comparative Example 1 6.0 0.0 10.0 0.156 0.316 Comparative Example 2 0.0 3.0 / / /

[0097] gel time binding Figure 1 In the performance comparison, after gelation, the mechanical strength and adhesion strength of Comparative Example 1 were not as good as those of Examples 2-4.

[0098] Furthermore, the hemostatic effect of the hemostatic powder SCT from Example 1 was compared with that of the gauze group and the blank control group. Figure 2 Comparison of hemostatic efficiency: (a) Schematic diagram of rat tail vein bleeding model establishment; (b) Representative images of tail bleeding sites and hemostasis after treatment with gauze and SCT powder respectively; (c, d) Quantitative analysis results of tail bleeding volume in different groups (SCT powder group, gauze group and blank control group); (e) Schematic diagram of rat liver bleeding model establishment; (f) Representative images of liver bleeding sites and hemostatic effects after treatment with gauze and SCT powder respectively; (g, h) Quantitative analysis results of liver bleeding volume in different groups; The hemostatic powder of the present invention has significant efficiency.

[0099] Figure 3 Powder uptake ratio and in vitro coagulation efficiency: (a) Blood uptake rate of powder samples from Comparative Example 1 and Examples 2-4; (b) Photographs of blood clots formed after adding calcified whole blood to different samples; Examples were significantly better than Comparative Examples.

[0100] More importantly, Figure 4 In vitro antimicrobial properties of powders: (a) evaluation of antimicrobial activity of SF / RfCNF, S2 and TPs; (b) and (c) statistical analysis results of antimicrobial activity of corresponding samples; Example 2 is superior to Comparative Example 3.

[0101] In summary, this invention provides: 1. Rapid hemostasis: It can rapidly absorb blood upon contact and trigger a coagulation cascade reaction, achieving rapid clotting and shortening bleeding time; 2. Excellent adhesion: Even on moist tissue surfaces or irregular wound sites, it can form a firm adhesion with bleeding tissue, preventing material displacement or detachment and ensuring the stability of the hemostatic effect; 3. Superior mechanical properties: It possesses sufficient tensile strength and toughness to resist damage from external forces such as blood flushing, tissue friction, and arterial blood pressure, maintaining the integrity of the material at the wound site; 4. Antibacterial and healing-promoting functions: It effectively inhibits wound infection while promoting rapid wound healing, making it a hemostatic powder with superior overall performance compared to existing hemostatic methods, suitable for infected wounds or bleeding scenarios requiring long-term care.

[0102] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a multi-layered nanostructured hemostatic powder, characterized in that, Includes the following steps: 1) Extraction and dissolution of silk fibroin (SF): Take degummed silkworm cocoons, cut them into small pieces, and place them in a 9.0-9.5M lithium bromide solution prepared with deionized water to dissolve them; The resulting mixed solution was dialyzed through a dialysis bag; using reverse osmosis, the dialyzed silk fibroin solution was concentrated by adding 10 wt% polyethylene glycol to obtain a clear SF solution. 2) Preparation of regenerated chitin nanofibers RChNF: The purified chitin powder was placed in sodium hydroxide solution to obtain a mixture, and then allowed to stand. The mixture was then repeatedly frozen and thawed 2-4 times. Impurities were removed by high-speed centrifugation. The supernatant was allowed to stand, then heated, and dialyzed with deionized water through a dialysis bag until all salts were removed to obtain an RChNF aqueous solution. 3) Preparation of hemostatic powder: The prepared RChNF aqueous solution and SF solution were mixed in proportion, and water was used as a diluent. The mixture was then slowly added dropwise to a 10-20 wt% TPs solution under stirring to initiate a gelation reaction. Collect the gel that has settled at the bottom and wash it with deionized water to remove excess TPs; The washed gel was freeze-dried to remove moisture; the dried gel was then ground into powder to obtain SCT, a multi-layered nanostructured hemostatic powder with self-gelling properties.

2. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, In step 1), the dissolution conditions are 55-65℃ for 0.5-1.5h; the polyethylene glycol is PEG-20000, and the concentration of the SF solution is 10-15w / v.

3. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, The molecular weight cutoff of the dialysis bag mentioned in step 1) is 8000-14000.

4. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, In step 2), the concentration of the sodium hydroxide solution is 0.3-0.5 g / mL; the mass ratio of chitosan powder to sodium hydroxide solution is 1:6 to 1:

10.

5. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 4, characterized in that, In step 2), the operation of the repeated freeze-thaw unit is as follows: after the mixture is left to stand at room temperature for 8 hours, it is frozen at -40℃ for 5 hours; after complete thawing, 70g of deionized water is added, and it is frozen again at -40℃ for 5 hours, then thawed at room temperature and stirred vigorously. One unit is one cycle.

6. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, In step 2), the obtained supernatant was allowed to stand at 25°C for 24 hours, and then heated at 60°C for 2 hours; the concentration of the obtained RChNF aqueous solution was 2-4 wt%.

7. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, In step 3), the prepared RChNF aqueous solution and SF solution are mixed in a ratio of 1:3 to 1:

11.

8. The method for preparing a multi-layered nanostructured hemostatic powder according to claim 1, characterized in that, In step 3), the freezing conditions are -40°C to -50°C; the particle size of the freeze-dried powder is 1-5µm.

9. A method for using a multi-layered nanostructured hemostatic powder, characterized in that, The hemostasis steps are as follows: SCT powder is directly and evenly applied to the bleeding tissue surface at the bleeding wound site; the powder expands and gels rapidly upon contact with blood, closely adhering to the wound contour to form a physical barrier to prevent blood from flowing out; at the same time, TPs components interact with proteins and cells in the blood, accelerating the coagulation process and achieving rapid hemostasis.

10. The method of using a multi-layered nanostructured hemostatic powder according to claim 9, characterized in that, The powder expands and gels rapidly upon contact with blood within 10 seconds.

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