A rapid hemostatic powder material for massive bleeding, its preparation and application

Abstract

This invention discloses a rapid hemostatic powder material for massive bleeding, which is tannic acid-modified carboxymethyl chitosan. This invention also provides a preparation method and application of the rapid hemostatic powder material for massive bleeding, as well as a rapid hemostatic microgel composition containing the powder material. The rapid hemostatic powder material and microgel composition of this invention possess excellent water solubility, hemostatic properties, and biocompatibility, while also exhibiting multiple functions such as bactericidal, anti-inflammatory, and wound-healing promotion, which is beneficial for the rapid healing of the wound site after hemostasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a rapid hemostatic powder material for massive bleeding and its preparation and application. Background Technology

[0002] Uncontrolled bleeding is a significant cause of increased mortality. For patients undergoing surgery, civilians suffering accidental injuries, and soldiers wounded on the battlefield, timely and effective hemostasis can reduce mortality rates. Currently, commonly used hemostatic materials on the domestic and international markets fall into three categories: polysaccharides, proteins, and inorganic minerals. Polysaccharide hemostatic materials include chitosan and cellulose; protein hemostatic materials include thrombin, collagen, and fibrin; and inorganic mineral hemostatic materials include zeolite, montmorillonite, porous silica, and kaolin. Different types of hemostatic materials have different hemostatic functions and advantages and disadvantages. For example, the protein hemostatic material thrombin can accelerate the natural coagulation cascade reaction, but it has disadvantages such as high processing costs, susceptibility to foreign body reactions, and demanding storage and transportation conditions. Inorganic mineral hemostatic materials utilize their porous nature to absorb water from the blood and concentrate clotting factors and platelets, thereby promoting blood coagulation, but they also have side effects such as fever, which can damage wounds and hinder wound healing.

[0003] Polysaccharides are natural organic compounds with advantages such as wide availability, good biocompatibility, biodegradability, and non-immunogenicity. Chitosan is a product of chitin through deacetylation and is mainly derived from crustaceans. With technological advancements, chitosan has been developed into a hemostatic material. However, the effectiveness of chitosan is not yet stable. While it can effectively stop bleeding under ideal laboratory conditions, in reality, such as in cases of deep wounds or on the battlefield, it encounters many problems and cannot stop bleeding in a timely and effective manner. Furthermore, chitosan is poorly soluble in water, and single-use chitosan hemostatic materials are expensive, hindering large-scale application.

[0004] Despite significant progress in the research of existing hemostatic materials, achieving ideal hemostasis for massive bleeding remains challenging. In cases of massive bleeding wounds, hemostatic materials not only need to effectively stop the outflowing blood promptly but also need to inhibit wound infection, reduce inflammation, and promote wound healing. A single hemostatic material is unlikely to possess multiple functions simultaneously. Therefore, it is necessary to research multi-component rapid hemostatic materials for massive bleeding, applicable to various emergency and special scenarios involving large wound areas and significant bleeding, such as sudden massive bleeding in battlefields, accidents, fieldwork, outdoor settings, and surgical procedures, while also possessing bactericidal, anti-inflammatory, and wound-healing properties. Summary of the Invention

[0005] One objective of this invention is to address the above-mentioned technical problems by providing a rapid hemostatic powder material for massive bleeding that combines multiple functions such as hemostasis, sterilization, anti-inflammation, and wound healing promotion.

[0006] Another object of the present invention is to provide a method for preparing the aforementioned rapid hemostasis powder material for massive bleeding.

[0007] Another object of the present invention is to provide the application of the aforementioned rapid hemostasis powder material for massive bleeding.

[0008] To achieve the above-mentioned objectives, the present invention provides a rapid hemostatic powder material for massive bleeding, wherein the rapid hemostatic powder material for massive bleeding is tannic acid modified carboxymethyl chitosan.

[0009] On the other hand, the present invention also provides a method for preparing the aforementioned rapid hemostatic powder material for massive bleeding, which includes the following steps: adding a 4% tannic acid solution dropwise into a 4% carboxymethyl chitosan aqueous solution, wherein the volume ratio of the tannic acid solution to the carboxymethyl chitosan aqueous solution is 50:1, the pH is maintained at 8.5, the reaction is carried out at 60°C for 3 hours, air is bubbled in during the reaction, dialyzed, freeze-dried, and ground to obtain tannic acid modified carboxymethyl chitosan powder.

[0010] On the other hand, the present invention also provides the application of the aforementioned rapid hemostatic powder material for massive bleeding in any of the following (a) to (d):

[0011] (a) Used to promote blood clotting;

[0012] (b) Used in the preparation of hemostatic materials;

[0013] (c) Used to enhance the hemostatic effect of other hemostatic products;

[0014] (d) Used for antibacterial purposes;

[0015] (e) Used to promote wound healing;

[0016] (f) Used for anti-inflammatory purposes.

[0017] Preferably, the other hemostatic powder is any one or more of cross-linked starch, chitosan, or composite microporous polysaccharide hemostatic powder. More preferably, the other hemostatic powder is cross-linked starch.

[0018] On the other hand, the present invention also provides a rapid hemostatic microgel composition comprising the rapid hemostatic powder material for massive bleeding as described in the present invention and another different hemostatic material.

[0019] Preferably, the weight ratio of the rapid hemostatic powder material for massive bleeding to the other different hemostatic material is 2:1 to 1:2.

[0020] Preferably, the hemostatic material is any one or more of cross-linked starch, chitosan, or composite microporous polysaccharide hemostatic powder.

[0021] Preferably, the hemostatic material is cross-linked starch.

[0022] The cross-linked starch is prepared by the following steps:

[0023] (1) Dissolve 21g of potato starch in 150 mL of 0.2M acetic acid-sodium acetate buffer solution with pH 4. The volume ratio of acetic acid solution to sodium acetate solution is 41:9. Stir at 50℃ for 20 minutes. Then add 2% by mass of mixed enzyme, which is a mixture of saccharifying enzyme and α-amylase, with a mass ratio of 4:1. React at 50℃ for 8 hours. Finally, wash the product with ultrapure water, filter, and vacuum dry at 50℃ to obtain enzymatically hydrolyzed starch.

[0024] (2) Sodium hydroxide was dissolved in 90% v / v ethanol to prepare a 2.5% solution. Then, an equal mass of enzymatically hydrolyzed starch was added. The mixture was refluxed and stirred at 50°C for 30 minutes. Chloroacetic acid was added to the reaction solution. The mass ratio of chloroacetic acid to enzymatically hydrolyzed starch was 18:25. The reaction was carried out at 50°C for 3 hours. Finally, the product was washed with anhydrous ethanol, filtered, and dried under vacuum at 50°C to obtain carboxymethyl enzymatically hydrolyzed starch.

[0025] (3) Dissolve 1 g of carboxymethyl hydrolyzed starch in 10 mL of pure water, adjust the pH of the solution to 10, add sodium trimetaphosphate at a mass ratio of 3% of carboxymethyl hydrolyzed starch, mix to form an aqueous phase, then mix 50 mL of liquid paraffin and Span 80 at a mass ratio of 1% of liquid paraffin to form an oil phase, stir the oil phase at 60 °C, add the aqueous phase dropwise into the oil phase, react at 60 °C for 6 hours, wash, vacuum dry, grind and sieve to obtain cross-linked starch.

[0026] On the other hand, the present invention also provides the use of the rapid hemostatic microgel composition in any of the following (a) to (d):

[0027] (i) Used to promote blood clotting;

[0028] (j) Used in the preparation of hemostatic materials;

[0029] (k) Used for antibacterial purposes;

[0030] (l) Used to promote wound healing;

[0031] (m) is used for anti-inflammatory purposes.

[0032] In this invention, carboxymethyl chitosan possesses excellent water solubility, hemostatic properties, and biocompatibility; therefore, it was chosen as the raw material for synthesizing a universal assembly agent. Since tannic acid has antibacterial, anti-inflammatory, and free radical scavenging effects, tannic acid was grafted onto carboxymethyl chitosan via a one-step Michael addition reaction. After dialysis, freeze-drying, and grinding, tannic acid-modified carboxymethyl chitosan (CTA) assembly agent powder was obtained. CTA can be blended with other commercial hemostatic powders such as chitosan-based Celox, composite microporous polysaccharides SKSS, and cross-linked starch CMS to form three microgel assemblies: Celox-CTA, SKSS-CTA, and CMS-CTA. Upon contact with blood, these assemblies absorb water to form microgels and form hydrogen bonds with hydroxyl and amino groups on red blood cells, platelets, and skin tissue, adhering firmly to the wound site and achieving hemostasis through physical sealing and promoting blood coagulation. The phenolic hydroxyl groups on CTA form hydrogen bonds with the functional groups (hydroxyl, carboxyl, and amino) on Celox and SKSS. The tissue adhesion and mechanical strength of the microgel assembly mainly come from the interaction between the phenolic hydroxyl groups on CTA and the tissue surface. The porous structure can quickly absorb water from the blood to form a microgel that concentrates clotting factors, thereby promoting coagulation. Tannic acid contains a large number of phenolic hydroxyl structures, which can bind to proteins on bacterial cell membranes or inhibit the activity of proteases, changing the structure and function of the membrane, leading to increased cell membrane permeability, leakage of cell contents, and ultimately bacterial death. Tannic acid can also inhibit bacterial cell wall synthesis and scavenge free radicals, reducing bacterial proliferation and damage caused by oxidative stress, thus promoting rapid wound healing after hemostasis. The material forms hydrogen bonds with skin, red blood cells, and platelets. This microgel system can firmly seal the bleeding site, preventing blood from escaping, while also playing an antibacterial and anti-inflammatory role, promoting wound healing.

[0033] In addition, the raw materials for processing are readily available and inexpensive, the operation process is simple, and the products do not require strict storage and transportation conditions, making them suitable for large-scale production and widespread use.

[0034] Specifically, in the CMS-CTA system, the introduction of cross-linked starch avoids the need for a relatively expensive single component like carboxymethyl chitosan. Cross-linked starch has abundant and inexpensive raw materials, is relatively simple to process, and exhibits good biocompatibility, avoiding side effects from foreign body reactions during use, making it suitable for large-scale application. Starch is a natural high-molecular-weight organic compound widely found in plants, with abundant sources and low prices. It possesses good adsorption, degradability, and biocompatibility. Through enzymatic action, starch forms a porous structure on its surface, increasing its surface area and facilitating water absorption. Furthermore, the introduction of carboxyl groups under alkaline conditions alters the original structure of the starch molecular chain, thus achieving a hydrophilic effect. However, single carboxymethyl starch lacks sufficient mechanical strength. Carboxymethyl starch can be cross-linked using sodium trimetaphosphate as a cross-linking agent to obtain cross-linked starch, thereby improving its physical strength. As a hemostatic material, cross-linked starch can be degraded in vivo by plasma amylase into oligosaccharides, maltose, and glucose, avoiding residue buildup.

[0035] Compared with existing technologies, the novel rapid hemostatic powder material and microgel composition of this invention can quickly absorb water in the blood to form a microgel that concentrates clotting factors, thereby promoting coagulation. Furthermore, it can bind to proteins on bacterial cell membranes or inhibit protease activity, altering the membrane structure and function, leading to increased cell membrane permeability, leakage of cell contents, and ultimately bacterial death, thus playing an antibacterial and healing-promoting role. The raw materials are abundant and inexpensive, and the processing is relatively simple. It has good biocompatibility, avoiding the side effects of foreign body reactions during use, making it suitable for large-scale promotion and application. It can be applied to various emergency and special scenarios with large wound areas and heavy bleeding, such as battlefields, accidents, the field, outdoor activities, and sudden massive bleeding during surgical operations. It also has bactericidal, anti-inflammatory, and wound-healing-promoting effects. Attached Figure Description

[0036] Figure 1 The synthesis and properties of tannic acid-modified carboxymethyl chitosan (CTA) are shown. (a) Synthetic route of CTA; (b) Powder appearance and SEM images of CTA and its microgel assemblies with Celox, SKSS, and CMS; (c) Compressive modulus of Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (d) Tissue adhesion strength of Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (e) Coagulation properties of Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies in different ratios (2:1, 1:1, 1:2).

[0037] Figure 2 The 1H NMR and FTIR spectra of CMCS and CTA are shown.

[0038] Figure 3 The synthetic route of cross-linked starch CMS is shown.

[0039] Figure 4 The FTIR spectrum of CMS is shown.

[0040] Figure 5 SEM images of Celox, SKSS, and CMS powders are shown.

[0041] Figure 6 The gelation properties, mechanical properties, adhesion properties, and burst pressure test results of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies are shown. (a) Experimental results of gelation properties of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (b) Compressive strength of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (c) Tensile test of pigskin of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (d) Burst pressure test results of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (e) Compressive modulus of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (f) Adhesive pressure of Celox, Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies; (g) Burst pressure test results.

[0042] Figure 7 The rheological measurements of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA are shown.

[0043] Figure 8 A schematic diagram of an explosion pressure device is shown.

[0044] Figure 9 The degradation performance results of Celox-CTA, Skss-CTA, and CMS-CTA are shown.

[0045] Figure 10 The in vitro coagulation properties of Celox, Celox-CTA, SKSS, SKSS-CTA, and CMS, CMS-CTA microgel assemblies are shown. (a) In vitro blood coagulation index (BCI) test; (b) BCI values ​​of each group; (c) In vitro coagulation time test; (d) APTT test results; (e) SEM images of the adhesion effect of samples to erythrocytes and platelets; (f) Percentage of erythrocyte adhesion; (g) Percentage of platelet adhesion.

[0046] Figure 11 The in vitro clotting times of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA are shown.

[0047] Figure 12 The zeta potentials of Celox-CTA, SKSS-CTA, and CMS-CTA are shown.

[0048] Figure 13 The antibacterial properties of Celox, Celox-CTA, SKSS, SKSS-CTA, and CMS and CMS-CTA microgel assemblies are shown. (a) Colony growth experiment of Escherichia coli and Staphylococcus aureus; (b) SEM image of bacterial morphology after co-culturing with the material samples; (c) Bactericidal rate of each material; (d) Hemolysis and lysis rate of each material; (e) Growth and cell viability of L929 cells.

[0049] Figure 14 The in vitro hemostatic effects of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA are shown. (a) Rat liver hemorrhage experiment; (b) Rat femoral artery hemorrhage experiment; (c) Rabbit liver hemorrhage experiment; (d) Porcine spleen and liver hemorrhage experiment.

[0050] Figure 15 The hemostasis time of Celox, Celox-CTA, CMS, and CMS-CTA is shown: (a) rat liver hemorrhage model; (b) rat femoral artery hemorrhage model; (c) rabbit liver hemorrhage model.

[0051] Figure 16 The healing-promoting properties of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA are shown. (a) Schematic diagram of the liver repair experimental route; (b) Hemostatic effect on rat liver; (c) Qualitative analysis results of liver repair; (d) Wound healing rate; (e) Degree of wound tissue infiltration; (f) Quantitative analysis results of single HNF-4α staining; (g) Quantitative analysis results of HNF-4α / DAPI staining. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0053] 1. Experimental Section

[0054] 1.1 Materials and Reagents

[0055] Carboxymethyl chitosan (Shanghai Aladdin Reagent Co., Ltd., catalog number C304738-25g), potato starch, tannic acid, saccharifying enzyme, chloroacetic acid, sodium hydroxide, liquid paraffin, and Span 80 were purchased from Aladdin (Shanghai, China). α-Amylase and sodium trimetaphosphate were purchased from Maclean's (China). Ethanol, acetone, and petroleum ether were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Chloroacetic acid was purchased from Tianjin Fuchen Chemical Reagent Factory (Tianjin, China). All chemical reagents were analytical grade and were used without further purification.

[0056] 1.2 Synthesis of Tannic Acid Modified Carboxymethyl Chitosan (CTA)

[0057] Carboxymethyl chitosan was dissolved in water to prepare a solution (4%, w / w), and stirred at 60°C until dissolved. The pH was adjusted to 8.5. Tannic acid solution (4%, w / w) was slowly added dropwise to the above carboxymethyl chitosan solution at a volume ratio of 50:1 (tannic acid solution: carboxymethyl chitosan solution = 50:1), and the reaction was carried out at 60°C for 3 hours. The pH of the solution was monitored using a pH meter and maintained at 8.5. Air was bubbled into the solution during the reaction. The reaction solution was dialyzed against deionized water for five days using a dialysis bag with a molecular weight cutoff (MWCO) of 1000 Da. After freeze-drying and grinding, tannic acid-modified carboxymethyl chitosan (CTA) powder was obtained.

[0058] 1.3 Synthesis of Cross-linked Starch (CMS)

[0059] 21 g of potato starch was dissolved in 150 mL of a 0.2 M, pH 4 acetate-sodium acetate buffer solution (acetic acid solution (0.2 mol / L) to sodium acetate solution (0.2 mol / L) volume ratio 41:9), and stirred at 50 °C for 20 minutes. Then, 0.4 g of a mixed enzyme (a mixture of saccharifying enzyme and α-amylase), with a saccharifying enzyme to α-amylase mass ratio of 4:1, was added, and the reaction was carried out at 50 °C for 8 hours. Finally, the product was washed with ultrapure water, filtered, and dried under vacuum at 50 °C to obtain enzymatically hydrolyzed starch.

[0060] Sodium hydroxide was dissolved in 90% v / v ethanol to prepare a solution (2.5%, w / w). Then, an equal mass of enzymatically hydrolyzed starch was added, the mixture was refluxed, and stirred at 50°C for 30 minutes. Chloroacetic acid (at a mass ratio of 18:25 to the enzymatically hydrolyzed starch) was added to the reaction solution, and the reaction was carried out at 50°C for 3 hours. Finally, the product was washed with anhydrous ethanol, filtered, and dried under vacuum at 50°C to obtain carboxymethyl hydrolyzed starch.

[0061] Dissolve 1 g of carboxymethyl hydrolyzed starch in 10 mL of pure water, adjust the pH of the solution to 10, and add sodium trimetaphosphate at 3% of the mass of carboxymethyl hydrolyzed starch to form an aqueous phase. Then mix 50 mL of liquid paraffin (oil phase) with Span 80 at 1% of the mass of the oil phase. After stirring the oil phase at 60°C for a period of time, add the aqueous phase dropwise to the oil phase and react at 60°C for 6 hours. Wash twice consecutively with excess (generally 2-3 times the volume of the reaction solution) of acetone, petroleum ether, 9% w / w sodium chloride solution, and ethanol. After vacuum drying at 50°C, grind and sieve (100 mesh, 0.154 mm pore size) to obtain cross-linked starch (CMS).

[0062] 1.4 Structural Characterization of CTA and CMS

[0063] The prepared CTA and unmodified carboxymethyl chitosan were dissolved in deuterated water (15 mg powder dissolved in 1.5 mL of deuterated water) and characterized by 1H NMR spectroscopy (Bruker AVANCE 400 MHz, Switzerland). The successful synthesis of CTA and CMS was then confirmed by FTIR (Bruker Vertex 70, Germany).

[0064] 1.5 Preparation and Proportion Optimization of Celox-CTA, SKSS-CTA and CMS-CTA

[0065] Different commercially available hemostatic powders, Celox (a commercial chitosan hemostatic powder), SKSS (a commercial composite microporous polysaccharide hemostatic powder, purchased from Cysec), and the aforementioned cross-linked starch CMS were uniformly mixed with tannic acid-modified carboxymethyl chitosan CTA at different mass ratios (2:1, 1:1, 1:2) to obtain microgel assemblies with different proportions. Compression tests, adhesion tests, and blood coagulation index (BCI) tests were performed on various microgel assemblies; detailed experimental methods are described below.

[0066] 1.6 Structural characterization of Celox-CTA, SKSS-CTA and CMS-CTA

[0067] CTA, Celox, SKSS, CMS, Celox-CTA (a mixture of Celox and CTA), SKSS-CTA (a mixture of SKSS and CTA), and CMS-CTA (a mixture of CMS and CTA) were characterized by field emission scanning electron microscopy (FESEM, Carl Zeiss Merlin, Germany) to determine their particle size and structure.

[0068] 1.7 Characterization of gelation behavior

[0069] Each powder sample (CTA, Celox, SKSS, CMS, Celox-CTA, SKSS-CTA, CMS-CTA) was added to PBS or anticoagulated blood (sample powder to PBS or anticoagulated blood mass ratio of 1:3), and its gelation was observed using tweezers. To determine the gelation time of each sample, rheological tests were performed using a rheometer (Anton Paar MCR 302, Austria), with the temperature set at 37°C, a fixed strain of 1.0%, a fixed frequency of 1 Hz, and a sample height of 10 mm. A fixed mass of powder sample was added to a parallel plate mold (diameter of 25 mm). After starting the test, a fixed amount of PBS or anticoagulated blood was added to the mold, and the oscillation time scan curve was obtained.

[0070] 1.8 Mechanical property characterization

[0071] Powdered samples (powder to PBS or anticoagulated blood mass ratio of 1:3) were prepared into cylinders (height: 5 mm; diameter: 10 mm) by adding PBS or anticoagulated blood. Different masses of weights (20 g, 50 g, 100 g) were applied to each cylinder, and the deformation was observed to determine its mechanical properties. Subsequently, compression tests were conducted on each cylinder using a dynamic thermodynamic analyzer (DMA, Q800DE, TA, USA). The strain rate was 50% / min, the maximum strain was set to 90%, and the initial force was 0.0010 N. The stress-strain curves for each experimental group were obtained, and the specific compressive modulus was calculated.

[0072] 1.9 Adhesion performance characterization

[0073] Each powder sample (powder to PBS or anticoagulated blood mass ratio of 1:3) was prepared into a gel by adding PBS or anticoagulated blood. Then, a 0.5 cm × 1 cm gel was applied to each 1 cm × 2 cm piece of pigskin, and the maximum area of ​​another piece of pigskin that could adhere to it was observed. Next, two 1 cm × 2 cm pieces of pigskin were bonded together with a 0.5 cm × 1 cm gel, and a tensile test was performed using a dynamic thermodynamic analyzer (DMA, Q800DE, TA, USA). The strength at which the two pieces of pigskin completely separated was taken as the adhesion strength of the powder gel.

[0074] 1.10 Burst Pressure Test

[0075] A 2 mm diameter incision was made in a 10 mm diameter porcine aorta. 30 mg of the hemostatic powder to be tested was deposited on the incision, and an equal volume of anticoagulated blood was added to allow a hydrogel layer to form in situ. After 5 minutes, PBS stained with red dye was introduced into the porcine aorta, and PBS leakage in different groups was observed to evaluate its occlusion performance. Next, the porcine aorta was connected to an infusion pump and a pressure monitor. The infusion pump gradually injected PBS into the device at a rate of 10 mL / min. When occlusion failed, the rupture pressure was used as the burst pressure.

[0076] 1.11 Degradation performance test

[0077] 0.05 g of Celox-CTA, SKSS-CTA, and CMS-CTA were added to 1.5 ml of SBF simulated body fluid. Degradation was observed after shaking at 37°C for 1, 3, 5, 7, and 14 days. The samples were then dried and the remaining mass was weighed. The SBF simulated body fluid was replaced every two days. Four parallel samples were prepared for each day group. Degradation rate = (W0 - W...) t ) / W0, where W0 is the initial mass of the sample, W t The mass of samples after drying for different degradation days.

[0078] 1.12 Whole Blood Coagulation Index (BCI) Test

[0079] Add 10 mg of the test powder sample to 200 μL of recalcified blood (each 10 μL of blood contains 10 μL of 0.2 M calcium chloride). After incubating at 37 °C with shaking (100 rpm) for 5 minutes, add 10 mL of deionized water to dissolve any uncoagulated blood clots. Measure the hemoglobin content of the supernatant at 540 nm using a microplate reader (Tecan, Switzerland). The coagulation index (BCI) is calculated as follows: BCI (%) = (Is - I0) / (Ic - I0) × 100%, where Is represents the absorbance value of the sample, Ic represents the absorbance value of the positive control group (10 mL of deionized water was directly added to 200 μL of recalcified blood), and I0 represents the absorbance value of the blank plate.

[0080] 1.13 In vitro coagulation time test

[0081] Add 5 mg of the test powder sample to a centrifuge tube containing 100 μL of recalcified whole blood. Add 400 μL of PBS at a predetermined time and observe the blood diffusion behavior. The time during which no blood diffuses into the PBS is defined as the clotting time.

[0082] 1.14 Zeta potential test

[0083] The 0.5 mg / mL, 1 mg / mL, and 5 mg / mL powder samples to be tested were placed in the test mold. The electrolyte circulated through the sample cell, creating a flow pressure difference. The movement of charges relative to the electrochemical double layer generated a flow potential, which was measured by the electrodes at both ends of the sample.

[0084] 1.15 Red blood cell and platelet adhesion

[0085] Fresh sheep whole blood anticoagulated with sodium citrate was centrifuged at 200×g for 15 minutes to obtain red blood cell suspension and platelet-rich plasma.

[0086] Red blood cell adhesion and quantification analysis: 100 µL of red blood cell suspension was added to 10 mg of the test powder sample and incubated at 37°C for 5 minutes. The sample was then washed 5 times with DPBS (Dubor's phosphate buffer) to remove non-adhering red blood cells, followed by the addition of 10 mL of deionized water. The absorbance of the supernatant at 540 nm was measured using a microplate reader. A red blood cell suspension without added material served as a blank control, and each group was repeated 3 times. The percentage of adherent red blood cells was calculated using the following formula: The absorbance of the sample and the reference ratio were respectively measured using... and express.

[0087] Platelet adhesion and quantification analysis: 100 µL of platelet-rich plasma was added to 10 mg of the test powder sample and incubated at 37 °C for 5 minutes. The sample was then washed 5 times with DPBS to remove non-adhering platelets. Adhering platelets were first lysed using 1% Triton X-100, and the number of adherent platelets was finally determined using a lactate dehydrogenase cytotoxicity assay kit (Beyotime, catalog number C0016). The absorbance of the supernatant at 490 nm was measured using a microplate reader. Platelets without added material served as a blank control, and each group was repeated 3 times. The percentage of adherent platelets was calculated using the following formula: .

[0088] To observe the morphology and number of attached red blood cells and platelets, samples were fixed with 4% paraformaldehyde for 2 hours, followed by gradient dehydration (ethanol concentrations of 50%, 60%, 70%, 80%, 90%, and 100%). The dehydrated samples were then dried, sputter-coated with gold, and observed under a scanning electron microscope.

[0089] 1.16 Characterization of antibacterial properties

[0090] Escherichia coli (ATCC 8739, Gram-negative bacteria) and Staphylococcus aureus (ATCC 6538, Gram-positive bacteria) were selected as test strains to evaluate the antibacterial properties of the test powder. 100 μL of a 10... 7CFU mL −1 A solution of *E. coli* or *Staphylococcus aureus* was added dropwise to the powder gel to be tested and incubated at 37°C for 2 hours. 900 μL of PBS was added and mixed thoroughly. Then, 10 μL of the solution was taken and evenly spread onto a pre-prepared agar plate and incubated at 37°C for 12 hours. Finally, the number of bacterial colonies on the agar plate was observed and recorded. Similarly, a 100 μL solution of *E. coli* or *Staphylococcus aureus* at a concentration of 10... 7 CFU mL −1 The samples were incubated at 37°C for 2 hours, then submerged in 900 μL of Luria-Bertani (LB) broth and incubated for another 12 hours. Finally, 200 μL of bacterial suspension from each sample tube was transferred to a 96-well plate, and the absorbance at 600 nm was measured using a microplate reader. Each experiment was repeated three times. The sterilization rate (%) was expressed as (ODc-ODs) / ODc×100%, where ODs is the absorbance of the sample and ODc is the absorbance of the blank control without the sample.

[0091] The morphology of bacteria after co-culturing with the material samples was observed using scanning electron microscopy. The bacterial culture was co-cultured with the material using bacterial culture medium. After 4 hours, the bacteria were resuspended by sonication to detach from the material. The material was removed, the bacterial culture was collected, and centrifuged (6000 rpm, 5 minutes). The supernatant was discarded, and the bacteria were washed three times with PBS. Then, 4% paraformaldehyde solution was added for fixation for 1 hour. After centrifugation, the paraformaldehyde was aspirated, and the bacteria were dehydrated using gradient concentrations of ethanol and tert-butanol. The samples were then lyophilized and finally observed using scanning electron microscopy.

[0092] 1.17 Hemolytic Performance Test

[0093] Anticoagulated sheep whole blood was centrifuged at 200 × g for 10 minutes to collect red blood cells (RBCs), which were then diluted 10-fold (v / v) with PBS. 5 mg of the test powder sample was added to 0.8 mL of PBS, followed by 0.2 mL of the diluted RBC suspension. The DW group and PBS group served as positive and negative controls, respectively, with three replicates per group. After incubating the RBCs with the sample for 1 hour, the mixture was centrifuged at 3000 rpm for 10 minutes. 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader. The hemolysis rate was calculated using the formula: .

[0094] 1.18 Cell compatibility characterization

[0095] Each powder sample was sterilized by irradiation with ultraviolet light for 24 hours before the experiment. PBS (powder sample to PBS mass ratio of 1:3) was added to the powder sample to gel it, forming cylinders (10 mm in diameter and 2 mm in height). After sterilization by immersion in alcohol for 24 hours, the cylinders were further immersed in culture medium for 24 hours to wash away excess alcohol. L929 cells were seeded at a density of 20,000 cells per well and cultured for 24 hours and 48 hours, respectively. Cell viability was assessed using a live / dead staining method, and cell counts were observed using an inverted fluorescence microscope (DMi1, Leica, Germany). The control group consisted of pure culture medium without any added materials.

[0096] 1.19 Characterization of in vivo hemostatic properties

[0097] First, in vivo hemostasis experiments were conducted using rat liver and femoral artery hemorrhage models and rabbit liver hemorrhage models to preliminarily characterize the hemostasis.

[0098] In a rat liver hemorrhage experiment, male Spragge-Dawley (SD) rats (0.3-0.4 kg) were randomly divided into 5 groups (n=5 per group). Zoletil® 50 and xylazine hydrochloride were mixed at a 1:1 mass ratio, then diluted 10-fold with sterile water, and the rats were anesthetized by intramuscular injection at 1 μL / g. A 5 mm diameter, 5 mm deep wound was created using a punch, and 20 mg of the test powder sample was added to the bleeding area. Weighed filter paper was placed under the wound, and the amount and time of bleeding were recorded.

[0099] For the rat femoral artery bleeding experiment, the anesthesia method is the same as above. After locating the rat's femoral artery, a 10 mm long wound is made with an incision. 20 mg of the test powder sample is added to the bleeding part, and a weighed filter paper is placed under the wound. The amount of bleeding and the bleeding time are recorded.

[0100] In a rabbit liver hemorrhage experiment, male New Zealand white rabbits (3.0–3.5 kg) were randomly divided into 5 groups (n=5 per group). Zoletil® 50 and xylazine hydrochloride were mixed at a mass ratio of 4:1 and administered intramuscularly at 300 μL / kg to anesthetize the rabbits. After locating the liver, a weighed filter paper was placed underneath the liver, and the liver lesion was made with scissors. Immediately afterward, 200 mg of the test powder was placed, and the amount of bleeding and the time to hemostasis were recorded.

[0101] Next, pig liver and spleen hemorrhage models were used to further characterize in vivo hemostatic performance. Bama pigs (female, 25-35 kg) were anesthetized by intramuscular injection of a 1:1 mixture of Zoletil® 50 and xylazine hydrochloride at a mass ratio of 0.2 mL / kg, followed by continuous anesthesia using isoflurane and an oxygen-assisted breathing machine. In the pig liver hemorrhage experiment, after locating the liver, a dust-proof paper was placed underneath, and a 20 mm long and 10 mm deep wound was made with a scalpel. After bleeding, the test powder sample was scattered, and gentle pressure was applied to observe hemostasis. Additional hemostatic powder could be added as needed. The amount and time of bleeding were recorded. In the pig spleen hemorrhage experiment, after locating the spleen, a dust-proof paper was placed underneath, and a 20 mm long and 10 mm deep wound was made with a scalpel. After bleeding, the test powder sample was scattered, and gentle pressure was applied to observe hemostasis. Additional hemostatic powder could be added as needed. The amount and time of bleeding were recorded. All animal experimental procedures were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals of South China University of Technology" and approved by the Animal Ethics Committee of South China University of Technology (Approval No.: 2024138).

[0102] 1.20 Liver Repair Experiment

[0103] After the above-mentioned rat liver hemostasis experiment was completed, excess powder on the liver surface was rinsed off with physiological saline, the liver was then returned to its original position, and the rat's skin wound was sutured. For 3 days post-surgery, the rats were injected daily with 1 mL of ampicillin (Servicebio, catalog number G4018-10ML). 14 days post-surgery, the livers treated for the hemostasis experiment were removed from each rat, and the liver repair process was observed.

[0104] To further evaluate the liver repair performance of different materials, histological staining analysis was performed on various liver samples. Liver samples were fixed with 4% (w / v) paraformaldehyde, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E), PAS glycogen staining, and immunofluorescence staining. The staining results were scanned using a digital pathology scanning system, and the images were saved and analyzed. All animal procedures were performed in accordance with the "Guidelines for the Care and Use of Laboratory Animals of South China University of Technology" and approved by the Animal Ethics Committee of South China University of Technology.

[0105] 2 Results and Discussion

[0106] 2.1 Preparation and characterization of CTA and three microgel assemblies

[0107] Carboxymethyl chitosan possesses excellent water solubility, hemostatic properties, and biocompatibility; therefore, it was chosen as a raw material for synthesizing a universal assembly agent. According to... Figure 1 The synthetic route shown ( Figure 1(a) Tannic acid was grafted onto carboxymethyl chitosan via a one-step Michael addition reaction, and CTA powder was obtained after dialysis, freeze-drying and grinding.

[0108] Collect 1H NMR and FTIR spectra to verify the synthesis of CTA. Figure 2 ).

[0109] Cross-linked starch CMS was also synthesized. Figure 3 The FTIR spectrum of CMS confirmed its successful synthesis. Figure 4 ).

[0110] Two commercially available hemostatic powders, Celox and SKSS, were introduced. Celox, SKSS, and CMS were blended with CTA to obtain three microgel assemblies: Celox-CTA, SKSS-CTA, and CMS-CTA. As shown in the figure, the CTA powder is light yellow (…). Figure 1 (b) SEM images show that the particle size is around 10-40 μm. Figure 5 Celox powder is a pale yellow with a larger particle size, while SKSS and CMS are pure white. Their particle sizes are all around 100 μm, which is larger than CTA. After blending with CTA, Celox-CTA becomes more yellow, while SKSS-CTA and CMS-CTA show a slightly pale yellow color. SEM images show that small-sized CTA particles are distributed within the mixture, proving the successful addition of CTA.

[0111] The ratio of microgels (Celox, SKSS, and CMS) to assembly agents (CTA) in microgel assemblies is crucial for hemostatic properties. Mechanical strength, tissue adhesion strength, and coagulation index were selected as indicators for ratio optimization.

[0112] The compressive modulus and tissue adhesion strength of microgel assemblies are shown when the mass ratio of microgels (Celox, SKSS, and CMS) to assembly agents (CTA) varies (2:1, 1:1, 1:2). Figure 1 (c and d). Clearly, higher CTA content results in greater compressive modulus and tissue adhesion strength of the microgel assemblies. This is because the mechanical strength of the microgel assemblies primarily comes from the hydrogen bonding between the phenolic hydroxyl groups on CTA and the functional groups hydroxyl, carboxyl, and amino groups on the microgel, while the tissue adhesion strength mainly comes from the interaction between the phenolic hydroxyl groups on CTA and the tissue surface. However, it can be observed that the increase from 1:1 to 1:2 is much smaller than the increase from 2:1 to 1:1, indicating that increasing the CTA content from 1:1 to 1:2 has a relatively small impact on the mechanical strength and tissue adhesion strength of the microgel assemblies. Figure 1 It also showed the changes in the coagulation index at different microgel / assembly ratios. Figure 1(e) When the microgel / assembly mass ratio is 1:1, all three microgel assemblies exhibit lower coagulation indices, indicating better coagulation performance. This is likely because the two components have the best synergistic and combined effect on blood coagulation at a 1:1 ratio. Based on the fact that the three microgel assemblies exhibit the best coagulation performance at a 1:1 ratio, and that the mechanical strength and tissue adhesion strength change little when changing from 1:1 to 1:2, and considering the comprehensive performance of the materials, a 1:1 mass ratio of microgel / assembly agent was chosen for subsequent experiments.

[0113] 2.2 Enhancement of the mechanical strength, tissue adhesion, and blocking strength of microgels by the universal assembly agent CTA

[0114] like Figure 6 As shown, individual Celox, SKSS, and CMS powders, when added to PBS or blood, could not be picked up with tweezers and exhibited irregular shapes; however, when blended with CTA, Celox-CTA, SKSS-CTA, and CMS-CTA could all form a gel upon addition to PBS or blood and could be picked up with tweezers, and each could individually form a good gel. Figure 6 ,a).

[0115] The effect of CTA on the gelling properties of hemostatic powder was further investigated by rheological testing. Figure 7 As can be seen, without CTA, the loss modulus (G'') of all three hemostatic powders, whether added to PBS or blood, was consistently greater than the storage modulus (G'), exhibiting typical fluid characteristics, indicating that they did not gel. However, after blending with CTA, within 20 seconds, the storage modulus (G') of Celox-CTA, SKSS-CTA, and CMS-CTA all exceeded the loss modulus (G''), indicating that the addition of CTA, whether added to PBS or blood, facilitated the formation of microgel tissue gels. Furthermore, in the blood-added test, Celox-CTA, SKSS-CTA, and CMS-CTA all exhibited shorter gelation times and higher storage moduli. This demonstrates that the addition of CTA involved erythrocytes and platelets in the blood in the assembly process, filling the gaps between microgels and resulting in stronger gelation properties in the microgel assemblies.

[0116] The compressive strength of the gel was investigated by applying weights over it. Figure 6 (b) It was found that the results of the three groups of tests were consistent: the hemostatic powder before the addition of CTA showed the greatest compressive deformation under the weights, while the microgel assemblies formed by blood gelation after the addition of CTA showed the least compressive deformation. The compressive modulus of each group also showed this trend through testing. Figure 6 ,e).

[0117] Next, different gels were applied to pigskin to adhere pigskin of different sizes. Figure 6(c) After adding CTA, the microgel assemblies could adhere to a larger area of ​​pigskin, and the blood-gelled microgel assemblies had better adhesion performance than those gelled with PBS, which was further confirmed by the pigskin stretch test. Figure 6 f).

[0118] The above tests show that the three groups of hemostatic powders without CTA exhibited weak action and essentially lacked mechanical strength and tissue adhesion strength after being added to PBS. With the addition of CTA, the hydrogen bonding between CTA and the microgel, as well as the interaction between CTA and the tissue surface, endowed the microgel assemblies with superior mechanical strength and adhesion properties. Furthermore, because CTA also involved erythrocytes and platelets in the assembly, the microgel assemblies exhibited even better performance after gelation with added blood.

[0119] A 2mm diameter notch was created in the pig's aorta. Various powdered samples were gelled with blood and attached to the notch. Red-stained PBS was then introduced into the aorta, and leakage of the PBS was observed. Figure 6 As can be seen, PBS leakage occurred in all three groups without the addition of CTA, but no PBS leakage occurred after the addition of CTA, proving that the addition of CTA endowed the microgel with better blocking performance. Figure 6 d). Next, construct the explosive device ( Figure 8 This was used to test the burst pressure of each powder sample. As shown in the figure, the burst pressure of each individual microgel powder was much lower than that of normal human arterial blood pressure (…). Figure 6 The results showed that these powders (e.g., g) had poor application prospects in closure of massive hemorrhages in human bodies. However, after the addition of CTA, Celox-CTA, SKSS-CTA, and CMS-CTA all exhibited burst pressures greater than 120 mmHg, specifically 150 mmHg, 200 mmHg, and 250 mmHg, respectively. Clearly, the addition of CTA makes it possible to apply these powders to larger hemorrhage scenarios.

[0120] Results of in vitro degradation performance test of hemostatic powder as follows Figure 9 As shown, all three powders continued to degrade over time, exhibiting good degradation performance.

[0121] 2.3 Enhancement of in vitro coagulation properties of microgels by the addition of CTA

[0122] First, the in vitro blood coagulation index (BCI) of each group of powder samples was tested. 10 mg of powder sample was added to 200 μL of recalcified blood, incubated at 37°C for 5 minutes, and then 10 mL of deionized water was added to dissolve the uncoagulated blood clots. The hemoglobin content of the supernatant was measured by the absorbance at 540 nm, and the BCI of each sample was calculated using a formula. Figure 8As shown, before the addition of CTA, the blood coagulation effect of the three groups of microgel powders under the test conditions was poor, the blood clots were unstable, and the blood clots ruptured after the addition of a large amount of deionized water, resulting in the leakage of red blood cells and a pale red supernatant. However, after the addition of CTA, the coagulation performance of all three groups was significantly enhanced, the supernatant became clear, and there was almost no leakage of red blood cells. Figure 10 (a), the BCI value also proves this result ( Figure 10 b).

[0123] Next, 5 mg of powder sample was added to a centrifuge tube containing 100 μL of recalcified whole blood. At a predetermined time, 400 μL of PBS was added, and the blood diffusion behavior was observed to test in vitro clotting time. The results showed that after the addition of CTA, all three groups had shorter clotting times. Figure 10 c; Figure 11 ).

[0124] The two tests above demonstrate that the addition of CTA significantly improves the in vitro coagulation properties of the microgel. Celox primarily promotes blood coagulation through a strong positive charge, while SKSS and CMS primarily promote blood coagulation by absorbing blood and concentrating coagulation factors. CTA, however, primarily promotes blood coagulation through hydrophobic interactions attracting erythrocytes and electrostatic interactions attracting platelets. It is speculated that the coagulation properties of the microgel itself synergistically interact with CTA. Furthermore, the stronger mechanical properties of the assembly prevent clot diffusion, resulting in the superior in vitro coagulation properties of the microgel assembly formed after the addition of CTA.

[0125] To investigate the mechanism by which CTA promotes blood coagulation, a Zeta potential test was first performed. It can be seen that Celox itself is positively charged, while SKSS and CMS are negatively charged. CTA, due to its carboxyl and tannic acid groups, is also negatively charged. Therefore, the addition of CTA makes the material surface negatively charged. SKSS-CTA and CMS-CTA both exhibit more pronounced negative potentials, while Celox-CTA remains positively charged. This is likely due to the strong positive charge of Celox itself. Figure 12 ).

[0126] APTT test results showed that the addition of CTA resulted in a lower APTT time for the material, indicating that the material has a stronger ability to activate intrinsic coagulation pathways. Figure 10 (d) This is because the strong negative charge of CTA itself affects the intrinsic coagulation pathway.

[0127] Next, the adhesion effect of the samples on erythrocytes and platelets was observed using scanning electron microscopy. It can be seen that only a small number of erythrocytes and platelets were adhered to the Celox, SKSS, and CMS powders under the field of view. However, the microgel assemblies after adding CTA showed that more erythrocytes and platelets were adhered. Figure 10 ,e). Quantitative test data also proves this point ( Figure 10 (f, g). CTA has a hydrophobic effect on erythrocytes and an electrostatic effect on platelets, which allows more erythrocytes and platelets to adhere to the material and participate in the assembly of the assembly.

[0128] In summary, CTA improves the coagulation performance of materials by enhancing their activation of intrinsic coagulation pathways and their adhesion to erythrocytes and platelets.

[0129] 2.4 The addition of CTA enhances the antibacterial properties and biocompatibility of microgels.

[0130] After hemostasis is achieved with hemostatic materials, the moist and oxygen-deficient environment at the wound site easily promotes bacterial growth, leading to wound inflammation. Therefore, in practical applications, hemostatic materials need to have excellent antibacterial properties to prevent inflammation and promote subsequent wound healing. (Introduction of Escherichia coli...) E. coli Gram-negative bacteria) and Staphylococcus aureus ( Staphylococcus aureus This study investigated the effect of CTA addition on the antibacterial properties of microgels, specifically targeting two of the most common bacteria (Gram-positive bacteria). First, powdered samples were co-cultured with *Escherichia coli* and *Staphylococcus aureus* for 2 hours using an agar method. Then, 10 μL of the suspension was evenly spread onto agar plates and incubated at 37°C for 12 hours. The number of bacterial colonies on the plates was then observed. Fewer colonies indicated stronger antibacterial properties. As shown in the figure, Celox, due to its strong positive charge, exhibited some antibacterial effect; SKSS and CMS showed virtually no difference compared to the control group, indicating poor antibacterial performance. However, after the addition of CTA, all three assemblies showed significant antibacterial activity against both bacteria, with no bacterial colonies visible on the plates, demonstrating that the addition of CTA significantly enhances the antibacterial properties of the microgels. Figure 13 (a) To investigate the antibacterial mechanism of CTA, the microscopic morphology of bacteria in each group after co-culture was observed using SEM. The bacteria in the SKSS and CMS groups showed no morphological difference from the control group, exhibiting intact and smooth bacterial structures, proving that they did not affect the bacteria. The bacteria in the Celox group showed some morphological changes, indicating some inhibitory effect on the bacteria. After the addition of CTA, both types of bacteria in all three groups underwent severe deformation and even rupture, proving that CTA acts on the bacterial surface, thereby inhibiting and killing the bacteria. In fact, CTA exerts its antibacterial effect by acting on the bacterial membrane through the numerous phenolic hydroxyl groups on its surface. Figure 13b).

[0131] The specific sterilization rate of each material was calculated by measuring the absorbance (600 nm) of the co-culture solution and combining it with a formula. Figure 13 (c) After the addition of CTA, the sterilization rate of each group was close to 100%, proving that the addition of CTA can endow the material with excellent antibacterial properties.

[0132] Excellent hemostatic materials also need to possess superior biocompatibility to ensure they do not pose a risk to the human body. First, the powder sample was co-cultured with a red blood cell suspension to test the hemolytic properties of the material. As shown in the figure, the hemolysis rates of the three microgel assemblies and CTA itself are all below 5%, demonstrating excellent blood compatibility and indicating no risk of hemolysis to the human body. Figure 13 Next, to further verify the cell compatibility of the materials, each powder sample was co-cultured with fibroblasts (L929 cells). After 24 and 48 hours of co-culture, the growth and cell viability of the three microgel assemblies and CTA-treated L929 cells were almost indistinguishable from the control group, demonstrating that the materials have excellent cell compatibility. Figure 13 (e). The excellent blood compatibility and cell compatibility of each group after the addition of CTA proves that the addition of CTA does not cause biological harm to the material and can achieve a balance between antibacterial and cell compatibility. It also proves that the CTA material itself has excellent cell compatibility.

[0133] 2.5 The addition of CTA enhances the in vivo hemostatic effect of the microgel.

[0134] First, hepatic and femoral artery hemorrhage models were established in small animal rats. Then, powdered samples were used to verify the hemostatic effect. For rat liver hemorrhage, Celox and CMS alone could not achieve timely hemostasis, although they were better than the control group, they still caused some bleeding. With the addition of CTA, both Celox-CTA and CMS-CTA could stop the liver hemorrhage promptly, with almost no visible blood flow. Figure 14 , a; Figure 15 (a) For femoral artery hemorrhage in rats, the occlusion performance of the material is more demanding. Celox and CMS alone could not stop the femoral artery hemorrhage in time, and a large amount of blood could be seen flowing out. However, after adding CTA, both Celox-CTA and CMS-CTA could stop the femoral artery hemorrhage in time, proving that the material not only has excellent hemostatic performance, but also excellent occlusion performance. Figure 14 b; Figure 15 b).

[0135] Next, a liver hemorrhage model was constructed using medium-sized rabbits. Removing a large portion of the rabbit's liver resulted in significant bleeding, posing a significant challenge to the hemostatic and occlusive properties of the materials. Celox and CMS alone failed to stop the rabbit liver hemorrhage, resulting in substantial blood loss. However, with the addition of CTA, Celox-CTA stopped the bleeding after a certain time with only a small amount of blood loss, while CMS-CTA stopped the bleeding promptly with almost no blood loss. This is consistent with the better occlusive and coagulation properties of CMS-CTA in in vitro tests. Figure 14 c; Figure 15 c).

[0136] Finally, to better verify the actual hemostatic performance of the material and its application prospects in human hemostasis, a liver and spleen hemorrhage model was constructed in the large animal Bama pig to further explore the actual hemostatic performance of the material. After locating the liver of the Bama pig, a 20mm long and 10mm deep wound was constructed with a scalpel, and a large amount of blood was observed to flow out. In the CMS group, after adding the powder, blood was still flowing out after 8 minutes. After rinsing with water, the powder dispersed directly, and a large amount of blood gushed out, proving that the hemostatic and sealing performance of CMS was insufficient. In the CMS-CTA group, after blood flow, the powder was quickly added, and after 3 minutes, no more blood flow was observed. After rinsing with water, the microgel assembly remained firmly adhered to the wound. The same phenomenon was observed for spleen hemorrhage in Bama pigs. The CMS group failed to stop the bleeding in the spleen, while the CMS-CTA group stopped the bleeding within three minutes, and no more blood flowed out after rinsing with water. The microgel assembly at the wound site was not dispersed. Figure 14 (d). The enhanced occlusion and hemostatic properties brought by CTA to CMS enable CMS-CTA to achieve excellent hemostatic effects on bleeding wounds in large animals, further demonstrating the universal and enhanced significance of CTA.

[0137] 2.6 Enhancement of Hemostatic Powder's Healing-Promoting Properties by CTA

[0138] In actual hemostasis procedures, removing the hemostatic material may lead to secondary bleeding from the wound, causing further injury to the patient. However, if the material does not have the ability to promote in-situ tissue regeneration at the site of injury and carries the risk of causing other complications, then the hemostatic material must be forcibly removed.

[0139] To address this issue, an in situ regeneration model of rat liver was constructed to investigate the effect of CTA on the hemostatic properties of the hemostatic powder. A 2mm deep wound was created in the rat liver using a 5mm in vivo sampling device. The sample powder was then used for hemostasis. After complete cessation of bleeding, the material and liver were returned to the body, and the incision was sutured. For the first three days, the rats were injected with antibiotics and analgesics to ensure their survival. Fourteen days later, the animals were sacrificed, and the repair of the injured liver was observed. Figure 16 ,a).

[0140] In a rat model of liver hemorrhage, CTA continued to demonstrate its enhancing effect on the hemostatic performance of hemostatic materials. Figure 16 (b) Celox and CMS alone could not stop liver bleeding in rats in a timely manner. Although they were more effective than the control group, they still caused a certain amount of bleeding. After the addition of CTA, both Celox-CTA and CMS-CTA could stop liver bleeding in a timely manner, and almost no blood flow was visible.

[0141] A unified qualitative analysis of liver repair was conducted. Firstly, after 14 days, the control group showed some healing, but it still had the most obvious wound and the largest defect. In the Celox group, some material and fibrotic cysts were clearly visible, likely due to a foreign body reaction caused by the Celox powder, leading to fibroblast aggregation and fibrosis at the wound site. In the Celox-CTA group, the wound was almost completely repaired, but some material remained at the wound site, indicating that Celox degradation is slow and not completely degraded in vivo after 14 days. CMS showed excellent degradation properties, with no visible material, but significant defects remained. The CMS-CTA group showed almost complete repair of the defect, exhibiting the best repair effect. Figure 16 Next, immunohistochemical analysis was performed on these samples to assess the healing status of the liver, and quantitative analysis was performed using ImageJ software. H&E results showed that abnormal areas were indeed present in the Celox and Celox-CTA groups, most likely residual material and fibrotic areas, but the Celox-CTA group had fewer abnormal areas compared to the Celox group. The wounds in the CMS-CTA group healed almost completely, demonstrating that CTA significantly enhanced the healing-promoting performance of CMS. Approximately 30% of the wounds in both the Celox and CMS groups remained unhealed, while with the addition of CTA, only about 10% remained unhealed in the Celox-CTA group, and the wounds in the CMS-CTA group healed almost completely. Figure 16 (d) Regeneration at the site of injury requires greater host cell infiltration. PAS stains cellular glycogen. With the addition of CTA, both Celox-CTA and CMS-CTA showed improved tissue infiltration, implying better wound repair. Figure 16This is most likely attributed to the construction of the microgel assembly, which creates a more moist environment at the wound site, promoting cell migration and nutrient transport. Meanwhile, fibrosis was still clearly visible in the Celox and Celox-CTA groups during PAS staining. Finally, immunofluorescence staining with HNF-4α and DAPI was performed. HNF-4α (green) specifically stained hepatocyte cytokines, while DAPI (blue) stained the cell nucleus. The HNF-4α results alone showed that the abnormal areas in the Celox and Celox-CTA groups were indeed not liver tissue; those areas showed almost no green light, indicating the absence of hepatocytes. The results showed that after adding CTA, the Celox-CTA and CMS-CTA groups had more hepatocytes, clearly indicating stronger hepatocyte activity. Figure 16 (f, g). HNF-4α / DAPI showed that the defect in the liver sample was mainly composed of hepatocytes, but the Celox and Celox groups had more obvious single blue areas. This may be because the presence of Celox caused inflammation in the wound, resulting in the presence of inflammatory cells. The single blue area of ​​Celox was more than that of the Celox group, which proves that the addition of CTA can reduce the negative impact of Celox on tissue.

[0142] In summary, by constructing a liver repair model, it was demonstrated that the addition of CTA to the hemostatic powder system, through its antibacterial effect and the construction of microgel assemblies, promotes cell migration and nutrient transport at the wound site, thereby enhancing the wound healing performance of the hemostatic powder. Furthermore, the addition of CTA also has a certain enhancing effect on hemostatic powders such as Celox, which are difficult to degrade and can cause foreign body reactions in tissues.

Claims

1. A rapid hemostatic microgel composition, characterized in that, The invention includes a rapid hemostatic powder material for massive bleeding and another different hemostatic material; the rapid hemostatic powder material for massive bleeding is tannic acid-modified carboxymethyl chitosan, and the other different hemostatic material is any one or more of cross-linked starch, chitosan, or composite microporous polysaccharide hemostatic powder.

2. The rapid hemostatic microgel composition according to claim 1, characterized in that, The weight ratio of the rapid hemostatic powder material for massive bleeding to the other different hemostatic material is 2:1 to 1:

2.

3. The rapid hemostatic microgel composition according to claim 1 or 2, characterized in that, The rapid hemostatic powder material for massive bleeding is prepared by the following steps: a 4% tannic acid solution is added dropwise to a 4% carboxymethyl chitosan aqueous solution, with a volume ratio of 50:1 between the tannic acid solution and the carboxymethyl chitosan aqueous solution, the pH is maintained at 8.5, and the reaction is carried out at 60°C for 3 hours. Air is bubbled into the reaction process, followed by dialysis, freeze drying, and grinding to obtain tannic acid-modified carboxymethyl chitosan powder.

4. The rapid hemostatic microgel composition according to claim 1 or 2, characterized in that, The other different hemostatic material is cross-linked starch.

5. The rapid hemostatic microgel composition according to claim 1 or 2, characterized in that, The cross-linked starch is prepared by the following steps: (1) Dissolve 21g of potato starch in 150 mL of 0.2M acetic acid-sodium acetate buffer solution with pH 4. The volume ratio of acetic acid solution to sodium acetate solution is 41:

9. Stir at 50℃ for 20 minutes. Then add 2% by mass of mixed enzyme, which is a mixture of saccharifying enzyme and α-amylase, with a mass ratio of 4:

1. React at 50℃ for 8 hours. Finally, wash the product with ultrapure water, filter, and vacuum dry at 50℃ to obtain enzymatically hydrolyzed starch. (2) Sodium hydroxide was dissolved in 90% v / v ethanol to prepare a 2.5% solution. Then, an equal mass of enzymatically hydrolyzed starch was added. The mixture was refluxed and stirred at 50°C for 30 minutes. Chloroacetic acid was added to the reaction solution. The mass ratio of chloroacetic acid to enzymatically hydrolyzed starch was 18:

25. The reaction was carried out at 50°C for 3 hours. Finally, the product was washed with anhydrous ethanol, filtered, and dried under vacuum at 50°C to obtain carboxymethyl enzymatically hydrolyzed starch. (3) Dissolve 1 g of carboxymethyl hydrolyzed starch in 10 mL of pure water, adjust the pH of the solution to 10, add sodium trimetaphosphate at a mass ratio of 3% of carboxymethyl hydrolyzed starch, mix to form an aqueous phase, then mix 50 mL of liquid paraffin and Span 80 at a mass ratio of 1% of liquid paraffin to form an oil phase, stir the oil phase at 60 °C, add the aqueous phase dropwise into the oil phase, react at 60 °C for 6 hours, wash, vacuum dry, grind and sieve to obtain cross-linked starch.

6. The use of the rapid hemostatic microgel composition according to any one of claims 1 to 5 in the preparation of a medicament for treating any one of the following (i) to (m): (i) Used to promote blood clotting; (j) Used in the preparation of hemostatic materials; (k) Used for antibacterial purposes; (l) Used to promote wound healing; (m) is used for anti-inflammatory purposes.

Images