A self-gelling porous hemostatic microsheet, its preparation method and application

Abstract

This invention discloses a self-gelling porous hemostatic microsheet, its preparation method, and its application; belonging to the field of hydrogel technology. The method first prepares two raw materials in a sponge-like state: quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid. Then, the two raw materials are separately pulverized into microsheets using a pulverizer. After controlling the size of the microsheets through a sieve, the two types of microsheets are mixed to prepare a porous hemostatic microsheet. This hemostatic microsheet combines the advantages of sponge, hydrogel, and powder, achieving rapid hemostasis and efficient wound healing. The hemostatic microsheet has a large specific surface area and excellent hydrophilicity, enabling rapid blood absorption. It achieves self-gelling through electrostatic interaction and Schiff base crosslinking, forming a dense, porous hydrogel adhesive. The porous hemostatic microsheet possesses excellent mechanical properties, adhesive strength, and ultra-high burst pressure, while also exhibiting biocompatibility, biodegradability, and procoagulant and antibacterial properties.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a self-gelling porous hemostatic microsheet, its preparation method, and its application. Background Technology

[0002] Every year, more than 2 million people worldwide die from uncontrolled bleeding. While surgical suturing is a common method in the emergency treatment of massive blood loss, it is highly dependent on the skill of the specialist and the suturing process is time-consuming, which can severely delay rescue time in emergency situations. Surgical suturing becomes particularly difficult in sudden emergencies such as battlefields, traffic accidents, and natural disasters. This urgently requires researchers to develop effective hemostatic materials suitable for pre-hospital emergency care.

[0003] Based on their form, existing hemostatic materials can be categorized into bandages, gauze, hydrogels, sponges, and powders. However, bandages generally require continuous pressure, and frequent changes can cause secondary injury and increase patient suffering. Hydrogels adhere to wounds through physical / chemical cross-linking with tissue surfaces, but their mechanical properties and adhesion strength weaken as blood flows out. Sponges, with their porous structure, possess excellent blood-absorbing capabilities, but are generally unsuitable for wounds with complex shapes. Traditional hemostatic powders adapt to wound shapes, but they can disperse or dissolve in the blood, posing a risk of blood clots if they enter the body.

[0004] Given that each hemostatic material has its own advantages and disadvantages, researchers have gradually shifted their attention to materials that combine the advantages of multiple material forms. In recent years, self-gelling hemostatic powders have received increasing attention. They combine the advantages of hydrogels in terms of mechanical support, tissue adhesion, and wound closure, while retaining the high blood absorption capacity and ease of filling irregular wounds of powders. However, self-gelling powders have limitations in terms of sustained blood absorption. This may result in self-gelling only occurring in the initial area of ​​direct contact with blood, leading to a relatively thin hydrogel layer that cannot provide strong adhesive strength, burst pressure, or other mechanical properties. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a self-gelling porous hemostatic microsheet, its preparation method and application, to solve the problems in the prior art such as the mechanical strength of hydrogel being weakened by blood flow, the poor adaptability of sponges to complex wounds, and the limitations of self-gelling hemostatic powder in continuous blood absorption.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] A method for preparing a self-gelling porous hemostatic microsheet includes the following steps:

[0008] Step 1: Dissolve quaternized chitosan grafted with gallic acid in water to obtain a first solution; dissolve oxidized hyaluronic acid in water to obtain a second solution; freeze-dry the first and second solutions respectively to obtain sponge-like quaternized chitosan grafted with gallic acid and sponge-like oxidized hyaluronic acid.

[0009] Step 2: The sponge-like quaternized chitosan grafted with gallic acid and the sponge-like oxidized hyaluronic acid are mechanically pulverized and sieved to obtain quaternized chitosan grafted with gallic acid micro flakes and oxidized hyaluronic acid micro flakes.

[0010] Step 3: Mix quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets to obtain self-gelling porous hemostatic microsheets;

[0011] The self-gelling porous hemostatic microsheet forms a porous hydrogel upon contact with blood, and the porous hydrogel can adsorb and store blood.

[0012] A further improvement of the present invention is that:

[0013] Preferably, in step 1, the concentrations of both the first solution and the second solution are 20-30 mg / mL.

[0014] Preferably, in step 1, the freeze-drying temperature is -80℃.

[0015] Preferably, in step 2, the mesh size of the sieve is 50 mesh.

[0016] Preferably, in step 3, the mixing mass ratio of quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets is (10-40):13.

[0017] Preferably, in step 1, the preparation process of quaternized chitosan grafted with gallic acid is as follows: quaternized chitosan is dissolved in water, 1-hydroxybenzotriazole is added, and after stirring, gallic acid is added to obtain reaction system C; 1-ethyl-(3-dimethylaminopropyl)carbodiimide is dissolved in anhydrous ethanol and then added dropwise to reaction system C to obtain reaction system D; reaction system D is dialyzed and freeze-dried to obtain quaternized chitosan grafted with gallic acid.

[0018] Preferably, the preparation process of the quaternized chitosan is as follows: chitosan is dissolved in water, glacial acetic acid is added to obtain reaction system A; glycidyltrimethylammonium chloride is added to reaction system A, and after stirring, the undissolved polymer is removed by centrifugation to obtain supernatant B; supernatant B is precipitated in pre-cooled acetone, and the precipitate is dried to obtain quaternized chitosan.

[0019] Preferably, in step 1, the preparation process of oxidized hyaluronic acid is as follows: under dark conditions, sodium periodate solution is added dropwise to hyaluronic acid solution, stirred, and ethylene glycol is added to terminate the reaction. Sodium chloride is added to form reaction system E. Reaction system E is poured into anhydrous ethanol to precipitate, and the precipitate is dried to obtain oxidized hyaluronic acid.

[0020] A self-gelling porous hemostatic microsheet prepared by any one of the above preparation methods includes quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets.

[0021] One application of the above-mentioned self-gelling porous hemostatic microparticles involves placing the self-gelling porous hemostatic microparticles at the wound site. After the quaternized chitosan-grafted gallic acid microparticles and oxidized hyaluronic acid microparticles come into contact with the blood, they expand into a hydrogel, which can absorb and store blood.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention discloses a method for preparing self-gelling porous hemostatic microsheets. The preparation process first involves freeze-drying two raw materials in a sponge-like state: quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid. Then, the two raw materials are separately pulverized into microsheets using a pulverizer. After controlling the size of the microsheets with a sieve, the two types of microsheets are mixed in a specific ratio to prepare a series of porous hemostatic microsheets. The freeze-drying method preserves the abundant pores in the two sponge-like raw materials. The pulverization of the two sponges further preserves the porous structure to the maximum extent, which is crucial for the material's subsequent expansion at the wound site to form a hydrogel and its continuous absorption of blood. Mechanical pulverization is superior to traditional grinding methods that compress and deform the sponge's pores. The size of the microsheets is then controlled using a sieve, and the quaternized chitosan grafted with gallic acid microsheets and oxidized hyaluronic acid microsheets are mixed according to a predetermined formula to obtain the self-gelling porous hemostatic microsheets. The entire preparation process is simple, low-cost, and suitable for large-scale production.

[0024] Furthermore, quaternized chitosan (QCS) was prepared by reacting biocompatible chitosan with glycidyltrimethylammonium chloride, which improved the water solubility of chitosan and introduced antibacterial properties. By modifying QCS with gallic acid, quaternized chitosan grafted with gallic acid (QCS-GA) was prepared, further enhancing its procoagulant ability and improving the material's adhesion to wet tissues.

[0025] Furthermore, sodium periodate was used as an oxidant to prepare oxidized hyaluronic acid (OHA) with excellent biocompatibility and anti-inflammatory properties.

[0026] This invention also discloses a self-gelling porous hemostatic microsheet, which combines the advantages of sponge, hydrogel, and powder. Specifically, it combines the porosity of sponge, the rapid adsorption of hydrogel, and the good adaptability of powder to complex wounds, enabling rapid hemostasis and efficient wound healing when applied. This porous hemostatic microsheet possesses excellent mechanical properties, adhesive strength, and ultra-high burst pressure, while also exhibiting biocompatibility, biodegradability, and procoagulant and antibacterial properties. It can also deliver lidocaine for analgesia in emergency situations. Furthermore, it can promote wound healing in mice with full-thickness skin defects. This easily manufactured porous hemostatic microsheet can adapt to irregular wounds, providing a new solution for rapid hemostasis and wound healing.

[0027] This invention also discloses the application of a self-gelling porous hemostatic microsheet. This self-gelling porous hemostatic microsheet has a large specific surface area and excellent hydrophilicity. When applied to wounds, the powder form allows it to adapt to complex wounds. After contact with blood, the two types of microsheets can form a hydrogel with the help of blood. At the same time, the formed porous hydrogel can rapidly absorb blood, allowing blood and hydrogel to interact with each other and achieve self-gelling through electrostatic interaction and Schiff base crosslinking, thereby forming a dense, porous hydrogel adhesive.

[0028] This self-gelling porous hemostatic microsheet utilizes two raw materials, quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid, both of which possess excellent water solubility. Therefore, upon contact with blood, the microsheet rapidly gels within 5 seconds, preventing blood from entering the bloodstream and thus reducing the risk of potential thrombosis. Simultaneously, during the in-situ self-gelling process, the abundant aldehyde, hydroxyl, and amino groups on the quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid not only form physical interactions within the material and between tissues, such as electrostatic interactions, hydrogen bonds, and cation π interactions, but also form Schiff base chemical crosslinks with amino groups on the tissue, achieving effective tissue sealing. Through this dual action, the material's adhesion strength and mechanical properties are significantly enhanced. Attached Figure Description

[0029] Figure 1 In the figure, (a) is a schematic diagram of the preparation process of the self-gelling porous hemostatic microplate QCS-GA+OHA; (b) is the mechanism of action of the QCS-GA+OHA microplate.

[0030] Figure 2 In the image, (a) is the 1H NMR spectrum of quaternized chitosan grafted with gallic acid (QCS-GA). 1 (a) HNMR spectrum; (b) oxidized hyaluronic acid (OHA). 1 H NMR spectrum;

[0031] Figure 3 Fourier transform infrared (FT-IR) spectra of QCS, QCS-GA, OHA, QCS-GA / OHA hydrogels and QCS-GA+OHA microsheet hydrogels.

[0032] Figure 4 Images obtained by scanning electron microscopy (SEM): (a) QCS microsheets; (b) OHA microsheets; (c) QCS-GA / OHA hydrogel; (d) QCS-GA / OHA hydrogel microsheets; (e) QCS-GA+OHA microsheets; (f) Hydrogel formed by mixing QCS-GA+OHA microsheets with water.

[0033] Figure 5 Rheological properties of QCS-GA+OHA microplates at 37℃;

[0034] Figure 6 The axial compressive stress-strain curve of the QCS-GA+OHA microsheet;

[0035] Figure 7 The shear adhesion strength of QCS-GA+OHA microplates to moist pigskin;

[0036] Figure 8 The bursting pressure of QCS-GA+OHA microplates on pigskin;

[0037] Figure 9 The coagulation time was determined by adding different hemostatic materials to whole blood at 37°C.

[0038] Figure 10 The blood coagulation index (BCI) of various materials at different time points;

[0039] Figure 11 SEM images of blood cells and platelets adhering to Celox, QCS-GA+OHA4 microplates and QCS-GA+OHA4 microgels;

[0040] Figure 12 In the image, (a) shows the cell compatibility assessment of L929 cells with QCS-GA / OHA hydrogel and QCS-GA+OHA microflake extract. (b) shows the live / dead staining images of L929 cells after co-incubation with QCS-GA / OHA hydrogel and QCS-GA+OHA microflake extract for 24 hours.

[0041] Figure 13 The hemolysis rate of QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, QCS-GA+OHA microsheets and Triton X-100 positive control group upon contact with blood cells;

[0042] Figure 14 Images of hematoxylin and eosin (H&E) staining on day 7 and day 28 for QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, and QCS-GA+OHA microsheets;

[0043] Figure 15 The in vitro degradation curves of QCS-GA+OHA microplates are shown.

[0044] Figure 16 The drug release profile of lidocaine loaded onto QCS-GA+OHA microplates (LID@QCS-GA+OHA microplates);

[0045] Figure 17 This is a statistical analysis of the number of wriggling movements and pain inhibition rate (PIR) in mice during the acetic acid wriggling test.

[0046] Figure 18 The hemostatic effect of a mouse liver bifurcated hemorrhage model; (a) blood loss; (b) hemostasis time;

[0047] Figure 19 To standardize the hemostatic effect in a rat hepatic disc defect hemorrhage model; (a) blood loss; (b) hemostasis time;

[0048] Figure 20 The hemostatic effect in a rat model of complete femoral artery transection and hemorrhage; (a) blood loss; (b) hemostasis time;

[0049] Figure 21 The wound contraction rate of each treatment group on days 3, 7 and 14 in the full-thickness skin wound model was statistically analyzed.

[0050] Figure 22 H&E stained images of wound regeneration in each treatment group on days 3, 7 and 14;

[0051] Figure 23 Mason trichrome staining images of wound regeneration in each treatment group on day 14;

[0052] Figure 24 To quantify the relative collagen content based on the results of Masson trichrome staining on day 14. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to the accompanying drawings:

[0054] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0055] In this article, unless otherwise specified, the terms “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of”. For example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a”.

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0057] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0058] The first aspect of this invention discloses a method for preparing self-gelling porous hemostatic microsheets, comprising the following steps:

[0059] (1) Quaternized chitosan (QCS) was prepared by reacting glycidyltrimethylammonium chloride and chitosan. Specifically, 5 g of chitosan (viscosity less than 200 mPa·s) was dispersed in 180 mL of deionized water, and 900 μL of glacial acetic acid was added. The reaction was continued at 55 °C for 2–3 hours, followed by the addition of 5.8 mL of glycidyltrimethylammonium chloride (GTMAC) and the reaction was continued for 18 hours. After the reaction was completed, the resulting product was poured into pre-cooled acetone to precipitate. QCS precipitated as flocculent matter, which was collected and dried for 3–5 days to obtain the dried flocculent matter.

[0060] (2) Quaternized chitosan-grafted gallic acid (QCS-GA) was prepared by reacting 1-hydroxybenzotriazole, gallic acid, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. 3.03 g of QCS was dissolved in 300 mL of deionized water, and then 2.82 g of 1-hydroxybenzotriazole (HOBt) was added. The mixture was stirred at room temperature for 12 hours. Then, 2.33 g of gallic acid was weighed and added to the solution, and the mixture was stirred until completely dissolved. 2.61 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and dissolved in 10 mL of anhydrous ethanol, and then added dropwise to the solution. The reaction was carried out at room temperature for 24 hours. Afterward, the mixture was dialyzed for 3–5 days, and purified quaternized chitosan-grafted gallic acid (QCS-GA) was obtained by lyophilization. The reaction principle is shown in equation (I):

[0061]

[0062] (3) Oxidized hyaluronic acid (OHA) was prepared by reacting sodium periodate and hyaluronic acid under dark conditions. Under dark conditions, 2.675 g of sodium periodate (NaIO4) was dissolved in 25 mL of deionized water, and then added dropwise to 500 mL of 1% high molecular weight hyaluronic acid solution (HA). The mixture was mechanically stirred for 2 hours in the dark. The reaction was terminated by adding 2.5 mL of ethylene glycol, and a small amount of NaCl was added and stirred for 1 hour. Finally, the mixture was added to anhydrous ethanol at a volume ratio of 1:5 to precipitate the precipitate. After filtration, the precipitate was dried in a vacuum drying oven for about 3 days to obtain block-shaped oxidized hyaluronic acid. The reaction principle is as follows: (II)

[0063]

[0064] (4) Dissolve quaternized chitosan grafted with gallic acid in water to prepare a solution of 20-30 mg / mL, which is the first solution; dissolve oxidized hyaluronic acid in water to prepare a solution of 20-30 mg / mL, which is the second solution; freeze-dry the above two solutions into a sponge state at a freezing temperature of -80℃ for 2-3 days to obtain sponge-like quaternized chitosan grafted with gallic acid and sponge-like oxidized hyaluronic acid; after this step, the two sponge-like raw materials are rich in pores.

[0065] (5) The freeze-dried quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid were crushed by a pulverizer and the size of the micro flakes was controlled by a 50-mesh sieve.

[0066] In this step, crushing with a pulverizer will not damage the pores in the sponge-like material. If grinding is used, the particles after grinding will be too fine and will damage the pores in the sponge-like material.

[0067] Furthermore, this step involves sieving through a 50-mesh sieve to ensure the particle size of the micro-flakes, allowing them to fully fill the wound during application.

[0068] (6) Mixing quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets at a mass ratio of (10-40):13 yields self-gelling porous hemostatic microsheets. The mass ratio is determined based on the amino groups on the quaternized chitosan-grafted gallic acid and the aldehyde groups on the oxidized hyaluronic acid. The process is as follows: Figure 1 As shown in a.

[0069] The second aspect of this invention discloses a self-gelling porous hemostatic microsheet prepared by the above-described method. This microsheet combines the advantages of sponges, hydrogels, and powders. It integrates the rapid and continuous blood absorption capacity of sponges, the adaptability of powders to deep, narrow, and irregularly shaped wounds, and the bioadhesion and effective sealing of moist tissue advantages of hydrogels, achieving rapid hemostasis and efficient wound healing. This has been demonstrated in mouse models of hepatic suture bleeding, standardized rat models of hepatic discoid defects, and rat models of complete femoral artery transection bleeding. The mechanism of action is as follows: Figure 1 As shown in b, it has a large specific surface area and excellent hydrophilicity, enabling it to be rapidly absorbed into blood and achieve self-gelling through the synergistic effect of physical and chemical cross-linking, including electrostatic interactions, cation π interactions, and Schiff base cross-linking.

[0070] This self-gelling porous hemostatic microsheet can adapt to irregularly shaped wounds and rapidly self-gelles upon contact with blood. During in-situ self-gelation, the abundant aldehyde, hydroxyl, and amino groups on the quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid not only form physical interactions within the material and between tissues, such as electrostatic interactions, hydrogen bonds, and cation π interactions, but also form Schiff base chemical crosslinks with amino groups on the tissue, thereby achieving effective tissue sealing. Through this dual action, the material's adhesive strength and mechanical properties are greatly enhanced. Simultaneously, both the microsheet itself and the resulting gel have a porous structure, enabling continuous blood absorption.

[0071] Furthermore, the material is biodegradable and antibacterial, making it safer and more effective in practical applications. It also possesses the ability to deliver local analgesia through the release of lidocaine. The self-gelling porous hemostatic microparticles are easy to use, requiring no complex pretreatment steps in emergency situations, allowing even non-professionals to use them for first aid. Whether applied directly to the wound or using a spatula, syringe, or spray bottle, the various flexible application methods meet the needs of different emergency scenarios, ensuring the stability and reliability of the self-gelling porous hemostatic microparticles in complex hemostatic environments.

[0072] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0073] Example 1

[0074] (1) Quaternized chitosan (QCS) was prepared by reacting glycidyltrimethylammonium chloride and chitosan. 5 g of chitosan (viscosity less than 200 mPa·s) was dispersed in 180 mL of deionized water, and 900 μL of glacial acetic acid was added. The reaction was continued at 55 °C for 2–3 hours, followed by the addition of 5.8 mL of glycidyltrimethylammonium chloride (GTMAC) and a further reaction for 18 hours. After the reaction was complete, the resulting product was poured into pre-cooled acetone to precipitate. QCS precipitated as flocculent matter, which was collected and dried for 3–5 days.

[0075] (2) Quaternized chitosan-grafted gallic acid (QCS-GA) was prepared by reacting 1-hydroxybenzotriazole, gallic acid, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. 3.03 g of QCS was dissolved in 300 mL of deionized water, and then 2.82 g of 1-hydroxybenzotriazole (HOBt) was added. The mixture was stirred at room temperature for 12 hours. Then, 2.33 g of gallic acid was weighed and added to the solution, and the mixture was stirred until completely dissolved. 2.61 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and dissolved in 10 mL of anhydrous ethanol, and then added dropwise to the solution. The mixture was reacted at room temperature for 24 hours. Afterward, the mixture was dialyzed for 3–5 days, and purified QCS-GA was obtained by lyophilization.

[0076] (3) Oxidized hyaluronic acid (OHA) was prepared by reacting sodium periodate and hyaluronic acid under dark conditions. Under dark conditions, 2.675 g of sodium periodate (NaIO4) was dissolved in 25 mL of deionized water, and then added dropwise to 500 mL of 1% high molecular weight hyaluronic acid (HA) solution. The mixture was mechanically stirred for 2 hours in the dark. The reaction was terminated by adding 2.5 mL of ethylene glycol, and a small amount of NaCl was added and stirred for 1 hour. Finally, the precipitate was collected by adding anhydrous ethanol at a volume ratio of 1:5, filtered, and dried in a vacuum drying oven for approximately 3 days.

[0077] (4) Quaternized chitosan grafted with gallic acid was dissolved in water to prepare a solution of 25 mg / mL, and oxidized hyaluronic acid was dissolved in water to prepare a solution of 21.6 mg / mL. The two solutions were then freeze-dried into a sponge state.

[0078] (5) The freeze-dried quaternized chitosan grafted with gallic acid and oxidized hyaluronic acid were crushed by a pulverizer and the size of the micro flakes was controlled by a 50-mesh sieve.

[0079] (6) Mixing quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets at a mass ratio of 15:13 yields a self-gelling porous hemostatic microsheet, which is named QCS-GA+OHA4.

[0080] Example 2

[0081] Unlike Example 1, the mass ratio of quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets in step (6) was adjusted to 40:13, and the resulting self-gelling porous hemostatic microsheets were named QCS-GA+OHA1.

[0082] Example 3

[0083] Unlike Example 1, the mass ratio of quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets in step (6) was adjusted to 26.67:13, and the resulting self-gelling porous hemostatic microsheets were named QCS-GA+OHA2.

[0084] Example 4

[0085] Unlike Example 1, the mass ratio of quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets in step (6) was adjusted to 20:13, and the resulting self-gelling porous hemostatic microsheets were named QCS-GA+OHA3.

[0086] Example 5

[0087] Unlike Example 1, the mass ratio of quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets in step (6) was adjusted to 10:13, and the resulting self-gelling porous hemostatic microsheets were named QCS-GA+OHA5.

[0088] Example 6

[0089] Unlike Example 1, the QCS-GA+OHA4 obtained in step (6) was mixed with deionized water to form a hydrogel, which was named QCS-GA+OHA microsheet hydrogel.

[0090] Example 7

[0091] Unlike Example 1, the concentration of the quaternized chitosan-grafted gallic acid solution in step (4) was prepared to be 50 mg / mL, and the concentration of the oxidized hyaluronic acid solution was prepared to be 43.2 mg / mL. The two solutions were then mixed in equal volumes using a vortex mixer, and the resulting hydrogel was named QCS-GA / OHA hydrogel.

[0092] Example 8

[0093] Unlike Example 7, the QCS-GA / OHA hydrogel was freeze-dried, then pulverized using a pulverizer and the micro-flake size was controlled by a 50-mesh sieve, and it was named QCS-GA / OHA hydrogel micro-flakes.

[0094] Example 9

[0095] Unlike Example 1, quaternized chitosan grafted with gallic acid was dissolved in water to prepare a solution of 20 mg / mL, and oxidized hyaluronic acid was dissolved in water to prepare a solution of 20 mg / mL. Both solutions were then freeze-dried into a sponge-like state.

[0096] Example 9

[0097] Unlike Example 1, quaternized chitosan grafted with gallic acid was dissolved in water to prepare a solution of 30 mg / mL, and oxidized hyaluronic acid was dissolved in water to prepare a solution of 30 mg / mL. Both solutions were then freeze-dried into a sponge-like state.

[0098] The substances obtained in Example 1 were analyzed. 1 H NMR spectrum ( Figure 2 ) and FT-IR spectroscopy ( Figure 3 The successful synthesis of QCS-GA and OHA was confirmed. Regarding QCS-GA... 1 In the 1H NMR spectrum, the characteristic peaks at 3.1 and 3.4 ppm correspond to the trimethylammonium group and the -NH-CH2- group, respectively, indicating that CS reacted successfully with GTMAC. The characteristic peaks between 6.8 and 7.3 ppm are attributed to the protons on the pyrogallol. Furthermore, in the FT-IR results, QCS-GA, relative to QCS, showed significant differences in peak values ​​between 1600 and 1450 cm⁻¹. -1 (C=C stretching vibration of benzene) and 880-680cm -1 The fluctuations in the C-H bending vibration of benzene demonstrate the successful grafting of gallic acid. OHA 1 The 1H NMR spectrum showed a characteristic peak for the hemiacetal proton of OHA at 4.8 ppm. Simultaneously, the FT-IR spectrum showed a peak at 1726 cm⁻¹. -1 The peak at [value missing] cm⁻¹ represents the stretching vibration of the aldehyde-CO- bond, confirming successful oxidation of HA. For hydrogel crosslinking, the FT-IR spectra of QCS-GA / OHA hydrogel and QCS-GA+OHA microsheet hydrogel are essentially the same. The FT-IR spectra of QCS and QCS-GA are in the 3500-3200 cm⁻¹ range. -1 Strong absorption was observed in the range of 1644 cm⁻¹, attributed to N-H and O-H stretching, while this absorption was significantly reduced in both hydrogels, indicating that the functional groups were consumed. Meanwhile, both hydrogels showed absorption at 1644 cm⁻¹. -1The characteristic peaks appear at the specified location, consistent with the characteristic peaks of Schiff base bonds. These results demonstrate that the synthesis experiment and gelation process are consistent with expectations, and that the functional groups in the QCS-GA / OHA hydrogel and the hydrogel formed from QCS-GA+OHA microsheets are essentially the same, both exhibiting Schiff base reactions.

[0099] Figure 4 The images are from a scanning electron microscope (SEM). QCS-GA microflakes exhibit a large, porous, sheet-like structure (a), while OHA microflakes exhibit a slender, porous, sheet-like structure (b). When their solutions are mixed to form a QCS-GA / OHA hydrogel, the pores are rounded and full (c), indicating that in the hydrogel state, it is rich in water, which is subsequently lost due to freeze-drying. Figure 4 As shown in Figure (d), after the hydrogel was pulverized, it was observed that compared to the QCS-GA+OHA micro-flakes prepared by simply mixing QCS-GA and OHA micro-flakes (Figure (e)), the porous structure of the QCS-GA / OHA hydrogel micro-flakes was somewhat lost. Furthermore, compared to the QCS-GA / OHA hydrogel, the hydrogel formed by mixing QCS-GA+OHA micro-flakes with water had denser, sharper, and more irregular pores. This indicates that the QCS-GA+OHA micro-flakes were prepared by mixing... Figure 4 The relative positions in Figure (f) indicate the gelation process. The pores originate from the microsheets themselves and rapid gelation. Therefore, it can be inferred that the hydrogel formed from the microsheets may have better water absorption than the hydrogel formed from solution mixing, which can be macroscopically manifested as a stronger ability to absorb blood. In addition, its dense and porous structure may endow it with higher burst pressure resistance, indicating excellent blood sealing potential.

[0100] Figure 5 The rheological properties of QCS-GA+OHA microsheets were analyzed. After mixing the microsheets with deionized water, the storage modulus of all five QCS-GA+OHA microsheets exceeded the loss modulus within 5 seconds, demonstrating that they could rapidly gel within that timeframe. Simultaneously, the storage modulus of the hydrogel continued to increase during the standing process, indicating a sustained strengthening of the cross-linking effect within the material, which stabilized after approximately 1 hour. Upon complete gelation, group 4 of the QCS-GA+OHA microsheets exhibited better physical properties, with a storage modulus approaching 15 kPa, indicating the formation of a denser hydrogel network.

[0101] Figure 6 The axial compressive stress-strain curves of the QCS-GA+OHA microsheets are shown. All five microsheets formed a solid three-dimensional network structure after absorbing water, capable of resisting a certain amount of pressure. Among them, QCS-GA+OHA4 exhibited the highest compressive strength, at 3.33 MPa.

[0102] Figure 7The adhesion strength of five QCS-GA+OHA microplates to pigskin was evaluated using an overlap shear test. As shown in the figure, even the lowest adhesion strength of QCS-GA+OHA1 reached 25.58 kPa. With increasing OHA content, the adhesion strengths of QCS-GA+OHA2 and QCS-GA+OHA3 gradually increased, reaching 30.40 kPa and 34.59 kPa, respectively. QCS-GA+OHA4 exhibited the highest adhesion strength at 40.98 kPa. However, the adhesion strength of QCS-GA+OHA5, with the highest OHA content, decreased to 27.26 kPa. These results indicate that an appropriate excess of aldehyde groups can better improve tissue adhesion. This may be because, in addition to electrostatic interactions, hydrogen bonding, and cation π interactions, excess aldehyde groups can bind to amino groups on the skin tissue surface, increasing adhesion strength. However, a significantly excessive amount of aldehyde groups affects the crosslinking density, weakening the material's cohesion and thus reducing adhesion.

[0103] Figure 8 The burst pressures of QCS-GA+OHA1, QCS-GA+OHA2, QCS-GA+OHA3, QCS-GA+OHA4, and QCS-GA+OHA5 microplates on pigskin were 823 mmHg, 830 mmHg, 908 mmHg, 848 mmHg, and 763 mmHg, respectively. The burst pressures of all five microplates were significantly higher than the normal systolic blood pressure in humans (90-140 mmHg), and this exceptional burst pressure is highly beneficial for controlling massive bleeding. This is likely due to the porous internal structure of the QCS-GA+OHA microplates and the unique microstructure of the stacking and gelation between the layers, giving them excellent cohesiveness. This prevents the hydrogel from rupturing and enhances its sealing properties. When QCS-GA+OHA microplates are applied to blood, the abundant positive charge on QCS-GA allows for electrostatic interactions with red blood cells and platelets, resulting in even higher adhesion strength and burst pressure of the hydrogel.

[0104] Whole blood clotting time was tested using the inverted test tube method. The ratio of hemostatic material to blood was approximately 1:20. Celox commercial hemostatic powder (with chitosan as the main component) was used as a positive control. Figure 9 The results showed that the clotting time of whole blood without hemostatic materials was as long as 3.74 minutes, while the clotting time in the Celox group was 3.12 minutes. For all five types of QCS-GA+OHA microplates, the clotting time decreased, especially for QCS-GA+OHA4, which had the shortest clotting time of 1.28 minutes, far lower than that of the blank group and the Celox group.

[0105] Figure 10The blood clotting index (BCI) of the materials at different time points was displayed. The control group showed the highest BCI in the first two minutes, which only decreased significantly at 4 minutes, indicating the lowest clotting rate. The BCI of the Celox group and the five QCS-GA+OHA microplates was already about half that of the control group at 0.5 minutes, but the BCI of the five QCS-GA+OHA microplates decreased more significantly with time than that of the Celox group. Furthermore, QCS-GA+OHA4 had the lowest BCI at 4 minutes, at 6.6%. The results indicate that QCS-GA+OHA microplates have effective clotting ability, with QCS-GA+OHA4 exhibiting the strongest clotting ability.

[0106] Figure 11 The study demonstrated the material's ability to aggregate blood cells and platelets. Hydrogels formed by mixing Celox commercial hemostatic powder, QCS-GA+OHA4 microplates, and QCS-GA+OHA4 microplates with PBS were immersed in ACD-anticoagulated whole blood or platelet-rich plasma. The adhesion of blood cells or platelets to these materials was then observed using SEM. The Celox group remained dispersed particles upon contact with anticoagulated whole blood / plasma, with blood cells and platelets merely adhering to the surface of the Celox particles. In contrast, the QCS-GA+OHA4 microplates formed a gel upon mixing with anticoagulated whole blood. Cutting the gel and observing its interior revealed that the raised areas indicated by arrows encapsulated aggregated blood cells / platelets. Furthermore, when the QCS-GA+OHA4 microplate hydrogel was immersed in anticoagulated whole blood / plasma, blood clots were clearly visible on its surface, indicating that the QCS-GA+OHA4 microplates actively adsorb blood cells and platelets. This is because the positive charge on QCS-GA enables the adsorption of blood cells. These results demonstrate that QCS-GA+OHA4 microplates can rapidly adsorb blood and form an in-situ gel upon contact, thus creating a stable and robust physical barrier. Simultaneously, it can aggregate blood cells and platelets, thereby enhancing hemostasis.

[0107] L929 cells were co-cultured with the material leachate to determine cell compatibility. L929 cells were co-cultured with QCS-GA / OHA hydrogel and QCS-GA+OHA microplate leachate for 24 hours, with tissue culture plates (TCP) serving as a control group. Figure 12 As shown in Figure (a), compared to the TCP group, cell viability exceeded 120% in both groups when the leachate concentration ranged from 1.25 mg / mL to 2.5 mg / mL and 5 mg / mL. Referring to Figure (b), the live / dead staining results are consistent with the cell viability results; L929 cells in both groups exhibited a green spindle shape with only a few dead cells in the field of view, similar to the TCP group. These results indicate that the hydrogels prepared by QCS-GA and OHA possess good cell compatibility and can be used as potential hemostatic agents.

[0108] Depend on Figure 13 As shown, the hemolysis rates of QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, and QCS-GA+OHA microsheets were all less than 5%, indicating that these materials have good blood compatibility.

[0109] The biocompatibility of the material was assessed by subcutaneous implantation in rats. Based on H&E staining results ( Figure 14 The results showed that all three samples—QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, and QCS-GA+OHA microsheets—induced some degree of inflammation one week after implantation. The QCS-GA+OHA microsheet group had a relatively thinner encapsulation layer and a milder inflammatory response. Four weeks after implantation, inflammatory cells were significantly reduced in all three groups, and new tissue formed in the blood clot spaces of the hydrogel / microsheets. In particular, the fibrous connective tissue in the QCS-GA+OHA microsheet surface layer group was thinner than the other two groups, and the infiltration of inflammatory cells was also weaker. The results indicate that QCS-GA+OHA microsheets have better in vivo biocompatibility.

[0110] Figure 15 The graph shows the in vitro degradation curve of QCS-GA+OHA microparticles. As can be seen, the material degraded by more than 50% within about a week, and by nearly 75% after 16 days. Therefore, while treating wounds, QCS-GA+OHA microparticles do not remain for a long time and will not significantly affect the user's normal physiological activities. Furthermore, the material's slow degradation also provides an effective physical barrier and protection during the vulnerable inflammatory and repair phases of the wound.

[0111] Figure 16 The figure shows the drug release curve. Introducing lidocaine (LID) into the QCS-GA+OHA microplate achieves rapid hemostasis while simultaneously alleviating patient pain. As shown, LID exhibits burst release within 1 hour, followed by continuous release over 3 hours, with a final total release rate of 86% after 24 hours. When LID@QCS-GA+OHA microplates are applied to fresh wounds, they rapidly bind with blood to form a hydrogel in situ. The porous structure of the hydrogel further facilitates the rapid release of LID, as the pores create solvent absorption channels, accelerating the dissolution of LID and its rapid release into the wound environment.

[0112] The acetic acid torsion test is a commonly used test to verify the pharmacodynamics of analgesic drugs. According to... Figure 17Statistical analysis showed that the control group mice experienced the most discomfort and writhed the most, at 22 times. In contrast, the mice in the LID@QCS-GA+OHA group exhibited greater activity, with a significantly reduced writhing frequency to 4 times, and a pain inhibition ratio (PIR) as high as 81.81%. This demonstrates that LID@QCS-GA+OHA has a good analgesic effect. It can provide rapid analgesia during wound hemostasis, reducing discomfort caused by pain in patients.

[0113] Figure 18 The results show the blood loss (a) and hemostasis time (b) in a mouse hemorrhage model with a hepatic tabular incision. The control group had the highest blood loss at 0.75 g. Blood loss in mice treated with Celox commercial hemostatic powder decreased to 0.50 g. When QCS-GA / OHA hydrogel microplates were used, blood loss was further reduced to 0.21 g. Notably, the blood loss in the QCS-GA+OHA4 microplate group was only 0.10 g, approximately one-seventh that of the control group. The hemostasis time in the control group and the Celox group was 271 seconds and 256 seconds, respectively, which was further shortened to 156 seconds in the QCS-GA / OHA hydrogel microplate group. However, the hemostasis time in the QCS-GA+OHA microplate group was significantly reduced to 99 seconds, approximately one-third that of the control group. These results indicate that QCS-GA+OHA microplates have the best hemostatic effect in this bleeding model. The main reason for the reduced performance of QCS-GA / OHA hydrogel microsheets is that the aldehyde groups in the hydrogel microsheets participate in the homogeneous reaction, which leads to a significant reduction in the number of aldehyde groups available for interfacial adhesion with tissue. Furthermore, the network of the hydrogel microsheets is uniformly and stably cross-linked, affecting the physical and chemical interactions between the QCS-GA / OHA hydrogel microsheets and the wet tissue interface. Therefore, its hemostatic performance is inferior to that of QCS-GA+OHA microsheets.

[0114] Figure 19 The results show the blood loss (a) and hemostasis time (b) in a standardized rat hepatic disc defect hemorrhage model. The blood loss in the control group was 2.00 g, which decreased to 1.05 g after applying Celox to the wound. Applying the same amount of QCS-GA+OHA microparticles significantly reduced the blood loss to 0.12 g, approximately one-twentieth of the control group. Regarding hemostasis time, the control group had 447 seconds, while the Celox group had 207 seconds. Therefore, QCS-GA+OHA microparticles are highly effective for hemostasis in large-area bleeding wounds.

[0115] Figure 20The figures (a) and (b) show the blood loss and hemostasis time in a rat model of complete femoral artery transection bleeding. The rat's femoral artery was completely severed with surgical scissors, resulting in gushing blood. Hemostasis was achieved with gauze, with a blood loss of 5.38 g. Applying QCS-GA+OHA microparticles to the wound resulted in only 1.68 g of blood loss, one-third that of the gauze group. The hemostasis time in the gauze group was 503 seconds, while it was reduced to 175 seconds in the QCS-GA+OHA microparticle group. Therefore, QCS-GA+OHA microparticles are also effective in stopping arterial bleeding.

[0116] A full-thickness skin wound model was used to evaluate the wound healing promoting effect. QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, and QCS-GA+OHA microsheets were used as experimental groups, and commercial Tegaderm was used. TM The thin film dressing served as a control. Based on the statistical results of wound contraction rate (…), Figure 21 After 3 and 7 days of treatment, the QCS-GA+OHA microplate group showed faster healing than the Tegaderm group. TM The study included three groups: a thin film group, a QCS-GA / OHA hydrogel group, and a QCS-GA / OHA hydrogel microplate group. Furthermore, the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microplate group showed faster healing speeds than Tegaderm. TM The membrane group. On day 14, the wound contraction rates of the four groups from left to right were 90%, 95%, 94%, and 96%, respectively, with no significant difference.

[0117] The ability to promote wound healing was further assessed through histological examination. Based on H&E staining results ( Figure 22 All groups showed varying degrees of inflammatory cell infiltration after 3 days of treatment. Among them, Tegaderm... TM The wound site in the membrane group showed a severe inflammatory response, while the other three groups recruited appropriate levels of inflammatory cells. Appropriate inflammation indicates that the body is responding to the wound, recruiting immune cells such as macrophages to clear invading bacteria. Therefore, an appropriate level of inflammation can promote wound healing. After 7 days of treatment, Tegaderm... TM The wound epithelium in the film group was incomplete, while the wound epithelial structure in the three experimental groups was better, and neovascularization was observed. Among them, the QCS-GA+OHA microsheet group had the most vessels observed at the wound site, followed by the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microsheet group. (Tegaderm) TMIn the membrane group, almost no new blood vessels were observed at the wound site. After 14 days of treatment, the wound in the QCS-GA+OHA microplate group almost completely healed, and numerous hair follicles formed, exhibiting the most regular epidermal structure. The wounds in the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microplate group also showed signs of hair follicle growth, demonstrating good re-epithelialization and connective tissue arrangement. Meanwhile, Tegaderm... TM The film group was in the late repair phase, with almost no visible hair follicles, blood vessels, or other differentiated tissues. The results indicate that all three materials have good healing-promoting effects. This is because chitosan in the components can promote cell proliferation and migration, while also producing collagen, hyaluronic acid, and other components. Their porous structure also contributes to the therapeutic effect, with the QCS-GA+OHA microplate group showing the best results.

[0118] Collagen deposition is associated with the mechanical strength of healed skin, and this was analyzed using Masson's trichrome staining. Figure 23 ). Statistical analysis shows that ( Figure 24 The QCS-GA+OHA microplate group showed the highest collagen deposition, followed by the QCS-GA / OHA hydrogel microplate group and the QCS-GA / OHA hydrogel group, while Tegaderm... TM The thin film group had the lowest collagen deposition.

[0119] Based on the above results, in mouse and rat liver hemorrhage models, the optimized formulation (QCS-GA+OHA4) showed superior hemostatic effects compared to Celox. Specifically, the QCS-GA+OHA4 microplates rapidly stopped bleeding at the femoral artery transection site in rats and provided analgesia via lidocaine delivery during emergency treatment. Furthermore, they promoted wound healing in mice with full-thickness skin defects.

[0120] In summary, this invention prepares self-gelling porous hemostatic microsheets composed of QCS-GA and OHA through simple freeze-drying and mechanical pulverization. This is an innovative form of material that combines the advantages of sponges, hydrogels, and powders. Through the synergistic effect of physical and chemical cross-linking, including electrostatic interactions, cation π interactions, and Schiff base bonds, they can rapidly absorb blood and quickly self-gel. This cross-linking and the formation of a dense porous structure simultaneously endow them with excellent mechanical properties, adhesive strength, burst pressure, and excellent sealing ability. In in vitro experiments, the microsheets also exhibited excellent procoagulant activity, blood compatibility, and antibacterial properties. In vivo hemostasis experiments showed that in mouse hepatic tabular incision bleeding models and standardized rat hepatic disc defect bleeding models, the hemostatic effect of QCS-GA+OHA microsheets was significantly better than that of commercial hemostatic powder Celox. In particular, it showed a rapid and effective hemostatic effect in the rat femoral artery complete transection model. Furthermore, the self-gelling porous hemostatic microsheets are biodegradable and biocompatible, can be safely integrated into the body, and effectively promote wound healing. It can also deliver lidocaine for immediate pain relief. This biocompatible QCS-GA+OHA self-gelling porous hemostatic microsheet, which is simple to prepare, easy to use, and has excellent hemostatic effects, may become an indispensable part of emergency care and surgery in the future, providing injured people with rapid and effective treatment and healing opportunities.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a self-gelling porous hemostatic microsheet, characterized in that, Includes the following steps: Step 1: Dissolve quaternized chitosan grafted with gallic acid in water to obtain a first solution; dissolve oxidized hyaluronic acid in water to obtain a second solution; freeze-dry the first and second solutions respectively to obtain sponge-like quaternized chitosan grafted with gallic acid and sponge-like oxidized hyaluronic acid; the freeze-drying temperature is -80℃. The concentrations of both the first and second solutions are 20-30 mg / mL; Step 2: The sponge-like quaternized chitosan grafted with gallic acid and the sponge-like oxidized hyaluronic acid are mechanically pulverized and sieved to obtain quaternized chitosan grafted with gallic acid micro flakes and oxidized hyaluronic acid micro flakes. Step 3: Mix quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets to obtain self-gelling porous hemostatic microsheets; The self-gelling porous hemostatic microsheet forms a porous hydrogel after contacting blood, and the porous hydrogel can adsorb and store blood. Upon contact with blood, the quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets rapidly absorb blood and form porous hydrogels in situ through Schiff base crosslinking reactions and physical interactions between the amino groups on the quaternized chitosan-grafted gallic acid and the aldehyde groups on the oxidized hyaluronic acid.

2. The method for preparing a self-gelling porous hemostatic microsheet according to claim 1, characterized in that, In step 2, the sieve mesh size is 50 mesh.

3. The method for preparing a self-gelling porous hemostatic microsheet according to claim 1, characterized in that, In step 3, the mass ratio of quaternized chitosan grafted gallic acid microsheets and oxidized hyaluronic acid microsheets is (10-40):

13.

4. The method for preparing a self-gelling porous hemostatic microsheet according to claim 1, characterized in that, In step 1, the preparation process of quaternized chitosan grafted with gallic acid is as follows: quaternized chitosan is dissolved in water, 1-hydroxybenzotriazole is added, and after stirring, gallic acid is added to obtain reaction system C; 1-ethyl-(3-dimethylaminopropyl)carbodiimide is dissolved in anhydrous ethanol and then added dropwise to reaction system C to obtain reaction system D; reaction system D is dialyzed and freeze-dried to obtain quaternized chitosan grafted with gallic acid.

5. The method for preparing a self-gelling porous hemostatic microsheet according to claim 4, characterized in that, The preparation process of the quaternized chitosan is as follows: chitosan is dissolved in water, glacial acetic acid is added to obtain reaction system A; glycidyltrimethylammonium chloride is added to reaction system A, and after stirring, the undissolved polymer is removed by centrifugation to obtain supernatant B; supernatant B is precipitated in pre-cooled acetone, and the precipitate is dried to obtain quaternized chitosan.

6. The method for preparing a self-gelling porous hemostatic microsheet according to claim 1, characterized in that, In step 1, the preparation process of oxidized hyaluronic acid is as follows: under dark conditions, sodium periodate solution is added dropwise to hyaluronic acid solution, stirred, and ethylene glycol is added to terminate the reaction. Sodium chloride is added to form reaction system E. Reaction system E is poured into anhydrous ethanol to precipitate, and the precipitate is dried to obtain oxidized hyaluronic acid.

7. A self-gelling porous hemostatic microsheet prepared by the preparation method according to any one of claims 1-6, characterized in that, This includes quaternized chitosan-grafted gallic acid microplates and oxidized hyaluronic acid microplates.

8. An application of the self-gelling porous hemostatic microsheet according to claim 7, characterized in that, When self-gelling porous hemostatic microsheets are placed at the wound, the quaternized chitosan-grafted gallic acid microsheets and oxidized hyaluronic acid microsheets expand into a hydrogel upon contact with the blood, and the hydrogel can absorb and store the blood.

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