Self-gelation porous hemostatic micro-tablet as well as preparation method and application thereof

Abstract

The invention discloses a self-gelation porous hemostatic micro-tablet as well as a preparation method and application thereof. Belongs to the technical field of hydrogel. The method comprises the following steps: firstly, preparing two raw materials of quaternized chitosan grafted gallic acid and oxidized hyaluronic acid in a sponge state by a freeze-drying method, and then respectively crushing the two raw materials into micro-tablets by using a crusher; after the size of the microchip is controlled through a screen, the two kinds of microchips are mixed, and the porous hemostatic microchip is prepared. The hemostatic microchip integrates three advantages of sponge, hydrogel and powder, and can realize rapid hemostasis and efficient wound healing. The hemostatic microchip has a large specific surface area and excellent hydrophilicity, can rapidly absorb blood, and realizes self-gelation through electrostatic interaction and Schiff base crosslinking, so that a compact and porous hydrogel adhesive is formed, and the porous hemostatic microchip has excellent mechanical properties, adhesive strength and ultrahigh bursting pressure, and can be used as a hemostatic material for medical application. And meanwhile, the hydrogel also has the characteristics of biocompatibility, biodegradability, procoagulant property and antibacterial property.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technical materials, and in particular relates to a self-gelling porous hemostatic micro-sheet and a preparation method and application thereof. Background Art

[0002] Every year, more than 2 million people worldwide die from uncontrolled bleeding. Although surgical suturing is a common method for emergency treatment of massive blood loss, it is highly dependent on the skills of professionals and the suturing process is time-consuming, which may seriously 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] According to the form of existing hemostatic materials, they can be divided into bandages, gauze, hydrogels, sponges and powders. However, bandages generally require continuous pressure, and frequent replacement may cause secondary injuries and increase the patient's pain. Hydrogels are able to adhere to wounds through physical / chemical cross-linking with the tissue surface, but their mechanical properties and adhesion strength will be weakened as blood flows out. The porous structure of the sponge gives it excellent blood absorption ability, but it is generally not suitable for wounds with complex shapes. Traditional hemostatic powders adapt to the shape of the wound, but they will disperse or dissolve in the blood, and there is a risk of causing thrombosis when entering the human body.

[0004] Given that each hemostatic material has its own advantages and disadvantages, researchers have gradually turned their attention to materials that combine the advantages of multiple material forms. In recent years, self-gelling hemostatic powder has received increasing attention. It combines the advantages of hydrogels in mechanical support, tissue adhesion and wound closure, while retaining the high blood absorption capacity of powders and the characteristics of easy filling of irregular wounds. However, self-gelling powders have limitations in continuous blood absorption. This may cause only the powder that is initially in direct contact with the blood to self-gel, resulting in the formation of a relatively weak hydrogel layer that cannot provide strong mechanical properties such as adhesion strength and burst pressure. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a self-gelling porous hemostatic micro-sheet and its preparation method and application, so as to solve the problems in the prior art that the mechanical strength of the hydrogel is weakened by blood flow, the sponge has poor adaptability to complex wounds, and the self-gelling hemostatic powder has limitations in continuously absorbing blood.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a self-gelling porous hemostatic micro-sheet comprises the following steps:

[0008] Step 1, dissolving quaternized chitosan grafted gallic acid in water to obtain a first solution; dissolving oxidized hyaluronic acid in water to obtain a second solution, and freeze-drying the first solution and the second solution respectively to obtain sponge-like quaternized chitosan grafted gallic acid and sponge-like oxidized hyaluronic acid;

[0009] Step 2, mechanically crushing the sponge-like quaternized chitosan grafted gallic acid and the sponge-like oxidized hyaluronic acid respectively and sieving them to obtain quaternized chitosan grafted gallic acid micro-sheets and oxidized hyaluronic acid micro-sheets;

[0010] Step 3, mixing the quaternized chitosan grafted gallic acid microsheets and the oxidized hyaluronic acid microsheets to obtain a self-gelling porous hemostatic microsheet;

[0011] The self-gelling porous hemostatic micro-sheet forms a porous hydrogel after contacting with blood, and the porous hydrogel can absorb and store blood.

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

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

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

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

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

[0017] Preferably, in step 1, the preparation process of quaternized chitosan grafted with gallic acid is as follows: dissolving quaternized chitosan in water, adding 1-hydroxybenzotriazole, stirring and then adding gallic acid to obtain a reaction system C; dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide in anhydrous ethanol and then dripping it into the reaction system C to obtain a reaction system D; dialyzing the reaction system D and freeze-drying it to obtain quaternized chitosan grafted with gallic acid.

[0018] Preferably, the preparation process of the quaternized chitosan is as follows: dissolving chitosan in water, adding glacial acetic acid to obtain a reaction system A; adding glycidyl trimethylammonium chloride to the reaction system A, stirring and centrifuging to remove undissolved polymer to obtain a supernatant B, precipitating the supernatant B in precooled acetone, and drying the precipitated product to obtain the quaternized chitosan.

[0019] Preferably, in step 1, the preparation process of oxidized hyaluronic acid is: under dark conditions, sodium periodate solution is dripped into hyaluronic acid solution, ethylene glycol is added after stirring to terminate the reaction, and sodium chloride is added to form a reaction system E; the reaction system E is poured into anhydrous ethanol for precipitation, and the precipitated product is dried to obtain oxidized hyaluronic acid.

[0020] A self-gelling porous hemostatic micro-sheet prepared by any one of the preparation methods described above comprises a quaternized chitosan grafted gallic acid micro-sheet and an oxidized hyaluronic acid micro-sheet.

[0021] An application of the self-gelling porous hemostatic micro-sheet is to place the self-gelling porous hemostatic micro-sheet on a wound, and the quaternized chitosan grafted gallic acid micro-sheet and the oxidized hyaluronic acid micro-sheet expand into a hydrogel after contacting blood, and the hydrogel can absorb and store blood.

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

[0023] The invention discloses a method for preparing a self-gelling porous hemostatic micro-sheet. In the preparation process, two raw materials, quaternized chitosan grafted gallic acid and oxidized hyaluronic acid, in a sponge state are first prepared by freeze drying, and then the two raw materials are crushed into micro-sheets by a pulverizer. After the size of the micro-sheet is controlled by a screen, the two micro-sheets are mixed in a certain proportion to prepare a series of porous hemostatic micro-sheets. Two sponge-like raw materials are prepared by freeze drying, and the raw materials themselves are kept to have abundant pores; and then the two sponges are crushed by a pulverizer to retain the porous structure to the greatest extent, which is the key to the subsequent expansion of the material at the wound to form a hydrogel and to continuously absorb blood. The mechanical crushing method is superior to the traditional grinding method that can compress and deform the sponge pores. Then, the size of the micro-sheet is controlled by a screen, and then the quaternized chitosan grafted gallic acid micro-sheet and the oxidized hyaluronic acid micro-sheet are mixed according to the setting to obtain the self-gelling porous hemostatic micro-sheet. The whole preparation process is simple, low in cost, and suitable for large-scale production.

[0024] Furthermore, quaternized chitosan (QCS) was prepared by reacting chitosan with good biocompatibility with glycidyl trimethylammonium 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, which further strengthened its ability to promote blood coagulation and enhanced the material's adhesion to wet tissue.

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

[0026] The present invention also discloses a self-gelling porous hemostatic micro-sheet, which combines the advantages of sponge, hydrogel and powder, and specifically combines the porosity of sponge, the rapid adsorption of hydrogel and the good adaptability of powder to complex wounds, so that the self-gelling porous hemostatic micro-sheet can achieve rapid hemostasis and efficient wound healing when used. The porous hemostatic micro-sheet has excellent mechanical properties, bonding strength and ultra-high bursting pressure, and also has biocompatibility, biodegradability, as well as procoagulant and antibacterial properties, and can deliver lidocaine to provide analgesia in emergency situations. In addition, it can also promote wound healing of full-thickness skin defects in mice. This easy-to-manufacture porous hemostatic micro-sheet can adapt to irregular wounds and provides a new solution for rapid hemostasis and wound healing.

[0027] The present invention also discloses an application of a self-gelling porous hemostatic micro-sheet. The self-gelling porous hemostatic micro-sheet has a large specific surface area and excellent hydrophilicity. When applied to a wound, the powder form enables it to adapt to complex wounds. After the two types of micro-sheets come into contact with blood, they can form a hydrogel with the help of the blood. At the same time, the porous hydrogel formed can quickly absorb the blood, so that the blood and the hydrogel can interact with each other, and achieve self-gelation through electrostatic action and Schiff base cross-linking, thereby forming a dense, porous hydrogel adhesive.

[0028] The self-gelling porous hemostatic micro-sheet, quaternized chitosan grafted gallic acid and oxidized hyaluronic acid, both raw materials have good water solubility. Therefore, after contacting with blood, the self-gelling porous hemostatic micro-sheet can quickly gel within 5 seconds to prevent it from entering the blood, thereby reducing the risk of possible thrombosis. At the same time, during the in situ self-gelling process, the abundant aldehyde groups, hydroxyl groups and amino groups on the quaternized chitosan grafted gallic acid and oxidized hyaluronic acid can not only form physical interactions between the material and the tissue, such as electrostatic interactions, hydrogen bonds and cation π interactions, but also form Schiff base chemical cross-linking with the amino groups on the tissue, thereby achieving effective tissue sealing. Through this dual effect, the adhesion strength and mechanical properties of the material are greatly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 In the figure, (a) is the H NMR spectrum of quaternized chitosan grafted with gallic acid (QCS-GA) ( 1 HNMR spectrum); (b) is 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 hydrogel and QCS-GA+OHA microsheet hydrogel.

[0032] Figure 4 Scanning electron microscope (SEM) images; (a) QCS microsheet; (b) OHA microsheet; (c) QCS-GA / OHA hydrogel; (d) QCS-GA / OHA hydrogel microsheet; (e) QCS-GA+OHA microsheet; (f) hydrogel formed by mixing QCS-GA+OHA microsheet with water;

[0033] Figure 5 is the rheological properties of QCS-GA+OHA microsheets at 37°C;

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

[0035] Figure 7 is the shear adhesion strength of QCS-GA+OHA microsheets to wet pig skin;

[0036] Figure 8 is the bursting pressure of QCS-GA+OHA microsheets on pig skin;

[0037] Fig. 9 The clotting time of different hemostatic materials added to whole blood at 37°C;

[0038] Fig.10 is the blood coagulation index (BCI) of each material at different time points;

[0039] Fig.11 SEM images of blood cells and platelets adhered to Celox, QCS-GA+OHA4 microsheets and QCS-GA+OHA4 microsheet hydrogels;

[0040] Fig.12 In the figure, (a) is the cytocompatibility evaluation of QCS-GA / OHA hydrogel and QCS-GA+OHA microsheet leaching solution on L929 cells. (b) is the live / dead staining image of L929 cells after co-incubation with QCS-GA / OHA hydrogel and QCS-GA+OHA microsheet leaching solution for 24 hours;

[0041] Fig.13 It is the hemolysis rate of QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheet, QCS-GA+OHA microsheet and Triton X-100 positive control group in contact with blood cells;

[0042] Fig.14 The hematoxylin-eosin (H&E) staining images of QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheet, and QCS-GA+OHA microsheet on the 7th and 28th days;

[0043] Fig.15 is the in vitro degradation curve of QCS-GA+OHA microsheets;

[0044] Fig.16 This is the drug release curve of lidocaine loaded on QCS-GA+OHA microtablets (LID@QCS-GA+OHA microtablets);

[0045] Fig.17 Statistics of the number of twisting movements and pain inhibition rate (PIR) of mice in the acetic acid twisting test;

[0046] Fig.18 The hemostatic effect of the mouse liver straight incision bleeding model; (a) blood loss; (b) hemostasis time;

[0047] Fig.19 The hemostatic effect of the standardized rat liver discoid defect bleeding model; (a) blood loss; (b) hemostasis time;

[0048] Fig. 20 The hemostatic effect of the rat femoral artery complete transection bleeding model; (a) blood loss; (b) hemostasis time;

[0049] Fig.21 The wound contraction rate statistics of each treatment group on days 3, 7 and 14 in the full-thickness skin wound model;

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

[0051] Fig.23 Masson's trichrome staining images of wound regeneration in each treatment group on day 14;

[0052] Fig.24 To quantify the relative collagen content based on Masson's trichrome staining results on day 14. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0054] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0055] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0056] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0057] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

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

[0059] (1) Quaternary ammonium chitosan (QCS) was prepared by using glycidyl trimethyl ammonium chloride and chitosan. The specific process was as follows: 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 to 3 hours, and then 5.8 mL of glycidyl trimethyl ammonium chloride (GTMAC) was added and the reaction was continued for 18 hours. After the reaction was completed, the obtained product was poured into precooled acetone for precipitation, and QCS was precipitated in the form of flocs. After being collected, it was dried for 3 to 5 days to obtain dried flocs.

[0060] (2) Preparation of quaternized chitosan grafted gallic acid (QCS-GA) by 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 and stirred at room temperature for 12 hours. Then 2.33 g of gallic acid was weighed and added to the above solution, and stirring was continued until it was 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 above solution and reacted at room temperature for 24 hours. Subsequently, it was dialyzed for 3 to 5 days, and purified quaternized chitosan grafted gallic acid (QCS-GA) was obtained by freeze-drying. The reaction principle is as follows (I):

[0061]

[0062] (3) Oxidized hyaluronic acid (OHA) was prepared by 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), and 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, anhydrous ethanol was poured into it at a volume ratio of 1:5 for precipitation. After being collected by suction, it was placed in a vacuum drying oven and dried for about 3 days to obtain block-shaped oxidized hyaluronic acid. The reaction principle is as follows (II):

[0063]

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

[0065] (5) The freeze-dried quaternized chitosan grafted gallic acid and oxidized hyaluronic acid were pulverized by a pulverizer, and the size of the micro-pieces was controlled by a 50-mesh screen.

[0066] In this step, the pulverizing by a pulverizer will not destroy the pores in the sponge-like raw material. If grinding is used, the pores in the sponge-like raw material will be destroyed because the particles after grinding are too fine.

[0067] Furthermore, this step uses 50-mesh sieving to ensure the particle size of the micro-sheets, so that the micro-sheets can be fully filled in the wound during the application process.

[0068] (6) The quaternized chitosan grafted gallic acid microsheets and the oxidized hyaluronic acid microsheets are mixed in a mass ratio of (10-40):13 to obtain 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 the present invention discloses a self-gelling porous hemostatic micro-sheet prepared by the above-mentioned preparation method. The micro-sheet combines the advantages of sponge, hydrogel and powder, including the ability of sponge to absorb blood rapidly and continuously, the adaptability of powder to deep, narrow and irregular wounds, and the advantages of hydrogel bioadhesion and effective sealing of wet tissue, so as to achieve rapid hemostasis and efficient wound healing, which can be reflected in the mouse liver straight incision bleeding model, the standardized rat liver discoid defect bleeding model and the rat femoral artery complete transection bleeding model. The mechanism of action is as follows Figure 1 b. It has a large specific surface area and excellent hydrophilicity, can quickly absorb blood, and achieves self-gelation through the synergistic effects of physical and chemical cross-linking, including electrostatic interaction, cationic π interaction, and Schiff base cross-linking.

[0070] The self-gelling porous hemostatic micro-sheet can adapt to irregular-shaped wounds when applied, and can quickly self-gel after contact with blood. During the in situ self-gelation process, the abundant aldehyde groups, hydroxyl groups and amino groups on the quaternized chitosan grafted gallic acid and oxidized hyaluronic acid can not only form physical interactions such as electrostatic interactions, hydrogen bonds and cationic π interactions within the material and between tissues, but also form Schiff base chemical cross-linking with the amino groups on the tissues, thereby achieving effective tissue sealing. Through this dual effect, the adhesion strength and mechanical properties of the material are greatly enhanced. At the same time, the micro-sheet itself and the formed gel have a porous structure that can continuously absorb blood.

[0071] In addition, the material is biodegradable and antibacterial, making it safer and more effective in practical applications, and it has the drug-carrying capacity to provide local analgesia through the release of lidocaine. The self-gelling porous hemostatic micro-sheet is easy to use and does not require complicated pretreatment steps in emergency situations. Even non-professionals can use it for first aid. Whether it is applied directly to the wound surface or with the help of a medicine spoon, syringe or spray bottle, a variety of flexible application methods can meet the first aid needs in different scenarios, making the self-gelling porous hemostatic micro-sheet stable and reliable in complex hemostatic environments.

[0072] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0073] Example 1

[0074] (1) Preparation of quaternized chitosan (QCS) by glycidyl trimethyl ammonium 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 to 3 hours, and then 5.8 mL of glycidyl trimethyl ammonium chloride (GTMAC) was added and the reaction was continued for 18 hours. After the reaction was completed, the obtained product was poured into precooled acetone for precipitation. QCS was precipitated in the form of flocs, which were collected and dried for 3 to 5 days.

[0075] (2) Preparation of quaternized chitosan grafted with gallic acid (QCS-GA) by 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 and stirred at room temperature for 12 hours. Then 2.33 g of gallic acid was weighed and added to the above solution, and stirring was continued until it was 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 above solution and reacted at room temperature for 24 hours. Subsequently, it was dialyzed for 3 to 5 days and purified QCS-GA was obtained by freeze-drying.

[0076] (3) Oxidized hyaluronic acid (OHA) was prepared by 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, then added dropwise to 500 mL of 1% high molecular weight hyaluronic acid solution (HA), and 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, anhydrous ethanol was poured into it at a volume ratio of 1:5 for precipitation, and after suction filtration, it was placed in a vacuum drying oven and dried for about 3 days.

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

[0078] (5) The freeze-dried quaternized chitosan grafted gallic acid and oxidized hyaluronic acid were pulverized by a pulverizer, and the size of the micro-pieces was controlled by a 50-mesh screen.

[0079] (6) Quaternized chitosan grafted gallic acid microsheets and oxidized hyaluronic acid microsheets were mixed in a mass ratio of 15:13 to obtain self-gelling porous hemostatic microsheets, which were named QCS-GA+OHA4.

[0080] Example 2

[0081] Different from 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 obtained self-gelling porous hemostatic microsheets were named QCS-GA+OHA1.

[0082] Example 3

[0083] Different from 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 obtained self-gelling porous hemostatic microsheets were named QCS-GA+OHA2.

[0084] Example 4

[0085] Different from 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 obtained self-gelling porous hemostatic microsheets were named QCS-GA+OHA3.

[0086] Example 5

[0087] Different from 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 obtained self-gelling porous hemostatic microsheets were named QCS-GA+OHA5.

[0088] Example 6

[0089] Different from Example 1, the QCS-GA+OHA4 prepared in step (6) is mixed with deionized water to prepare a hydrogel, which is named QCS-GA+OHA microsheet hydrogel.

[0090] Example 7

[0091] Different from 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. Then, the two were mixed evenly in equal volumes using a vortex oscillator, and the resulting hydrogel was named QCS-GA / OHA hydrogel.

[0092] Example 8

[0093] Different from Example 7, the QCS-GA / OHA hydrogel was freeze-dried, then crushed by a grinder and passed through a 50-mesh sieve to control the size of the microsheets, which were named QCS-GA / OHA hydrogel microsheets.

[0094] Example 9

[0095] Different from Example 1, quaternary ammonium chitosan grafted gallic acid was dissolved in water to prepare a 20 mg / mL solution, oxidized hyaluronic acid was dissolved in water to prepare a 20 mg / mL solution, and the two solutions were freeze-dried into a sponge state.

[0096] Example 9

[0097] Different from Example 1, quaternary ammonium chitosan grafted gallic acid was dissolved in water to prepare a 30 mg / mL solution, oxidized hyaluronic acid was dissolved in water to prepare a 30 mg / mL solution, and the two solutions were freeze-dried into a sponge state.

[0098] The various substances obtained in Example 1 were analyzed. 1 H NMR spectrum ( Figure 2 ) and FT-IR spectra ( Figure 3 ) confirmed the successful synthesis of QCS-GA and OHA. 1 In the H NMR spectrum, the characteristic peaks at 3.1 and 3.4 ppm correspond to the trimethylammonium group and -NH-CH2- group, respectively, which indicates that CS reacts successfully with GTMAC. The characteristic peaks between 6.8 and 7.3 ppm are attributed to the protons on pyrogallol. In addition, in the FT-IR results, QCS-GA has a higher peak at 1600-1450 cm-1 relative to QCS. -1 (C═C stretching vibration of benzene) and 880-680cm -1 (C─H bending vibration of benzene) fluctuates, which proves the successful grafting of gallic acid. 1 The H NMR spectrum showed that the characteristic peak of the hemiacetal proton of OHA was observed at 4.8 ppm. At the same time, in the FT-IR spectrum, the peak at 1726 cm -1 The peak at 3500-3200 cm-1 is the stretching vibration of the aldehyde-CO-bond, which proves that HA is successfully oxidized. For hydrogel crosslinking, the FT-IR spectra of QCS-GA / OHA hydrogel and QCS-GA+OHA microsheet hydrogel are basically the same. The FT-IR spectra of QCS and QCS-GA are at 3500-3200 cm-1. -1 The strong absorption in the range of 1644 cm-1 was attributed to N─H and O─H stretching, while this absorption was greatly reduced in the two hydrogels, indicating that the groups were consumed. -1The characteristic peaks appeared at , which were consistent with the characteristic peaks of Schiff base bonds. The above results proved that the synthesis experiment and the gelation process were consistent with expectations, and the groups in the hydrogel formed by QCS-GA / OHA hydrogel and QCS-GA+OHA microsheets were basically the same, and both underwent Schiff base reactions.

[0099] Figure 4 The scanning electron microscope (SEM) images show that QCS-GA microsheets show large porous sheet structures (a), while OHA microsheets show slender porous sheet structures (b); when the solutions of the two are mixed into QCS-GA / OHA hydrogel, its pores are round and full (c), indicating that in the hydrogel state, it is rich in water, and then the water is lost due to freeze-drying. Figure 4 As shown in Figure (d), after the hydrogel was crushed, it was observed that the porous structure of the QCS-GA / OHA hydrogel microsheets was lost compared to the QCS-GA+OHA microsheets (e) obtained by simply mixing QCS-GA and OHA microsheets. In addition, compared with the QCS-GA / OHA hydrogel, the pores of the hydrogel formed by mixing QCS-GA+OHA microsheets and water are denser, sharper and more irregular, which indicates that the QCS-GA+OHA microsheets are mixed. Figure 4 The relative position of the microsheet (f) is used for gelation. The holes are derived from the microsheet itself and the rapid gelation. Therefore, it can be inferred that the water absorption performance of the hydrogel formed by the microsheet may be better than that of the hydrogel formed by the solution mixture, which can be manifested as a stronger ability to absorb blood on a macro scale. In addition, its dense and porous structure may give it a higher burst pressure tolerance, indicating excellent blood sealing potential.

[0100] Figure 5 The rheological properties of QCS-GA+OHA microsheets. After the microsheets were mixed with deionized water, the storage modulus was greater than the loss modulus at 5 seconds, which proves that the five QCS-GA+OHA microsheets can quickly gel within 5 seconds. At the same time, the storage modulus of the hydrogel continued to increase during the static process, indicating that the cross-linking effect inside the material continued to increase and stabilized after about 1 hour. After complete gelation, the QCS-GA+OHA4 group showed better physical properties, and the storage modulus was close to 15kPa, indicating that it formed a denser hydrogel network.

[0101] Figure 6 The axial compressive stress-strain curve of QCS-GA+OHA microsheets. After absorbing water, the five microsheets can form a solid three-dimensional network structure that can withstand a certain pressure. Among them, QCS-GA+OHA4 has the highest compressive strength of 3.33Mpa.

[0102] Figure 7The adhesion strength of five QCS-GA+OHA microsheets to pig skin was evaluated by lap shear test. As shown in the figure, even the lowest adhesion strength of QCS-GA+OHA1 can reach 25.58 kPa. As the proportion of OHA gradually increases, the adhesion strength of QCS-GA+OHA2 and QCS-GA+OHA3 gradually increases, reaching 30.40 kPa and 34.59 kPa, respectively. QCS-GA+OHA4 has the highest adhesion strength of 40.98 kPa. However, the adhesion strength of QCS-GA+OHA5 with the highest OHA content drops to 27.26 kPa. These results indicate that a proper excess of aldehyde groups can better improve tissue adhesion. This may be because, in addition to electrostatic interactions, hydrogen bonds and cationic π interactions, excess aldehyde groups can combine with amino groups on the surface of skin tissue to increase adhesion strength. However, a large excess of aldehyde groups will affect the cross-linking density, making the material cohesion worse, resulting in decreased adhesion.

[0103] Figure 8 The burst pressures of QCS-GA+OHA1, QCS-GA+OHA2, QCS-GA+OHA3, QCS-GA+OHA4 and QCS-GA+OHA5 microsheets on pig skin were 823 mm Hg, 830 mm Hg, 908 mm Hg, 848 mm Hg and 763 mm Hg, respectively. The burst pressures of the five microsheets are much higher than the normal systolic blood pressure of the human body (90-140 mm Hg), and the outstanding burst pressure is very helpful in controlling massive bleeding. This may be due to the porous structure inside the QCS-GA+OHA microsheet and the special microstructure of the stacking and gelling between the sheets, which gives it excellent cohesion. This prevents the hydrogel from breaking and strengthens the sealing. When the QCS-GA+OHA microsheet is applied to the blood, due to the rich positive charges on the QCS-GA, it can form electrostatic interactions with red blood cells and platelets in the blood, which can make the adhesion strength and burst pressure of the hydrogel higher.

[0104] The whole blood coagulation time was tested using the test tube inversion method. The ratio of hemostatic material to blood was approximately 1:20. Celox commercial hemostatic powder (with chitosan as the main ingredient) was used as a positive control. Fig. 9 The results showed that the whole blood coagulation time without hemostatic material was as long as 3.74 minutes, while the coagulation time of Celox group was 3.12 minutes. For the five QCS-GA+OHA micro-tablets, the coagulation time decreased, especially QCS-GA+OHA4, which had the shortest coagulation time of 1.28 minutes, which was much lower than the blank group and Celox group.

[0105] Fig.10The blood coagulation index (BCI) of the materials at different time points is shown. The blank group showed the highest BCI in the first two minutes, which was significantly reduced at 4 minutes, indicating that its coagulation rate was the lowest. The BCI of the Celox group and the five QCS-GA+OHA microtablets at 0.5 minutes was about half of that of the blank group, but the BCI of the five QCS-GA+OHA microtablets decreased more significantly than that of the Celox group as time increased. And QCS-GA+OHA4 had the lowest BCI at 4 minutes, which was 6.6%. The results show that QCS-GA+OHA microtablets have effective coagulation ability, among which QCS-GA+OHA4 has the strongest coagulation ability.

[0106] Fig.11 The ability of the materials to aggregate blood cells and platelets was shown. Celox commercial hemostatic powder, QCS-GA+OHA4 microsheets, and hydrogels formed by mixing QCS-GA+OHA4 microsheets with PBS were immersed in ACD anticoagulated whole blood or platelet-rich plasma, and then the adhesion of blood cells or platelets on these materials was observed by SEM. The Celox group remained dispersed particles when in contact with anticoagulated whole blood / plasma, and blood cells and platelets were only attached to the surface of Celox particles. In contrast to the Celox group, QCS-GA+OHA4 microsheets formed a gel after mixing with anticoagulated whole blood. When the gel was cut open and observed inside, it can be inferred that the bulges shown by the arrows are encapsulated aggregated blood cells / platelets. In addition, the QCS-GA+OHA4 microsheet hydrogel was immersed in anticoagulated whole blood / plasma, and blood clots could be clearly seen on its surface, indicating that QCS-GA+OHA4 microsheets have the ability to actively adsorb blood cells and platelets. This is because the positive charge on QCS-GA can adsorb blood cells. These results indicate that QCS-GA+OHA4 microsheets can rapidly absorb blood and form an in situ gel upon contact, thereby forming a stable and strong physical barrier. At the same time, it can also aggregate blood cells and platelets, thereby enhancing the hemostatic effect.

[0107] L929 cells were co-cultured with the material leaching solution to determine the cell compatibility. QCS-GA / OHA hydrogel and QCS-GA+OHA microsheet leaching solution were co-cultured with L929 cells for 24 hours, and tissue culture plates (TCP) were used as the control group. Fig.12 As shown in Figure (a), compared with the TCP group, the cell survival rates of the two groups of materials exceeded 120% when the concentration of the leaching solution increased from 1.25 mg / mL to 2.5 mg / mL and 5 mg / mL. See Figure (b), the live / dead staining results are consistent with the cell survival results. The L929 cells in both groups of materials showed green spindles, and there were only a few dead cells in the field of view, which was similar to the situation in the TCP group. These results show that the hydrogels prepared by QCS-GA and OHA have good cell compatibility and can be used as potential hemostatic agents.

[0108] Depend on Fig.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 evaluated by subcutaneous implantation in rats. Fig.14 ) showed that the three samples of QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheet and QCS-GA+OHA microsheet all showed a certain degree of inflammation one week after implantation. The encapsulation layer thickness of the QCS-GA+OHA microsheet group was relatively small, and the inflammatory response was also milder. Four weeks after implantation, it can be found that the inflammatory cells in the three groups were significantly reduced, and new tissue was formed in the blood clot gaps of the hydrogel / microsheet. In particular, the fibrous connective tissue of the QCS-GA+OHA micro-surface group was thinner than that of the other two groups, and the infiltration of inflammatory cells was also weaker. The results show that QCS-GA+OHA microsheets have better in vivo biocompatibility.

[0110] Fig.15 This is the in vitro degradation curve of QCS-GA+OHA micro-sheets. As can be seen from the figure, the material degraded by more than 50% in about a week, and decomposed by nearly 75% after 16 days. Therefore, while treating wounds, QCS-GA+OHA micro-sheets will not remain for a long time and will not have a significant impact on the user's normal physiological activities. At the same time, the material will not degrade too quickly and can also play an effective physical barrier and protection role during the inflammatory and repair periods when the wound is more fragile.

[0111] Fig.16 is the drug release curve. Lidocaine (LID) is introduced into QCS-GA+OHA microtablets to achieve the effect of quickly stopping bleeding and relieving the patient's pain as soon as possible. As shown in the figure, LID bursts and releases within 1 hour, followed by continuous release for 3 hours, and the final total release rate of 24 hours can reach 86%. When LID@QCS-GA+OHA microtablets are applied to fresh wounds, they can quickly combine with blood to form a hydrogel in situ. The porous structure of the hydrogel contributes to the rapid release of LID because the pores form solvent absorption channels, which accelerate the dissolution of LID and quickly release it around the wound.

[0112] The acetic acid twist test is a commonly used test to verify the pharmacodynamics of analgesics. Fig.17According to statistics, the mice in the control group felt uncomfortable and twisted the most, 22 times. The mice in the LID@QCS-GA+OHA group showed a more active state, with the number of twists significantly reduced to 4 times, and the pain inhibition ratio (PIR) was as high as 81.81%. This proves that LID@QCS-GA+OHA has a good analgesic effect. It can provide rapid analgesia during wound hemostasis and relieve the discomfort caused by pain in patients.

[0113] Fig.18 The blood loss (a) and hemostasis time (b) of the mouse liver incision bleeding model are shown. The blank group had the highest blood loss of 0.75 g. The blood loss of mice treated with Celox commercial hemostatic powder was reduced to 0.50 g. When QCS-GA / OHA hydrogel microsheets were used, the blood loss was further reduced to 0.21 g. It is worth noting that the blood loss in the QCS-GA+OHA4 microsheet group was only 0.10 g, which is about one-seventh of the blank group. The hemostasis time of the blank group and Celox group was 271 seconds and 256 seconds, respectively, and the QCS-GA / OHA hydrogel microsheet group was further shortened to 156 seconds. However, the hemostasis time of the QCS-GA+OHA microsheet group was greatly shortened to 99 seconds, which is about one-third of the blank group. These results show that in this bleeding model, QCS-GA+OHA microsheets have the best hemostatic effect. 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 aldehyde groups on the surface that can be used for interfacial adhesion with tissues. In addition, the network of the hydrogel microsheets has been uniformly and stably cross-linked, and the physical and chemical interactions formed between the QCS-GA / OHA hydrogel microsheets and the wet tissue interface are also affected, so its hemostatic performance is not as good as that of QCS-GA+OHA microsheets.

[0114] Fig.19 The blood loss (a) and hemostasis time (b) of the standardized rat liver discoid defect bleeding model are shown. The blood loss of the blank group was 2.00 g, and after Celox was sprinkled on the wound, the blood loss was reduced to 1.05 g. After sprinkling the same amount of QCS-GA+OHA microtablets, the blood loss was greatly reduced to 0.12 g, which is about one twentieth of the blank group. In terms of hemostasis time, the blank group was 447 seconds, and the Celox group was 207 seconds. Therefore, QCS-GA+OHA microtablets are very effective in stopping bleeding in large-area bleeding wounds.

[0115] Fig. 20The blood loss (a) and hemostasis time (b) of the rat femoral artery complete transection bleeding model are shown. The femoral artery of the rat was completely cut with surgical scissors, and blood spurted out. Gauze was then used to stop the bleeding, and the blood loss was 5.38g. QCS-GA+OHA microtablets were sprinkled on the wound, and the blood loss was only 1.68g, which was one-third of the gauze group. The hemostasis time of the gauze group was 503 seconds, while the hemostasis time of the QCS-GA+OHA microtablet group was shortened to 175 seconds. Therefore, QCS-GA+OHA microtablets can also effectively stop arterial bleeding.

[0116] The full-thickness skin wound model was used to evaluate the wound healing effect. QCS-GA / OHA hydrogel, QCS-GA / OHA hydrogel microsheets, and QCS-GA+OHA microsheets were used as experimental groups. TM Film dressing was used as control. From the statistical results of wound contraction rate ( Fig.21 ), after 3 and 7 days of treatment, the healing speed of QCS-GA+OHA micro-sheet group was faster than that of Tegaderm TM The healing speed of the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microsheet group was also faster than that of Tegaderm. TM Film group. On the 14th day, the wound contraction rates of the four groups were 90%, 95%, 94% and 96% from left to right, respectively, with no significant difference.

[0117] The wound healing ability was further evaluated by histomorphological examination. Fig. 22 ), all groups showed varying degrees of inflammatory cell infiltration after 3 days of treatment. TM The wound site of the film group showed a severe inflammatory response, while the wounds of the other three groups recruited appropriate levels of inflammatory cells. Appropriate inflammation indicates that the body responds to the wound and recruits immune cells such as macrophages to the wound to remove invading bacteria. Therefore, an appropriate level of inflammation can promote wound healing. After 7 days of treatment, Tegaderm TM The wound epithelium of the film group was incomplete, while the wound epithelium of the three experimental groups had better structure and new blood vessels were formed. Among them, the most blood vessels were observed in the wound site of the QCS-GA+OHA microsheet group, followed by the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microsheet group. TMIn the film group, new blood vessels were hardly observed at the wound site. After 14 days of treatment, the wounds in the QCS-GA+OHA microsheet group were almost completely healed, and many hair follicles were formed, showing the most regular epidermal structure. The wounds in the QCS-GA / OHA hydrogel group and the QCS-GA / OHA hydrogel microsheet group also showed signs of hair follicle growth and showed good re-epithelialization and connective tissue arrangement. TM The film group was at the end of the repair period, and the differentiated tissues such as hair follicles and blood vessels were almost invisible. The results showed that all three materials had good healing effects, which was due to the fact that the chitosan in the components could promote cell proliferation and migration, and produce collagen, hyaluronic acid and other components. It was also due to their porous structure, and the QCS-GA+OHA micro-sheet group had the best therapeutic effect.

[0118] Collagen deposition is related to the mechanical strength of the healed skin and was analyzed using Masson's trichrome staining ( Fig.23 ). Through statistics, we know that ( Fig.24 ), the QCS-GA+OHA microsheet group had the highest collagen deposition, followed by the QCS-GA / OHA hydrogel microsheet group and the QCS-GA / OHA hydrogel group, while Tegaderm TM The film group had the lowest collagen deposition.

[0119] Based on the above results, the optimized formula (QCS-GA+OHA4) showed better hemostatic effects than Celox in the mouse and rat liver bleeding models. In particular, QCS-GA+OHA4 microtablets can quickly stop bleeding at the femoral artery transection site in rats and deliver lidocaine to provide analgesia during emergency treatment. In addition, they can promote wound healing of full-thickness skin defects in mice.

[0120] In summary, the present invention prepares self-gelling porous hemostatic micro-sheets composed of QCS-GA and OHA by simple freeze-drying and mechanical pulverization, which 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 quickly absorb blood and quickly self-gel. This cross-linking and the formation of dense porous structures simultaneously give them excellent mechanical properties, adhesion strength and burst pressure, as well as excellent sealing ability. In in vitro experiments, the micro-sheets also exhibited excellent procoagulant activity, blood compatibility and antibacterial properties. In vivo hemostasis experiments showed that the hemostatic effect of QCS-GA+OHA micro-sheets was significantly better than that of commercial hemostatic powder Celox in the mouse liver incision bleeding model and the standardized rat liver discoid defect bleeding model. In particular, it exhibited a rapid and effective hemostatic effect in the rat femoral artery complete transection model. In addition, the self-gelling porous hemostatic micro-sheets 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 effect, may become an indispensable part of emergency and surgical operations in the future, providing the wounded with a rapid and effective treatment and healing opportunity.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a self-gelling porous hemostatic micro-sheet, characterized in that: The following steps are involved: Step 1, dissolving quaternized chitosan grafted gallic acid in water to obtain a first solution; dissolving oxidized hyaluronic acid in water to obtain a second solution, and freeze-drying the first solution and the second solution respectively to obtain sponge-like quaternized chitosan grafted gallic acid and sponge-like oxidized hyaluronic acid; Step 2, mechanically crushing the sponge-like quaternized chitosan grafted gallic acid and the sponge-like oxidized hyaluronic acid respectively and sieving them to obtain quaternized chitosan grafted gallic acid micro-sheets and oxidized hyaluronic acid micro-sheets; Step 3, mixing the quaternized chitosan grafted gallic acid microsheets and the oxidized hyaluronic acid microsheets to obtain a self-gelling porous hemostatic microsheet; The self-gelling porous hemostatic micro-sheet forms a porous hydrogel after contacting with blood, and the porous hydrogel can absorb and store blood.

2. The method for preparing a self-gelling porous hemostatic micro-sheet according to claim 1, characterized in that: In step 1, the concentrations of the first solution and the second solution are both 20-30 mg / mL.

3. The method for preparing a self-gelling porous hemostatic micro-sheet according to claim 1, characterized in that: In step 1, the freeze-drying temperature is -80°C.

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

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

13.

6. The method for preparing a self-gelling porous hemostatic micro-sheet according to claim 1, characterized in that: In step 1, the preparation process of quaternized chitosan grafted with gallic acid is as follows: dissolving quaternized chitosan in water, adding 1-hydroxybenzotriazole, stirring and then adding gallic acid to obtain a reaction system C; dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide in anhydrous ethanol, and then dripping it into the reaction system C to obtain a reaction system D; dialyzing the reaction system D, and freeze-drying it to obtain a quaternized chitosan grafted with gallic acid.

7. The method for preparing a self-gelling porous hemostatic micro-sheet according to claim 6, characterized in that: The preparation process of the quaternary ammonium chitosan is as follows: dissolving chitosan in water, adding glacial acetic acid to obtain a reaction system A; adding glycidyl trimethylammonium chloride to the reaction system A, stirring and centrifuging to remove undissolved polymers to obtain a supernatant B; precipitating the supernatant B in precooled acetone, and drying the precipitated product to obtain the quaternary ammonium chitosan.

8. The method for preparing a self-gelling porous hemostatic micro-sheet according to claim 1, characterized in that: In step 1, the preparation process of oxidized hyaluronic acid is: under dark conditions, sodium periodate solution is dripped into hyaluronic acid solution, ethylene glycol is added after stirring to terminate the reaction, and sodium chloride is added to form a reaction system E; the reaction system E is poured into anhydrous ethanol for precipitation, and the precipitated product is dried to obtain oxidized hyaluronic acid.

9. A self-gelling porous hemostatic micro-sheet prepared by the preparation method according to any one of claims 1 to 8, characterized in that: It includes quaternized chitosan grafted gallic acid micro-sheets and oxidized hyaluronic acid micro-sheets.

10. An application of the self-gelling porous hemostatic micro-sheet according to claim 9, characterized in that: The self-gelling porous hemostatic micro-sheet is placed at the wound, and the quaternized chitosan grafted gallic acid micro-sheet and the oxidized hyaluronic acid micro-sheet expand into a hydrogel after contacting the blood, and the hydrogel can absorb and store blood.

Images