Rapid hemostasis powder material for massive hemorrhage as well as preparation and application of rapid hemostasis powder material

Abstract

The invention discloses a rapid hemostasis powder material for massive hemorrhage. The rapid hemostasis powder material for massive hemorrhage is tannic acid modified carboxymethyl chitosan. The invention also provides a preparation method and application of the rapid hemostasis powder material for massive hemorrhage, and a rapid hemostasis microgel composition containing the rapid hemostasis powder material for massive hemorrhage. The rapid hemostasis powder material and microgel composition for massive hemorrhage have excellent water solubility, hemostasis performance and biocompatibility, have multiple effects of sterilization, anti-inflammation, wound healing promotion and the like, and are beneficial to rapid healing of wound parts after hemostasis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a rapid hemostatic powder material for massive hemorrhage, its preparation and application. Background Art

[0002] Uncontrolled bleeding is an important cause of increased mortality. For patients undergoing surgery, civilians suffering from accidental trauma, and soldiers injured on the battlefield, timely and effective hemostasis can reduce the mortality rate of the injured. Currently, the commonly used hemostatic materials on the domestic and international markets include three categories: polysaccharides, proteins, and inorganic minerals. Polysaccharide hemostatic materials include chitosan and cellulose, etc.; protein hemostatic materials include thrombin, collagen, and fibrin, etc.; inorganic mineral hemostatic materials include zeolite, montmorillonite, porous silica, and kaolin, etc. Different types of hemostatic materials have different hemostatic functions, 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 cost, easy to cause foreign body reactions, and harsh storage and transportation conditions; inorganic mineral hemostatic materials utilize their porous characteristics to absorb the water in the blood, concentrate the coagulation factors and platelets in the blood, thereby facilitating blood coagulation, but there are also side effects such as fever, which can cause wound damage and is not conducive to the wound healing of the injured area.

[0003] Polysaccharides are natural organic substances, which have the advantages of wide sources, good biocompatibility, biodegradability, and non-immunogenicity. Chitosan is the product of chitin after deacetylation, and its main source is crustaceans. With the development of technology, chitosan has been developed into a hemostatic material. However, the effectiveness of chitosan is not yet stable enough. It can effectively stop bleeding under ideal laboratory conditions, but in reality, for example, when encountering a deeper bleeding wound or on the real battlefield, many problems are encountered and it is impossible to stop the bleeding site in a timely and effective manner. In addition, chitosan is insoluble in water, and a single chitosan hemostatic material is expensive, which is not conducive to large-scale popularization and use.

[0004] Although great progress has been made in the research of existing hemostatic materials, it is still difficult to achieve an ideal massive hemorrhage hemostatic effect. At the massive hemorrhage wound site, the hemostatic material not only needs to stop the bleeding gushing out in a timely and effective manner, but also needs to be able to inhibit wound infection, have anti-inflammatory and wound healing promoting effects. A single hemostatic material is difficult to simultaneously possess multiple functions. Therefore, it is necessary to study a multi-component rapid hemostatic material for massive hemorrhage, which is applied to various emergency special scenarios with large wound areas and large bleeding volumes, such as on the battlefield, in accidents, in the wild, outdoors, and in the case of sudden massive hemorrhage during surgical operations, and at the same time has the functions of sterilization, anti-inflammatory, and promoting wound healing. Summary of the Invention

[0005] An object of the present invention is to provide a rapid hemostatic powder material for massive hemorrhage, which has multiple functions such as hemostasis, bactericidal, anti-inflammatory and promoting wound healing, aiming at the above technical problems to be solved.

[0006] Another object of the present invention is to provide a preparation method of the rapid hemostatic powder material for massive hemorrhage.

[0007] Still another object of the present invention is to provide an application of the rapid hemostatic powder material for massive hemorrhage.

[0008] To achieve the above invention objects, the present invention provides a rapid hemostatic powder material for massive hemorrhage, and the rapid hemostatic powder material for massive hemorrhage is tannic acid-modified carboxymethyl chitosan.

[0009] On the other hand, the present invention also provides a method for preparing the rapid hemostatic powder material for massive hemorrhage, which includes the following steps: dropping a tannic acid solution with a mass concentration of 4% into an aqueous carboxymethyl chitosan solution with a mass concentration of 4%, the volume ratio of the tannic acid solution to the aqueous carboxymethyl chitosan solution is 50:1, the pH is maintained at 8.5, reacting at 60 °C for 3 hours, introducing air during the reaction process, dialysis, freeze-drying, and grinding to obtain tannic acid-modified carboxymethyl chitosan powder.

[0010] On the other hand, the present invention also provides an application of the rapid hemostatic powder material for massive hemorrhage in any one of the following (a) to (d): (a) For promoting blood coagulation; (b) For preparing a hemostatic material; (c) For enhancing the hemostatic effect of other hemostatic products; (d) For antibacterial; (e) For promoting wound healing; (f) For anti-inflammatory.

[0011] 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.

[0012] On the other hand, the present invention also provides a rapid hemostatic microgel composition, which includes the rapid hemostatic powder material for massive hemorrhage according to the present invention and another different hemostatic material.

[0013] Preferably, the weight ratio of the rapid hemostatic powder material for massive hemorrhage to the another different hemostatic material is 2:1 to 1:2.

[0014] Preferably, the hemostatic material is any one or more of cross-linked starch, chitosan or composite microporous polysaccharide hemostatic powder.

[0015] Preferably, the hemostatic material is cross-linked starch.

[0016] The cross-linked starch is prepared through the following steps: (1) Dissolve 21 g of potato starch in 150 mL of acetic acid-sodium acetate buffer solution with a concentration of 0.2 M and a pH of 4. The volume ratio of the acetic acid solution to the sodium acetate solution is 41:9. Stir at 50 °C for 20 minutes, then add a mixed enzyme with a mass fraction of 2%. The mixed enzyme is a mixture of glucoamylase and α-amylase, and the mass ratio of glucoamylase to α-amylase is 4:1. React at 50 °C for 8 hours. Finally, wash the product with ultrapure water, filter by suction, and dry in vacuum at 50 °C to obtain enzymatically hydrolyzed starch; (2) Dissolve sodium hydroxide in 90% v / v ethanol to prepare a solution with a mass fraction of 2.5%. Subsequently, add enzymatically hydrolyzed starch with the same mass as sodium hydroxide, carry out condensation reflux, and stir at 50 °C for 30 minutes. Add chloroacetic acid to the reaction solution. The mass ratio of chloroacetic acid to enzymatically hydrolyzed starch is 18:25. React at 50 °C for 3 hours. Finally, wash the product with absolute ethanol, filter by suction, and dry in vacuum at 50 °C to obtain carboxymethyl enzymatically hydrolyzed starch; (3) Dissolve 1 g of carboxymethyl enzymatically hydrolyzed starch in 10 mL of pure water, adjust the pH of the solution to 10, add sodium trimetaphosphate accounting for 3% of the mass of carboxymethyl enzymatically hydrolyzed starch, and mix to form an aqueous phase. Then mix 50 mL of liquid paraffin and 1% of Span 80 based on the mass of liquid paraffin to form an oil phase. Stir the oil phase at 60 °C, and drop the aqueous phase into the oil phase drop by drop. React at 60 °C for 6 hours, wash, dry in vacuum, and then grind and sieve to obtain cross-linked starch.

[0017] On the other hand, the present invention also provides the application of the rapid hemostatic microgel composition in any one of the following (a) to (d): (i) For promoting blood coagulation; (j) For preparing a hemostatic material; (k) For antibacterial; (l) For promoting wound healing; (m) For anti-inflammatory.

[0018] In the present invention, carboxymethyl chitosan has excellent water solubility, hemostatic performance and biocompatibility. Therefore, carboxymethyl chitosan is selected as the raw material for synthesizing a general-purpose assembly agent. Since tannic acid has antibacterial, anti-inflammatory and free radical scavenging effects, tannic acid is grafted onto carboxymethyl chitosan through a one-step Michael addition reaction. After dialysis, freeze-drying and grinding, tannic acid-modified carboxymethyl chitosan (CTA) assembly agent powder is obtained. CTA can be blended with other commercial hemostatic powders such as chitosan-based Celox, composite microporous polysaccharide-based SKSS, and cross-linked starch CMS to form three microgel assemblies, namely Celox-CTA, SKSS-CTA and CMS-CTA. After contacting with blood, they absorb water to form microgels, and form hydrogen bonds with red blood cells, platelets in the blood, as well as hydroxyl and amino groups on the skin tissue, firmly adhering to the wound site, and playing a hemostatic role through physical occlusion and promoting blood coagulation. The phenolic hydroxyl groups on CTA form hydrogen bond interactions with the functional groups on Celox and SKSS: hydroxyl, carboxyl, and amino groups. The tissue adhesion strength 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 the water in the blood to form microgels to concentrate coagulation factors, thus promoting blood coagulation. Tannic acid contains a large number of phenolic hydroxyl group structures, which can bind to the proteins on the bacterial cell membrane or inhibit the activity of proteases, change the structure and function of the membrane, increase the cell membrane permeability, and cause the leakage of cell contents, ultimately leading to the death of bacteria. Tannic acid can also inhibit the synthesis of bacterial cell walls and scavenge free radicals, reduce bacterial reproduction and damage caused by oxidative stress, thus facilitating the rapid healing of the wound site after hemostasis. The material forms hydrogen bonds with the skin, red blood cells and platelets. This microgel system can firmly block the bleeding site, prevent blood from oozing out, and at the same time play antibacterial and anti-inflammatory roles, promoting the healing of the injured site.

[0019] In addition, the processing raw materials are easily available, inexpensive, the operation process is simple, and the product does not require strict storage and transportation conditions, making it suitable for large-scale production and popularization.

[0020] Specifically, in the CMS-CTA system, the introduction of cross-linked starch avoids the relatively expensive single component of carboxymethyl chitosan. The raw materials of cross-linked starch are rich in sources, inexpensive, relatively simple in processing operation, and have good biocompatibility, avoiding the side effects caused by foreign body reactions during use, and are suitable for large-scale popularization and use. Starch is a natural polymer organic matter, widely existing in plants, with wide sources and low prices, and has good adsorption, degradation and biocompatibility. Through the action of enzymes, the surface of starch forms a porous structure, increasing its surface area and being conducive to water absorption. In addition, carboxyl groups are introduced under alkaline conditions to change the original structure of the starch molecular chain, thus playing a hydrophilic role. However, single carboxymethyl starch lacks sufficient mechanical strength. Carboxymethyl starch can be cross-linked with a sodium trimetaphosphate cross-linking agent to obtain cross-linked starch, thereby improving its physical strength. As a hemostatic material, cross-linked starch can be degraded into oligosaccharides, maltose and glucose in the body by plasma amylase, avoiding the residue effect.

[0021] Compared with the prior art, the novel rapid hemostatic powder material and microgel composition of the present invention can rapidly absorb the water in the blood to form a microgel to concentrate coagulation factors, thereby promoting the coagulation effect, and can bind to the proteins on the bacterial cell membrane or inhibit the activity of proteases, changing the structure and function of the membrane, resulting in an increase in the permeability of the cell membrane and leakage of cell contents, ultimately leading to the death of bacteria, playing an antibacterial and wound-healing promoting role. The production raw materials are rich in sources, inexpensive, relatively simple in processing operation, and have good biocompatibility, avoiding the side effects caused by foreign body reactions during use, and are suitable for large-scale popularization and use. It can be applied to various emergency special scenarios with large wound areas and large bleeding volumes, such as battlefields, accidents, the wild, outdoors, and sudden large bleeding during surgical operations, and at the same time has the functions of sterilization, anti-inflammation and promoting wound healing. Brief Description of the Drawings

[0022] Figure 1 Shows the synthesis and properties of tannic acid-modified carboxymethyl chitosan (CTA). (a) Synthesis route of CTA; (b) Powder appearance and SEM images of CTA and its microgel assemblies with Celox, SKSS, CMS; (c) Compression 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 performance of Celox-CTA, SKSS-CTA and CMS-CTA microgel assemblies with different ratios (2:1, 1:1, 1:2).

[0023] Figure 2 Shows the 1H NMR and FTIR spectra of CMCS and CTA.

[0024] Figure 3 Shows the synthesis route of crosslinked starch CMS.

[0025] Figure 4 Shows the FTIR spectrum of CMS.

[0026] Figure 5 Shows the SEM images of Celox, SKSS, and CMS powders.

[0027] Figure 6 Shows the gelation properties, mechanical properties, adhesion properties, and burst pressure test results of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies. (a) Experimental results of the 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) Pig skin tensile experiment of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (d) Experimental results of the burst pressure of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (e) Compression modulus of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (f) Adhesion pressure of Celox, Celox-CTA, SKSS-CTA, and CMS-CTA microgel assemblies; (g) Experimental results of the burst pressure.

[0028] Figure 7 Shows the rheological measurement results of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA.

[0029] Figure 8 Shows the schematic diagram of the explosion pressure device.

[0030] Figure 9 Shows the degradation performance results of Celox-CTA, Skss-CTA, and CMS-CTA.

[0031] Figure 10 Shows the in vitro coagulation performance of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA microgel assemblies. (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 on red blood cells and platelets; (f) Red blood cell adhesion percentage; (g) Platelet adhesion percentage.

[0032] Figure 11 Shows the in vitro clotting times of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA.

[0033] Figure 12 Shows the Zeta potentials of Celox-CTA, SKSS-CTA, and CMS-CTA.

[0034] Figure 13 Shows the antibacterial properties of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA microgel assemblies. (a) Escherichia coli and Staphylococcus aureus colony growth experiments; (b) SEM images of bacterial morphology after co-culture with material samples; (c) bactericidal rates of each material; (d) hemolysis and dissolution rates of each material; (e) growth of L929 cells and cell viability.

[0035] Figure 14 Shows the in vitro hemostatic effects of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA. (a) Rat liver bleeding experiment; (b) Rat femoral artery bleeding experiment; (c) Rabbit liver bleeding experiment; (d) Pig spleen and liver bleeding experiment.

[0036] Figure 15 Shows the hemostasis times of Celox, Celox-CTA, CMS, and CMS-CTA: (a) Rat liver bleeding model; (b) Rat femoral artery bleeding model; (c) Rabbit liver bleeding model.

[0037] Figure 16 Shows the wound-healing promoting properties of Celox, Celox-CTA, SKSS, SKSS-CTA, CMS, and CMS-CTA. (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 manners

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0039] 1. Experimental Section 1.1 Materials and Reagents Carboxymethyl chitosan (Shanghai Aladdin Reagent Co., Ltd., product number C304738-25g), potato starch, tannic acid, glucoamylase, chloroacetic acid, sodium hydroxide, liquid paraffin, and Span 80 were purchased from Aladdin (Shanghai, China). α-Amylase and sodium trimetaphosphate were purchased from Macklin (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 of analytical grade and were used without further purification.

[0040] 1.2 Synthesis of Tannic Acid Modified Carboxymethyl Chitosan (CTA) Carboxymethyl chitosan was dissolved in water to prepare a solution (4%, w / w), and stirred at 60 °C until dissolved, and the pH was adjusted to 8.5. The tannic acid solution (4%, w / w) was slowly dropped into the above carboxymethyl chitosan solution at a volume ratio of 50:1 (tannic acid solution: carboxymethyl chitosan solution = 50:1), and reacted at 60 °C for 3 hours. The pH of the solution during the reaction was monitored using a pH meter, and the pH was maintained at 8.5, and air was bubbled during the reaction. The reaction solution was dialyzed in deionized water for five days using a dialysis bag with a molecular weight cut-off (MWCO) of 1000 Da. Freeze-dried and ground to obtain tannic acid-modified carboxymethyl chitosan (CTA) powder.

[0041] 1.3 Synthesis of Crosslinked Starch (CMS) 21 g of potato starch was dissolved in 150 mL of acetic acid-sodium acetate buffer solution with a concentration of 0.2 M and a pH of 4 (the volume ratio of acetic acid solution (0.2 mol / L) to sodium acetate solution (0.2 mol / L) was 41:9), and stirred at 50 °C for 20 minutes. Then 0.4 g of a mixed enzyme (a mixture of glucoamylase and α-amylase) was added, and the mass ratio of glucoamylase to α-amylase was 4:1, and reacted at 50 °C for 8 hours. Finally, the product was washed with ultrapure water, filtered by suction, and dried in vacuo at 50 °C to obtain enzymatically hydrolyzed starch.

[0042] Sodium hydroxide was dissolved in 90% v / v ethanol to prepare a solution (2.5%, w / w), and then enzymatically hydrolyzed starch with the same mass as sodium hydroxide was added, and refluxed under condensation, stirred at 50 °C for 30 minutes, and chloroacetic acid (mass ratio to enzymatically hydrolyzed starch was 18:25) was added to the reaction solution, and reacted at 50 °C for 3 hours. Finally, the product was washed with absolute ethanol, filtered by suction, and dried in vacuo at 50 °C to obtain carboxymethylated enzymatically hydrolyzed starch.

[0043] Dissolve 1 g of carboxymethyl enzymatically hydrolyzed starch in 10 mL of pure water, adjust the pH of the solution to 10, add sodium trimetaphosphate accounting for 3% of the mass of carboxymethyl enzymatically hydrolyzed starch, and mix to form an aqueous phase. Then mix 50 mL of liquid paraffin (oil phase) and 1% of Span 80 based on the mass of the oil phase. After stirring the oil phase at 60 °C for a period of time, slowly drip the aqueous phase into the oil phase and react at 60 °C for 6 hours. Wash twice continuously with an excessive amount (generally 2 - 3 times the reaction solution) of acetone, petroleum ether, 9% w / w sodium chloride solution, and ethanol, and dry in vacuum at 50 °C, then grind and sieve (100 mesh, pore size 0.154 mm) to obtain cross-linked starch (CMS).

[0044] 1.4 Structural Characterization of CTA and CMS Dissolve the prepared CTA and unmodified carboxymethyl chitosan in deuterated water (15 mg of powder dissolved in 1.5 mL of deuterated water), and characterize by 1H NMR spectrum (Bruker AVANCE 400 MHz, Switzerland). Then prove the successful synthesis of CTA and CMS by FTIR (Bruker Vertex 70, Germany).

[0045] 1.5 Preparation and Proportion Optimization of Celox-CTA, SKSS-CTA and CMS-CTA Uniformly mix different commercially available hemostatic powders Celox (a commercial chitosan hemostatic powder), SKSS (a commercial composite microporous polysaccharide hemostatic powder, purchased from Saikesaisi), the above cross-linked starch CMS, and tannic acid-modified carboxymethyl chitosan CTA at different mass ratios (2:1, 1:1, 1:2) to obtain microgel assemblies with different ratios. Conduct compression tests, adhesion performance tests, and blood coagulation index (BCI) tests on various microgel assemblies. The detailed experimental methods are as follows.

[0046] 1.6 Structural Characterization of Celox-CTA, SKSS-CTA and CMS-CTA Characterize 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) by field emission scanning electron microscopy (FESEM, Carl Zeiss Merlin, Germany) to determine their particle sizes and structures.

[0047] 1.7 Gelation Behavior Characterization For each powder sample (CTA, Celox, SKSS, CMS, Celox-CTA, SKSS-CTA, CMS-CTA), PBS or anticoagulated blood was added (the mass ratio of sample powder to PBS or anticoagulated blood was 1:3), and forceps were used to pick it up and observe its gelation. To determine the gelation time of each sample, a rheometer (Anton Paar MCR 302, Austria) was used for rheological tests, with the temperature set at 37 °C, the fixed strain at 1.0%, the fixed frequency at 1 Hz, and the sample height at 10 mm. A fixed mass of the powder sample was added to a parallel plate mold (diameter 25 mm). After the test started, a certain amount of PBS or anticoagulated blood was added to the mold to obtain an oscillation time scan curve.

[0048] 1.8 Mechanical Property Characterization PBS or anticoagulated blood was added dropwise to make each powder sample (the mass ratio of sample powder to PBS or anticoagulated blood was 1:3) into a cylinder (height: 5 mm; diameter: 10 mm). Weights of different masses (20 g, 50 g, 100 g) were pressed on each cylinder, and the magnitude of its deformation was observed to judge its mechanical properties. Subsequently, a dynamic thermomechanical analyzer (DMA, Q800DE, TA, USA) was used to conduct compression experiments on each cylinder. During the experiment, the strain rate was 50% / minute, the maximum strain was set at 90%, and the initial force was 0.0010 N. The stress-strain curves of each experimental group were obtained and their specific compression moduli were calculated.

[0049] 1.9 Adhesion Property Characterization PBS or anticoagulated blood was added dropwise to make each powder sample (the mass ratio of sample powder to PBS or anticoagulated blood was 1:3) into a gel. Subsequently, a 0.5 cm×1 cm gel was added to each piece of pigskin sized 1 cm×2 cm, and the maximum area of another piece of pigskin that it could adhere to was observed. Then, two pieces of pigskin sized 1 cm×2 cm were bonded with a 0.5 cm×1 cm gel, and a dynamic thermomechanical analyzer (DMA, Q800DE, TA, USA) was used to conduct tensile tests on it. The strength when the two pieces of pigskin were completely separated from each other was the adhesion strength of the test powder gel.

[0050] 1.10 Bursting Pressure Test A 2-mm incision was made on the aorta of a porcine heart with a diameter of 10 mm, and 30 mg of the hemostatic powder to be tested was deposited on the incision. An equal volume of anticoagulated blood was added dropwise to form a hydrogel layer in situ. After 5 minutes, PBS stained with red dye was introduced into the porcine heart aorta, and the leakage of PBS in different groups was observed to evaluate its sealing performance. Subsequently, the porcine heart aorta was connected to an injection pump and a pressure monitor. The injection pump gradually injected PBS into the device at a rate of 10 mL / min. When the seal failed, the bursting pressure was taken as the rupture pressure.

[0051] 1.11 Degradation Property Test 0.05 g of Celox-CTA, SKSS-CTA, and CMS-CTA were placed in 1.5 ml of SBF simulated body fluid. After oscillating at 37 °C for 1, 3, 5, 7, and 14 days, the degradation was observed, and then it was dried and the remaining mass was weighed. The fresh SBF simulated body fluid was replaced every two days. Four parallel samples were set for each day group. Degradation rate = (W 0 -W t ) / W 0 ,where W 0 is the initial mass of the sample, and W t is the mass of the sample after drying for different days of degradation.

[0052] 1.12 Whole Blood Coagulation Index (BCI) Test 10 mg of the powder sample to be tested was added to 200 μL of recalcified blood (10 μL of 0.2 M calcium chloride per 100 μL of blood). After incubating with shaking (100 rpm) at 37 °C for 5 minutes, 10 mL of deionized water was added to dissolve the uncoagulated blood clots. The hemoglobin content of the supernatant was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Switzerland). The blood coagulation index (BCI) was calculated as follows: BCI (%) = (Is - I 0 ) / (Ic - I 0 )×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 dropped into 200 μL of recalcified blood), and I 0 represents the absorbance value of the blank well plate.

[0053] 1.13 In Vitro Coagulation Time Test 5 mg of the powder sample to be tested was added to a centrifuge tube containing 100 μL of recalcified whole blood. 400 μL of PBS was added at a preset time, and the diffusion behavior of the blood was observed. The time when no blood diffused into the PBS was defined as the coagulation time.

[0054] 1.14 Zeta Potential Test Put the powder samples to be tested at 0.5 mg / mL, 1 mg / mL and 5 mg / mL into the test mold. The electrolyte circulates through the sample cell to form a flow pressure difference. The flow potential is generated by the charge movement relative to the electrochemical double layer and measured by the electrodes at both ends of the sample.

[0055] 1.15 Red Blood Cell and Platelet Adhesion Centrifuge fresh sodium citrate-anticoagulated sheep whole blood at 200×g for 15 minutes to obtain a red blood cell suspension and platelet-rich plasma.

[0056] Red blood cell adhesion and quantitative analysis: Drop 100 μL of the red blood cell suspension into 10 mg of the powder sample to be tested and incubate at 37 °C for 5 minutes. Further wash the sample 5 times with DPBS (Dulbecco's phosphate buffered saline) to wash away the non-adherent red blood cells, and then add deionized water (10 mL). Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of the supernatant at 540 nm. The red blood cell suspension group without the material added serves as the blank control, and each group is repeated 3 times. The number of red blood cell adhesions (%) is calculated by the following formula: . The absorbances of the sample and the reference ratio are represented by and respectively.

[0057] Platelet adhesion and quantitative analysis: Drop 100 μL of the platelet-rich plasma into 10 mg of the powder sample to be tested and incubate at 37 °C for 5 minutes. Further wash the sample 5 times with DPBS to wash away the non-adherent platelets. First, use 1% Triton X-100 to lyse the adherent platelets, and finally use a lactate dehydrogenase cytotoxicity detection kit (Beyotime, product number C0016) to detect the number of adherent platelets. Use an ELISA reader to measure the absorbance value of the supernatant at 490 nm. The platelet group without the material added serves as the blank control, and each group is repeated 3 times. The number of platelet adhesions (%) is calculated by the following formula: .

[0058] To observe the morphology and number of the attached red blood cells and platelets, the samples are fixed with 4% paraformaldehyde for 2 hours and then subjected to gradient dehydration (ethanol concentrations are 50%, 60%, 70%, 80%, 90% and 100%). Finally, the dehydrated samples are dried, sputter-coated with gold and observed under a scanning electron microscope.

[0059] 1.16 Antibacterial Property Characterization Select Escherichia coli (ATCC8739, Gram-negative bacterium) and Staphylococcus aureus (ATCC 6538, Gram-positive bacterium) as the test strains to evaluate the antibacterial performance of the powder to be tested. Add 100 μL of a concentration of 10 7 CFU mL−1 Drop the Escherichia coli or Staphylococcus aureus solution into the powder gel to be tested, and incubate at 37 °C for 2 hours. Add 900 μL of PBS and mix thoroughly. Subsequently, take out 10 μL of the solution, evenly spread it on the pre-prepared agar plate, and incubate at 37 °C for 12 hours. Finally, observe and record the number of bacterial colonies growing on the agar plate. Similarly, 100 μL of the Escherichia coli or Staphylococcus aureus solution with a concentration of 10 7 CFU mL −1 Incubate with the sample at 37 °C for 2 hours, then add 900 μL of Luria-Bertani (LB) broth to submerge the sample, and further incubate for 12 hours. Finally, transfer 200 μL of the bacterial suspension in each sample tube to a 96-well plate, and measure the absorbance value at 600 nm using a microplate reader. Each group of experiments is repeated 3 times. The bactericidal rate (%) is 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.

[0060] Observe the bacterial morphology after co-culture with the material sample by scanning electron microscopy. Co-culture the bacterial solution with the material using a bacterial medium. After 4 hours, ultrasonically resuspend to detach the bacteria from the material. Take out the material, collect the bacterial solution and centrifuge (6000 rpm, 5 minutes). Discard the supernatant, wash the bacteria three times with PBS, then add 4% paraformaldehyde solution to fix for 1 hour. Centrifuge, aspirate the paraformaldehyde, dehydrate with gradient concentration ethanol solutions and tert-butanol, freeze-dry, and finally prepare the sample for observation by scanning electron microscopy.

[0061] 1.17 Hemolysis Property Test Centrifuge the anticoagulated sheep whole blood at a speed of 200 ×g for 10 minutes to collect red blood cells. Then dilute the obtained red blood cells (RBCs) tenfold (v / v) with PBS solution. Add 5 mg of the powder sample to be tested to 0.8 mL of PBS solution, and then add 0.2 mL of the diluted RBCs suspension. The DW group and the PBS group are used as positive and negative controls respectively, and 3 parallel samples are set in each group. After the RBCs are co-incubated with the sample for 1 hour, centrifuge at a speed of 3000 rpm for 10 minutes. Take 100 μL of the supernatant and place it in a 96-well plate, and measure the absorbance value of the supernatant at a wavelength of 540 nm using a microplate reader. Calculate the hemolysis rate according to the formula: 。

[0062] 1.18 Cell Compatibility Characterization Each powder sample to be tested was sterilized by ultraviolet light irradiation for 24 hours and then subjected to experiments. PBS was dropped onto the powder sample to be tested (the mass ratio of the powder sample to PBS was 1:3) to gel it, and a cylinder (with a diameter of 10 mm and a height of 2 mm) was prepared. After being disinfected by soaking in alcohol for 24 hours, it was continuously soaked in the culture medium for 24 hours to wash away the excess alcohol. L929 cells were inoculated at a density of 20,000 cells per well and cultured for 24 hours and 48 hours respectively. The cell viability was evaluated using the live / dead staining method, and observed and counted through an inverted fluorescence microscope (DMi1, Leica, Germany). The control group was a pure culture medium group without adding materials.

[0063] 1.19 In Vivo Hemostasis Property Characterization First, in vivo hemostasis tests were performed using rat liver, femoral artery bleeding models and rabbit liver bleeding models for preliminary characterization.

[0064] In the rat liver bleeding experiment, Sprague-Dawley (SD) rats (male, 0.3 - 0.4 kg) were randomly divided into 5 groups (5 rats in each group). Zoletil® 50 and xylazine hydrochloride were blended at a mass ratio of 1:1, then diluted 10 times with sterile water, and the rats were anesthetized by intramuscular injection at 1 μL / g. A wound with a diameter of 5 mm and a depth of 5 mm was made with a puncher, and 20 mg of the powder sample to be tested was added to the bleeding part. A weighed filter paper was placed under the wound, and the blood loss and bleeding time were recorded.

[0065] For the rat femoral artery bleeding experiment, the anesthesia method was the same as above. After finding the femoral artery of the rat's thigh, a 10 mm long wound was made with a knife edge, 20 mg of the powder sample to be tested was added to the bleeding part, and a weighed filter paper was placed under the wound, and the blood loss and bleeding time were recorded.

[0066] In the rabbit liver bleeding experiment, New Zealand white rabbits (3.0 - 3.5 kg, male) were randomly divided into 5 groups (5 rabbits in each group). Zoletil® 50 and xylazine hydrochloride were blended at a mass ratio of 4:1 and the rabbits were anesthetized by intramuscular injection at 300 μL / kg. After finding the rabbit's liver, a weighed filter paper was placed under the liver, and a liver injury was made with scissors. Then 200 mg of the powder sample to be tested was immediately placed, and the blood loss and hemostasis time were recorded.

[0067] Next, a liver and spleen bleeding model of pigs was used to further characterize the in vivo hemostatic performance. Bama pigs (female, 25 - 35 kg) were mixed with Zoletil® 50 and xylazine hydrochloride in a mass ratio of 1:1 and anesthetized by intramuscular injection at 0.2 mL / kg. Then, the pigs were continuously anesthetized with an isoflurane and oxygen breathing anesthesia machine. In the pig liver bleeding experiment, after finding the pig's liver, place a dust-proof paper underneath, make a wound with a length of 20 mm and a depth of 10 mm using a scalpel. After bleeding, sprinkle the powder sample to be tested, gently press, observe the hemostasis situation, and additional hemostatic powder can be added. Record the blood loss and bleeding time. In the pig spleen bleeding experiment, after finding the pig's spleen, place a dust-proof paper underneath, make a wound with a length of 20 mm and a depth of 10 mm using a scalpel. After bleeding, sprinkle the powder sample to be tested, gently press, observe the hemostasis situation, and additional hemostatic powder can be added. Record the blood loss and bleeding time. All animal experimental procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals of South China University of Technology" and were approved by the Animal Ethics Committee of South China University of Technology (Approval No.: 2024138).

[0068] 1.20 Liver Repair Experiment After the above-mentioned rat liver hemostasis experiment was completed, the excess powder on the liver surface was rinsed clean with normal saline, then the liver was placed back in place, and the skin wound of the rat was sutured. Within 3 days after the operation, each rat was injected with 1 mL of ampicillin (Servicebio, catalog number G4018 - 10ML) every day. After 14 days, the liver treated in the hemostasis experiment of each rat was taken out to observe its liver repair situation.

[0069] To further evaluate the liver repair performance of different materials, histological section staining analysis was performed on each liver sample. The liver samples were fixed with 4% (mass fraction) paraformaldehyde, embedded in paraffin for sectioning, then stained with hematoxylin and eosin (H&E), PAS glycogen staining, and immunofluorescence staining. The staining results were scanned by a digital pathology scanning system, and finally the pictures were saved and analyzed. All animal procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals of South China University of Technology" and were approved by the Animal Ethics Committee of South China University of Technology.

[0070] 2 Results and Discussion 2.1 Preparation and Characterization of CTA and Three Microgel Assemblies Carboxymethyl chitosan has excellent water solubility, hemostatic performance, and biocompatibility. Therefore, carboxymethyl chitosan was selected as the raw material for synthesizing a general-purpose assembly agent. According to Figure 1 the synthetic route shown ( Figure 1 , a), tannic acid was grafted onto carboxymethyl chitosan through a one-step Michael addition reaction, and CTA powder was obtained after dialysis, freeze-drying, and grinding.

[0071] 1H NMR and FTIR spectra were collected to verify the synthesis of CTA ( Figure 2 ).

[0072] Meanwhile, cross-linked starch CMS ( Figure 3 ) was also synthesized, and the FTIR spectrum of CMS confirmed its successful synthesis ( Figure 4 ).

[0073] Two commercial hemostatic powders, Celox and SKSS, were introduced. Celox, SKSS, and CMS were respectively blended with CTA to obtain three microgel assemblies, namely Celox-CTA, SKSS-CTA, and CMS-CTA. As shown in the figure, the CTA powder was light yellow ( Figure 1 , b), and the SEM image showed that its particle size was about 10 - 40 μm ( Figure 5 ); the Celox powder was light yellow with a larger particle size, SKSS and CMS were pure white, and their particle sizes were about 100 μm, which were larger than that of CTA; after blending with CTA, the yellow color of Celox-CTA deepened, and SKSS-CTA and CMS-CTA showed a faint light yellow. The SEM image showed that small-sized CTA was distributed therein, proving the successful addition of CTA.

[0074] The content ratio of microgels (Celox, SKSS, and CMS) to the assembly agent (CTA) in the microgel assembly is very important for the hemostatic properties. The mechanical strength, tissue adhesion strength, and coagulation index were selected as the indicators for ratio optimization.

[0075] The compression modulus and tissue adhesion strength of the microgel assemblies when the mass ratios of different microgels (Celox, SKSS, and CMS) / assembly agent (CTA) changed (2:1, 1:1, 1:2) were shown ( Figure 1 , c and d). Obviously, the higher the CTA content, the greater the compression modulus and tissue adhesion strength of the microgel assembly. This is because the mechanical strength of the microgel assembly mainly comes from the hydrogen bond interaction between the phenolic hydroxyl group on CTA and the functional group hydroxyl, carboxyl, and amino groups on the microgel, while the tissue adhesion strength of the microgel assembly mainly comes from the interaction between the phenolic hydroxyl group on CTA and the tissue surface. However, it can be found that the increase from 1:1 to 1:2 is much smaller than the increase from 2:1 to 1:1, indicating that the increase in the CTA content from 1:1 to 1:2 has less impact on the mechanical strength and tissue adhesion strength of the microgel assembly. Figure 1 The change in the coagulation index at different microgel / assembly ratios was also shown ( Figure 1, e). When the mass ratio of the microgel / assembly is 1:1, all three groups of microgel assemblies have lower coagulation indices, that is, better coagulation performance. This may be because when the two components are in a ratio of 1:1, the two components have the best synergistic effect and composite effect on blood coagulation at this ratio. Based on the fact that the three groups of microgel assemblies have the best coagulation performance at 1:1, and when changing from 1:1 to 1:2, the mechanical strength and tissue adhesion strength change less. After considering the comprehensive performance of the materials, it is selected to use a mass ratio of 1:1 of microgel / assembler for subsequent experiments.

[0076] 2.2 Enhancement of the Mechanical Strength, Tissue Adhesion and Occlusion Strength of Microgels by the Universal Assembler CTA Such as Figure 6 shown, single Celox, SKSS, and CMS powders could not be picked up with tweezers after dropping PBS or blood, and their shapes were irregular; after blending with CTA, Celox-CTA, SKSS-CTA, and CMS-CTA could form gels and be picked up with tweezers whether PBS or blood was dropped, and they could each form good shapes ( Figure 6 , a).

[0077] The influence of CTA on the gel-forming performance of the hemostatic powder was further explored through rheological tests ( Figure 7 ). It can be seen that when CTA was not added, for all three groups of hemostatic powders, whether PBS or blood was dropped, the loss modulus (G'') was always greater than the storage modulus (G'), showing typical fluid characteristics, indicating that no gels were formed; 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 whether PBS or blood was dropped, the addition of CTA enabled the microgels to form microgel tissue gels. At the same time, it can be seen that when testing by dropping blood, Celox-CTA, SKSS-CTA, and CMS-CTA all had shorter gel-forming times and higher storage moduli, which proved that the addition of CTA allowed red blood cells and platelets in the blood to also participate in the assembly. By filling the gaps between the microgels, the microgel assemblies had stronger gel-forming performance.

[0078] The compressive strength of the gel was explored by applying weights above the gel ( Figure 6 , b), and it was found that the results of the three groups of tests were consistent. The hemostatic powder before adding CTA had the largest compressive deformation when compressed by the weight. After adding CTA, the microgel assemblies formed by blood gelation had the least compressive deformation. The compressive moduli of each group obtained through testing also showed the same trend ( Figure 6 , e).

[0079] Next, different gels were coated on pig skins to adhere to pig skins of different areas ( Figure 6, c), after adding CTA, the microgel assemblies can adhere to a larger area of porcine skin, and the microgel assemblies that form a gel with blood have better adhesion performance than those that form a gel with PBS, which is further proven by the porcine skin stretching test ( Figure 6 , f).

[0080] The above tests show that the three groups of hemostatic powders without CTA have weak forces and basically do not have mechanical strength and tissue adhesion strength after adding PBS. After adding CTA, the hydrogen bond interaction between CTA and the microgel and the interaction between CTA and the tissue surface endow the microgel assemblies with more excellent mechanical strength and adhesion performance. And because CTA allows red blood cells and platelets to also participate in the assembly, the microgel assemblies have more excellent performance after forming a gel by dropping blood.

[0081] A 2-mm diameter notch was made on the aorta of pigs. After each powder sample was gelled with blood, it was attached to the notch. The red-stained PBS was passed into the aorta, and the leakage of PBS was observed. As Figure 6 can be seen, when CTA was not added, PBS leaked out in all three groups. After adding CTA, no PBS leaked out, proving that the addition of CTA endows the microgel with better plugging performance ( Figure 6 , d). Then, a blasting device ( Figure 8 ) was built to test the blasting pressure of each powder sample. As shown in the figure, the blasting pressures of single microgel powders are all far less than the normal human arterial blood pressure ( Figure 6 , g), proving that they have a poor application prospect in plugging massive bleeding scenarios in the human body. However, after adding CTA, the blasting pressures of Celox-CTA, SKSS-CTA, and CMS-CTA are all greater than 120 mmHg, which are 150 mmHg, 200 mmHg, and 250 mmHg respectively. Obviously, the addition of CTA provides the possibility for each powder to be applied to larger bleeding scenarios.

[0082] The test results of the in vitro degradation performance of the hemostatic powder are as Figure 9 shown. All three powders continue to degrade over time, and the degradation performance is good.

[0083] 2.3 Enhancement of the In Vitro Coagulation Performance of Microgels by the Addition of CTA First, the in vitro blood coagulation index (BCI) of each group of powder samples was tested. 10 mg of the powder sample was added to 200 μL of recalcified blood. After incubating at 37 °C for 5 minutes, 10 mL of deionized water was added to dissolve the uncoagulated blood clots. The hemoglobin content of the supernatant was measured by the absorbance value at 540 nm, and the BCI of each sample was calculated through the formula. As Figure 8As shown, before the addition of CTA, the blood coagulation effects of the three groups of microgel powders were all poor under this test condition, the blood clots were unstable, the blood clots ruptured after adding a large amount of deionized water, red blood cells overflowed, and the supernatant was light red; however, after adding CTA, the coagulation performance of the three groups was significantly enhanced, the supernatant was clear, and basically no red blood cells overflowed ( Figure 10 , a), and the BCI value also proved such a result ( Figure 10 , b).

[0084] Then, 5 mg of the powder sample was added to a centrifuge tube containing 100 μL of recalcified whole blood, and 400 μL of PBS was added at the preset time to observe the diffusion behavior of the blood to test the in vitro clotting time. The results showed that after adding CTA, all three groups had shorter clotting times ( Figure 10 , c; Figure 11 ).

[0085] The above two tests proved that the addition of CTA could significantly improve the in vitro coagulation performance of the microgel. Celox mainly promotes blood coagulation through strong positive charges, SKSS and CMS mainly promote blood coagulation by absorbing blood and concentrating coagulation factors, while CTA mainly promotes blood coagulation by attracting red blood cells through hydrophobic interaction and attracting platelets through electrostatic interaction. It is speculated that there is a synergistic effect between the coagulation effect of the microgel itself and CTA. In addition, since the assembly has stronger mechanical properties to prevent the spread of blood clots, the microgel assembly formed after adding CTA has more excellent in vitro coagulation performance.

[0086] To explore the mechanism of CTA promoting blood coagulation, first, Zeta potential tests were carried out. It can be seen that Celox itself is positively charged, SKSS and CMS are negatively charged; while CTA is also negatively charged because it has carboxyl groups and tannic acid, so adding CTA will make the material surface negatively charged. Both SKSS-CTA and CMS-CTA have more obvious negative potentials, while Celox-CTA is still positively charged, which should be attributed to the strong positive charge of Celox itself ( Figure 12 ).

[0087] The APTT test results showed that the addition of CTA made the material have a lower APTT time, indicating that the material has a stronger ability to activate the intrinsic coagulation pathway ( Figure 10 , d), which is because of the strong negative charge of CTA itself on the intrinsic coagulation pathway.

[0088] Next, the adhesion effects of the samples on red blood cells and platelets were observed by scanning electron microscopy. It can be seen that only a few red blood cells and platelets were adhered in the fields of view of the three groups of powders, namely Celox, SKSS, and CMS. However, more red blood cells and platelets were adhered to the microgel assemblies after the addition of CTA ( Figure 10 , e). The quantitative test data also proved this point ( Figure 10 , f, g). CTA has a hydrophobic effect on red blood cells and an electrostatic effect on platelets, causing more red blood cells and platelets to be adhered to the material and participate in the assembly of the assemblies.

[0089] In summary, CTA improves the blood coagulation performance of the material by strengthening the activation effect of the material on the endogenous coagulation pathway and strengthening the adhesion of the material to red blood cells and platelets.

[0090] 2.4 Enhancement of the Antibacterial Property and Biocompatibility of Microgels by the Addition of CTA After the hemostatic material completes hemostasis, the moist and hypoxic environment at the wound site is prone to bacterial growth, which may lead to wound infection. Therefore, in practical applications, the hemostatic material is required to have excellent antibacterial properties to prevent inflammation and promote the healing of the subsequent wound site. Escherichia coli ( E. coli , Gram-negative bacteria) and Staphylococcus aureus ( Staphylococcus aureus , Gram-positive bacteria), two of the most common bacteria, were introduced to study the effect of the addition of CTA on the antibacterial properties of the microgels. First, the agar method was used. The powder samples were co-cultured with Escherichia coli and Staphylococcus aureus for 2 hours respectively. Then, 10 μL of the suspension was taken out and evenly spread on the agar plate. After incubating at 37 °C for 12 hours, the number of bacterial colonies growing on the plate was observed. The fewer the colonies on the plate, the stronger the antibacterial property of the material. As shown in the figure, Celox has a certain antibacterial effect because it carries a strong positive charge; SKSS and CMS have basically no difference compared with the blank group, and their antibacterial effects are very poor; however, after the addition of CTA, the three groups of assemblies have obvious antibacterial effects on both bacteria, and no bacterial colonies can be seen on the plate, proving that the addition of CTA can significantly enhance the antibacterial properties of the microgels ( Figure 13 , a). To explore the antibacterial mechanism of CTA, the microscopic morphology of the bacteria in each group after co-culture was observed by SEM. The bacteria in the SKSS and CMS groups showed no difference in morphology from the blank group, presenting the intact and smooth original appearance of the bacteria, proving that they have no effect on the bacteria; the bacteria in the Celox group showed certain morphological changes, proving that they have some inhibitory effects on the bacteria; after the addition of CTA, the two bacteria in the three groups all showed severe deformation and even rupture, proving that CTA has an effect on the surface of the bacteria to inhibit and kill the bacteria; actually, CTA antibacterial is through the large number of phenolic hydroxyl groups on its surface to act on the bacterial membrane ( Figure 13 , b).

[0091] The specific bactericidal rate of each material was calculated by measuring the absorbance (600 nm) of the co-culture solution and combining with the formula ( Figure 13 , c). After adding CTA, the bactericidal rate of each group was close to 100%, indicating that the addition of CTA could endow the material with excellent antibacterial properties.

[0092] An excellent hemostatic material also needs to have excellent biocompatibility to ensure that it will not cause harm to the human body. First, the powder samples were co-cultured with the red blood cell suspension to test the hemolytic properties of the materials. As shown in the figure, it can be seen that the hemolysis rates of the three microgel assemblies and CTA itself are all lower than 5%, showing excellent blood compatibility and not causing hemolytic harm to the human body ( Figure 13 , d). Then, in order to further verify the cytocompatibility of the materials, each powder sample was co-cultured with fibroblasts (L929 cells). After 24 hours and 48 hours of co-culture, the growth and cell viability of L929 cells of the three microgel assemblies and CTA were almost no different from those of the blank group, indicating that the materials have excellent cytocompatibility ( Figure 13 , e). The excellent blood compatibility and cytocompatibility of each group after adding CTA indicate that the addition of CTA will not cause biological harm to the material, can achieve the balance between antibacterial property and cytocompatibility, and also prove that the single CTA material itself has excellent cytocompatibility.

[0093] 2.5 Enhancement of the In Vivo Hemostasis Effect of Microgels by the Addition of CTA First, a liver and femoral artery bleeding model was constructed for small animals, rats, and then the powder samples were used to verify the hemostatic effect. For rat liver bleeding, single Celox and CMS could not achieve timely hemostasis of rat liver bleeding. Although the effect was better than that of the blank group, there was still a certain amount of bleeding; after adding CTA, both Celox-CTA and CMS-CTA could promptly stop the liver bleeding, and almost no blood flow was visible ( Figure 14 , a; Figure 15 , a). For rat femoral artery bleeding, femoral artery bleeding has higher requirements for the plugging performance of the material. Single Celox and CMS could not promptly stop the rat femoral artery bleeding, and a large amount of blood flow could be seen. However, after adding CTA, both Celox-CTA and CMS-CTA could promptly stop the femoral artery bleeding, indicating that the material not only has excellent hemostatic performance but also excellent plugging performance ( Figure 14 , b; Figure 15 , b).

[0094] Next, a liver bleeding model was constructed for medium-sized animals, rabbits. Most of the rabbit's liver was directly excised, which resulted in a large amount of bleeding, posing a great test to the hemostatic and sealing properties of the materials. Single Celox and CMS could not stop the bleeding in the rabbit's liver, and a large amount of blood flowed out. After adding CTA, Celox-CTA could stop the bleeding after a certain time, and only a small amount of blood flowed out. CMS-CTA could promptly stop the bleeding in the rabbit's liver, and almost no blood was seen flowing out. This is consistent with the better sealing and coagulation properties of CMS-CTA in in vitro tests ( Figure 14 , c; Figure 15 , c).

[0095] Finally, to better verify the actual hemostatic performance of the materials and their application prospects for human hemostasis, liver and spleen bleeding models were constructed for large animals, Bama pigs, to more deeply explore the actual hemostatic performance of the materials. After locating the liver of the Bama pig, a 20-mm long and 10-mm deep wound was created with a scalpel, and a large amount of blood was seen flowing out. In the CMS group, after adding the powder, blood was still seen flowing out after 8 minutes. After flushing with water, the powder directly dispersed, and a large amount of blood gushed out, which proved that both the hemostatic and sealing properties of CMS were insufficient. In the CMS-CTA group, after the blood flowed out, the powder was quickly added, and no blood was seen flowing out after 3 minutes. After flushing with water, the microgel assembly still firmly adhered to the wound. The same phenomenon was seen for the spleen bleeding in Bama pigs. The CMS group could not stop the bleeding in the spleen. In the CMS-CTA group, the bleeding was stopped within three minutes, and no blood flowed out after flushing with water, and the microgel assembly at the wound site was not washed away ( Figure 14 , d). Relying on the stronger sealing and hemostatic properties brought by CTA to CMS, CMS-CTA also had excellent hemostatic effects on the bleeding wounds of large animals, once again proving the general strengthening significance of CTA.

[0096] 2.6 Enhancement of the Promoting Healing Performance of Hemostatic Powders by CTA During the actual hemostasis process, removing the hemostatic material may lead to secondary bleeding of the wound, which will cause secondary injury to the patient. However, if the material does not have the ability to promote in-situ tissue regeneration at the injury site and has the risk of causing other complications, the hemostatic material has to be forcibly removed.

[0097] To solve this problem, an in-situ liver regeneration model of rats was constructed to explore the effect of CTA on the healing performance of hemostatic powders. First, a 2-mm deep wound was created on the rat liver using a 5-mm in-vivo sampler, and then the sample powder was used for hemostasis. After the bleeding was completely stopped, the material and the liver were placed back into the body, and the incision was sutured. Antibiotics and painkillers were injected into the rats in the first three days to ensure the healthy survival of the rats. Fourteen days later, the animals were sacrificed, and the repair of the injured liver was observed ( Figure 16 , a).

[0098] In the rat liver bleeding model, CTA continued to show an enhanced effect on the hemostatic performance of the hemostatic material ( Figure 16 , b). Single Celox and CMS could not achieve timely hemostasis of the bleeding in the rat liver. Although the effect was better than that of the blank group, a certain amount of bleeding would still occur. After adding CTA, both Celox-CTA and CMS-CTA could promptly stop the liver bleeding, and almost no blood flow was visible.

[0099] A unified qualitative analysis of liver repair was carried out. First, it could be seen that 14 days later, the liver in the blank group had a certain degree of healing, but it was still the group with the most obvious wound and the largest defect. In the Celox group, some materials and fibrotic cysts could be clearly seen. This was probably because the Celox powder caused a tissue foreign body reaction, resulting in the aggregation of fibroblasts at the wound and the generation of fibrosis. In the Celox-CTA group, the wound was almost completely repaired, but some materials could be seen remaining at the wound, indicating that the degradation of Celox was slow and it could not be completely degraded in the body within 14 days. CMS had excellent degradability, and no materials were visible, but there were still obvious defects. In the CMS-CTA group, the injured wound was almost completely repaired, showing the best repair effect ( Figure 16 , c). Then, immunohistochemical analysis was performed on these samples to evaluate the healing status of the liver, and quantitative analysis was carried out using ImageJ software. The H&E results showed that there were indeed abnormal areas in the Celox group and the Celox-CTA group, probably the remaining materials and fibrotic areas. However, the Celox-CTA group had fewer abnormal areas than the Celox group. The wound in the CMS-CTA group was almost completely healed, proving that CTA had a significant enhancement on the healing performance of CMS. Approximately 30% of the wounds in both the Celox group and the CMS group were still not healed. After adding CTA, only about 10% of the Celox-CTA wounds were not healed, and the CMS-CTA wounds were almost completely healed ( Figure 16 , d). The regeneration of the injured wound site requires more infiltration of host cells. PAS stains the glycogen of cells. After adding CTA, both Celox-CTA and CMS-CTA had better tissue infiltration, which means better wound repair effects ( Figure 16, e), which is probably attributed to the construction of microgel assemblies, creating a more humid environment at the wound site, promoting cell migration and nutrient transport. At the same time, fibrosis can still be clearly seen in the Celox and Celox-CTA groups in the PAS staining. Finally, immunofluorescence staining combining HNF-4α and DAPI was performed. HNF-4α (green) can specifically stain hepatocyte factors, and DAPI (blue) stains the cell nuclei. The results of single HNF-4α showed that the abnormal areas in the Celox and Celox-CTA groups were indeed not liver tissues, and there was almost no green light in those areas, proving the absence of hepatocytes. The results showed that after adding CTA, there were more hepatocytes in the Celox-CTA and CMS-CTA groups, indicating stronger hepatocyte activity ( Figure 16 , f, g). HNF-4α / DAPI showed that hepatocytes were basically present at the defect sites of the liver samples tested, but there were more obvious single blues in the Celox and Celox groups. It may be that the presence of Celox caused inflammation at the wound, and thus there were inflammatory cells. The single blue area of Celox was even more than that of the Celox group, proving that the addition of CTA could reduce the negative impact of Celox on the tissue.

[0100] In summary, by constructing a liver repair model, it was proved that after adding CTA to the hemostatic powder system, through its antibacterial effect and the construction of microgel assemblies: promoting cell migration and nutrient transport at the wound site, the hemostatic powder had better wound healing performance. And for hemostatic powders like Celox that are difficult to degrade and can cause foreign body reactions in tissues, the addition of CTA also had a certain enhancing effect.

Claims

1. A powder material for rapid hemostasis of heavy bleeding, characterized in that: The heavy bleeding quick hemostasis powder material is tannic acid modified carboxymethyl chitosan.

2. A method for preparing the rapid hemostatic powder material for heavy bleeding according to claim 1, characterized in that: The method comprises the following steps: dropping a tannic acid solution with a mass concentration of 4% into a carboxymethyl chitosan aqueous solution with a mass concentration of 4%, wherein the volume ratio of the tannic acid solution to the carboxymethyl chitosan aqueous solution is 50:1, the pH value is maintained at 8.5, reacting at 60°C for 3 hours, blowing air into the reaction process, dialysis, freeze drying, and grinding to obtain tannic acid modified carboxymethyl chitosan powder.

3. Use of the rapid hemostatic powder material for heavy bleeding according to claim 1 in any one of the following (a) to (d): (a) Used to promote blood coagulation; (b) for preparing hemostatic materials; (c) Used to enhance the hemostatic effect of other hemostatic products; (d) Antibacterial use; (e) for promoting wound healing; (f) Used for anti-inflammatory purposes.

4. The use according to claim 3, characterized in that: The other hemostatic powder is any one or more of cross-linked starch, chitosan or composite microporous polysaccharide hemostatic powder.

5. A rapid hemostatic microgel composition, characterized in that: The invention comprises the rapid hemostatic powder material for heavy bleeding as claimed in claim 1 and another different hemostatic material.

6. The rapid hemostatic microgel composition according to claim 5, characterized in that: The weight ratio of the heavy bleeding rapid hemostatic powder material to the another different hemostatic material is 2:1 to 1:

2.

7. The rapid hemostatic microgel composition according to claim 5, characterized in that: The hemostatic material is any one or more of cross-linked starch, chitosan or composite microporous polysaccharide hemostatic powder.

8. The rapid hemostatic microgel composition according to claim 5, characterized in that: The hemostatic material is cross-linked starch.

9. The rapid hemostatic microgel composition according to claim 5, characterized in that: The cross-linked starch is prepared by the following steps: (1) 21 g of potato starch was dissolved in 150 mL of 0.2 M acetic acid-sodium acetate buffer solution with a pH of 4, the volume ratio of acetic acid solution to sodium acetate solution being 41:9, and stirred at 50°C for 20 minutes. Then, a mixed enzyme with a mass fraction of 2% was added, wherein the mixed enzyme was a mixture of saccharifying enzyme and α-amylase, wherein the mass ratio of saccharifying enzyme to α-amylase was 4:1, and the mixture was reacted at 50°C for 8 hours. Finally, the product was washed with ultrapure water, filtered, and vacuum dried at 50°C to obtain enzymatic starch; (2) Sodium hydroxide was dissolved in 90% v / v ethanol to prepare a solution with a mass fraction of 2.5%, and then enzymatic starch of the same mass as sodium hydroxide was added, condensed and refluxed, stirred at 50°C for 30 minutes, chloroacetic acid was added to the reaction solution, the mass ratio of chloroacetic acid to enzymatic starch was 18:25, and the reaction was carried out at 50°C for 3 hours. Finally, the product was washed with anhydrous ethanol, filtered, and vacuum dried at 50°C to obtain carboxymethyl enzymatic starch; (3) Dissolve 1 g of carboxymethyl enzymatic starch in 10 mL of pure water, adjust the pH of the solution to 10, add sodium trimetaphosphate (3% by mass of carboxymethyl enzymatic starch) and mix to form an aqueous phase. Then, mix 50 mL of liquid paraffin and Span 80 (1% by mass 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, and grind and sieve to obtain cross-linked starch.

10. Use of the rapid hemostatic microgel composition according to any one of claims 5 to 9 in any one of the following (i) to (m): (i) Used to promote blood coagulation; (j) for preparing hemostatic materials; (k) Antibacterial use; (l) Used to promote wound healing; (m) Used for anti-inflammatory purposes.

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