Microgel assembly powder capable of rapidly stopping bleeding as well as preparation method and application of microgel assembly powder

Abstract

The invention belongs to the technical field of biomedical materials, and discloses microgel assembly powder capable of rapidly stopping bleeding as well as a preparation method and application of the microgel assembly powder. The microgel assembly powder is composed of microgel powder and assembly agent powder, and the mass ratio of the microgel powder to the assembly agent powder is (1: 10)-(10: 1); the microgel powder comprises carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder, and the assembling agent powder is polyphenol modified polymer powder. The microgel assembly powder can quickly absorb moisture in blood to concentrate the blood after being in contact with the blood, meanwhile, rich ortho-phenolic hydroxyl groups on the assembly agent can immediately form a hydrogen bond network among microgel, red blood cells and platelets, and the microgel assembly powder is assembled into hydrogel with excellent mechanical strength and adhesion performance. In addition, the powder can load bioactive molecule thrombin, activate multiple blood coagulation cascade reaction and promote formation of a fibrous protein network and blood clots, and through the synergistic effect of multiple hemostasis mechanisms, the powder shows rapid and effective hemostasis performance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a microgel assembly powder capable of rapidly stopping bleeding, and a preparation method and application thereof. Background Art

[0002] Currently, the four main categories of hemostatic materials commonly used in clinical practice are gauze, sponges, hydrogels, and powders. However, their performance limitations limit their hemostatic effectiveness. Gauze has become the most widely used hemostatic material due to its ease of operation and low cost. However, the high permeability of its fiber network can easily lead to rapid blood diffusion, causing the risk of secondary bleeding. Although sponge materials can achieve physical sealing through high fluid absorption and expansion, they are limited by their rigid structure and lack of interfacial adhesion, making them difficult to adapt to deep or irregular wounds. Hydrogel materials can fill complex wounds in situ due to their dynamic cross-linking properties, but the fluid shear stress generated by continuous bleeding from the wound will destroy interfacial adhesion, causing the material to structurally collapse under high-pressure bleeding scenarios. Powdered hemostatic materials have become an important option for preoperative emergency treatment and battlefield treatment due to their portability, easy storage, and good adaptability to irregular, incompressible wounds. However, traditional powder materials are limited by the weak interaction between powder particles. When they come into contact with blood, it is difficult to form a good physical barrier at the wound, which seriously restricts their application in heavy bleeding scenarios.

[0003] To overcome the above bottlenecks, self-gelling hemostatic powders have attracted much attention as an emerging system. When exposed to blood, they can trigger physical / chemical cross-linking reactions in situ to form large hydrogels, which have the dual functions of physical blocking and activation of coagulation factors, showing great potential for clinical transformation. Although such materials have shown significant advantages, their development still faces three major challenges: First, the complexity of the synthesis strategy. Existing systems mostly rely on multiple functional group modifications or multi-component blends, resulting in cumbersome and costly preparation processes; second, biocompatibility disputes. Potential risks such as chemical cross-linker residues and non-physiological degradation products have caused concerns about clinical application; third, the blood interference effect. The competitive adsorption of high-density red blood cells and platelets at the material-blood interface not only weakens the cohesion between the powders, but also forms a physical isolation layer that hinders material-tissue adhesion, ultimately resulting in a significant decrease in the effectiveness of self-gelling hemostatic powders in actual use. CN 115845121 A introduces the concept of microgel assembly to construct self-gelling hemostatic powder, but it requires multiple modifications of three original materials. At the same time, the synthesis process of its microgel powder also involves a light-triggered photopolymerization process, making its preparation method too complicated and costly.

[0004] Based on this, a microgel assembly powder with a simple preparation process and rapid hemostasis function is designed to achieve rapid hemostasis, which has broad application prospects. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a microgel assembly powder that can quickly stop bleeding.

[0006] Another object of the present invention is to provide a method for preparing a microgel assembly powder that can quickly stop bleeding. The microgel assembly hemostatic powder prepared by this method can quickly absorb water from the blood when it comes into contact with blood, concentrating the blood to achieve initial hemostasis. The chemical groups of the assembling agent then instantly form a cross-linked network between the microgel, red blood cells, and platelets through multiple physical / chemical reactions, and assemble them into a microgel assembly with excellent mechanical strength and adhesion strength to seal the wound and achieve secondary hemostasis. The microgel assembly powder exerts its hemostatic function by concentrating blood, aggregating red blood cells / platelets, and blocking the physical barrier. At the same time, the thrombin bioactive molecules loaded on the powder can activate multiple coagulation cascade reactions and promote the formation of fibrin networks and blood clots. Through the synergistic effect of multiple hemostatic mechanisms, the microgel assembly powder exhibits rapid and effective hemostatic performance.

[0007] Another object of the present invention is to provide an application of microgel assembly powder for rapid hemostasis.

[0008] To achieve the above objectives, the technical solutions provided by the present invention are:

[0009] A rapid hemostatic microgel assembly powder is composed of microgel powder and assembly agent powder; wherein the mass ratio of the microgel powder to the assembly agent powder is 1:10 to 10:1;

[0010] The microgel powder comprises a polymer powder that is double-modified by carboxymethylation and cross-linked with sodium trimetaphosphate, and the assembly agent powder is a polymer powder modified by polyphenol.

[0011] Preferably, the polymer in the carboxymethylation and sodium trimetaphosphate cross-linked dual-modified polymer is at least one selected from starch, chitosan, hyaluronic acid, cellulose, chitin, sodium alginate, and glycogen.

[0012] Preferably, the preparation method of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer is as follows: first, the polymer is carboxymethylated, and then the carboxymethylated product is cross-linked with sodium trimetaphosphate.

[0013] Preferably, the preparation method of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder is as follows:

[0014] 1) dissolving the polymer in an ethanol solution containing sodium hydroxide, adding chloroacetic acid under condensation reflux at 40-60° C. and reacting for 2-4 hours, filtering the product, washing, and drying to obtain a carboxymethylated modified product;

[0015] 2) dissolving the carboxymethyl modified product in water with a pH of 9 to 11, adding sodium trimetaphosphate to form an aqueous phase, and emulsifying the aqueous phase with a liquid paraffin oil phase containing an emulsifier for 0.5 to 3 hours, washing, and drying to obtain a carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder.

[0016] Preferably, the mass ratio of the chloroacetic acid to the polymer is 0.5-10:1, and the mass ratio of the sodium trimetaphosphate to the polymer is 0.01-0.5:1.

[0017] Preferably, the washing is washing with acetone, petroleum ether, 9% sodium chloride solution and ethanol solution.

[0018] Preferably, the emulsifier is Span 80;

[0019] The emulsification reaction is to preheat the oil phase to 50-70° C. and then add the water phase dropwise into the oil phase.

[0020] Preferably, the polymer in the polyphenol-modified polymer is at least one selected from gelatin, hyaluronic acid, polylysine, carboxymethyl chitosan, collagen, silk fibroin, and polyethyleneimine;

[0021] The polyphenol group is derived from a polyphenol compound, and the polyphenol compound is selected from at least one of gallic acid, ellagic acid, quercetin, catechin, vanillin, and tannic acid.

[0022] Preferably, the polyphenol-modified polymer powder is prepared as follows:

[0023] The polymer is dissolved in water to obtain a solution, the pH of the solution is adjusted to 8-9, a polyphenol compound is added, and air is introduced to react under a constant pH condition of 50-70°C. After the reaction is completed, the polyphenol-modified polymer powder is obtained after dialysis, freeze-drying and grinding.

[0024] Preferably, the concentration of the polymer in the solution is 0.2-5 wt%, and the concentration of the polyphenol compound in the solution is 2-30 wt%.

[0025] Preferably, the dialysis cut-off molecular weight is 1000 Da.

[0026] Preferably, the mass ratio of the microgel powder to the assembly agent powder is 1:3 to 3:1.

[0027] Preferably, the microgel powder further comprises thrombin, and the amount of thrombin added is 0.03 to 0.3 wt % of the mass of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder.

[0028] The particle sizes of the microgel powder and the assembly agent powder are both 1-1000 μm.

[0029] A method for preparing a microgel assembly powder for rapid hemostasis comprises the following steps:

[0030] (1) dissolving the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer in water, or adding thrombin solution, stirring evenly, standing, and freeze-drying to obtain microgel powder;

[0031] (2) Mixing the microgel powder and the assembly agent powder according to a certain ratio to obtain microgel assembly powder.

[0032] Preferably, the concentration of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer in the solution is 2-20 wt%;

[0033] Preferably, the thrombin concentration in the thrombin solution is 0.5-20 wt%.

[0034] Preferably, 10 to 100 μL of thrombin solution is added to every 1 g of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer.

[0035] Application of the above-mentioned rapid hemostatic microgel assembly powder in the preparation of hemostatic materials.

[0036] The microgel assembly powder prepared by the present invention will quickly absorb water from the blood and concentrate the blood after contact with blood. At the same time, the abundant ortho-phenolic hydroxyl groups on the assembly agent can instantly form a hydrogen bond network between the microgel, red blood cells and platelets, assembling them into a hydrogel with excellent mechanical strength. This hydrogen bonding effect will also occur between the material and the tissue, giving the material good tissue adhesion, thereby significantly improving the wound sealing effect. The microgel assembly powder exerts a hemostatic function by concentrating blood, aggregating red blood cells / platelets and blocking the physical barrier. At the same time, the thrombin bioactive molecules loaded on the powder can activate multiple coagulation cascade reactions and promote the formation of fibrin networks and blood clots. Through the synergistic effect of multiple hemostatic mechanisms, the microgel assembly powder exhibits rapid and effective hemostatic performance.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] 1. A microgel assembly powder was constructed. When the powder encounters liquid, it quickly absorbs the liquid and assembles into a large hydrogel with a tightly cross-linked network, which combines the advantages of hemostatic powder and hemostatic hydrogel.

[0039] 2. In order to give the material self-gelling properties, multiple physical and chemical interactions (hydrogen bonding, hydrophobic interaction, and electrostatic interaction) were designed between the assembler, microgel, and blood components (red blood cells / platelets), so that the microgel assembly powder has a gelation speed of seconds after contacting blood.

[0040] 3. The assembler of the microgel assembly can attract red blood cells through hydrogen bonding and hydrophobic interaction by relying on the polyphenol functional groups, and attract platelets through hydrogen bonding and endogenous activation, which can improve the physical and chemical properties of the microgel assembly in the blood and promote blood coagulation.

[0041] 4. The microgel assembly relies on the physical / chemical effects of its polyphenol groups on the microgel, red blood cells / platelets and tissue surfaces, and has excellent gelation, mechanical and adhesion properties, ultimately showing effective sealing properties and effectively sealing the wound.

[0042] 5. The microgel assembly powder exerts its hemostatic function by concentrating blood, aggregating red blood cells and platelets, and blocking a physical barrier. Simultaneously, the thrombin bioactive molecule loaded into the powder activates multiple coagulation cascades, promoting the formation of a fibrin network and blood clots. Through the synergistic action of multiple hemostatic mechanisms, the microgel assembly powder exhibits rapid and effective hemostatic properties.

[0043] 6. The preparation method of microgel assembly powder is simple and inexpensive. The powder form is also easy to store and has good biodegradability. It can be used as an excellent and multifunctional hemostatic material with good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Diagram of the assembly mechanism of microgel powder when it encounters blood.

[0045] Figure 2 This is the infrared spectrum of starch double-modified by carboxymethylation and sodium trimetaphosphate cross-linking.

[0046] Figure 3 This is the infrared spectrum of tannic acid modified carboxymethyl chitosan.

[0047] Figure 4 The actual objects of the assembly agent and microgel powder of Example 2 and the corresponding SEM images, a) is the assembly agent powder, b) is the microgel powder.

[0048] Figure 5 Graph showing the liquid absorption rate of the microgel assemblies of Examples 2, 6 and 7.

[0049] Figure 6 Figure 2 shows the compression modulus a) and adhesion strength b) of the microgel assemblies of Examples 2, 6 and 7.

[0050] Figure 7 Figure 2 shows the blood coagulation index of the microgel assembly in vitro, a) is the blood coagulation index, b) is the coagulation image.

[0051] Figure 8 Figure 3 shows the in vitro coagulation time of the microgel assembly.

[0052] Figure 9 Figure 2 shows the red blood cell adhesion test of the microgel assembly.

[0053] Figure 10 Figure 2 shows the hemostasis experiment of rat liver using microgel assembly. a) is the hemostasis image, b) is the amount of bleeding, and c) is the hemostasis time.

[0054] Figure 11 Figure 2 shows the hemostasis experiment of the rat femoral artery using the microgel assembly. a) is the hemostasis image, b) is the amount of bleeding, and c) is the hemostasis time.

[0055] Figure 12 Figure 2 shows the hemostasis experiment of rabbit liver using microgel assembly. a) is the hemostasis image, b) is the amount of bleeding, and c) is the hemostasis time.

[0056] Figure 13 Figure 2 is a degradation test diagram of the microgel assembly. Celox refers to a commercial chitosan hemostatic powder.

[0057] Figure 14 Figure 2 is the hemolysis test diagram of the microgel assembly. DETAILED DESCRIPTION

[0058] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0059] The preparation of the carboxymethylated and sodium trimetaphosphate cross-linked double-modified starch described below comprises the following steps:

[0060] Dissolve 2.5g of sodium hydroxide in 100mL of 90% ethanol, then add 2.5g of potato starch. Stir at 50°C under reflux for 30 minutes. Add 1.8g of chloroacetic acid to the reaction solution and allow to react at 50°C for 3 hours. The product is filtered, washed with anhydrous ethanol, and dried under vacuum at 50°C to obtain carboxymethylated starch (CES).

[0061] Dissolve 2g of carboxymethylated starch in 20mL of pure water, adjust the solution pH to 10, and add sodium trimetaphosphate (3% by weight of starch) to form an aqueous phase. Then, mix 100mL of liquid paraffin and 1g of Span 80 to form an oil phase. Stir the oil phase at 60°C for 30 minutes, then add the aqueous phase dropwise to the oil phase and react at 60°C for 6 hours. The product is then filtered and washed twice with acetone, petroleum ether, 9% sodium chloride solution, and ethanol, followed by vacuum drying at 50°C to obtain carboxymethylated and sodium trimetaphosphate cross-linked dual-modified starch (CLS), designated Material A.

[0062] The preparation of the tannic acid modified carboxymethyl chitosan described below comprises the following steps:

[0063] Dissolve 2g of carboxymethyl chitosan (CMCS) in deionized water at a concentration of 5wt%, and adjust the solution pH to 8.5. Take 0.2g of tannic acid and dissolve it in deionized water at a concentration of 20wt%, slowly drip the tannic acid solution into the carboxymethyl chitosan solution, and stir the reaction at 60℃ for 3h. During the reaction, keep the pH value constant at 8.5, and use a syringe pump to uniformly blow air into the system through a syringe at a rate of 2ml / min. After the reaction is completed, dialyze in deionized water at room temperature for five days using a dialysis bag with a molecular weight of 1000Da. Finally, freeze-dry the solution and grind it to obtain tannic acid-modified carboxymethyl chitosan powder (CCTA), which is recorded as material B.

[0064] Thrombin solution: 1000 U of thrombin is dissolved in 1 mL of normal saline. The weight of 1000 U of thrombin is 30 mg, that is, the mass fraction of thrombin in the thrombin solution is 3%.

[0065] Example 1

[0066] 1) Dissolve 1 g of carboxymethylated and sodium trimetaphosphate cross-linked dual-modified starch in 10 ml of normal saline, stir evenly, and place at 4°C overnight, then freeze-dry for 48 hours to obtain microgel powder.

[0067] 2) The obtained microgel powder was uniformly blended with material B as an assembling agent in a mass ratio of 1:1 to obtain a microgel assembly powder with a particle size of 50-300 μm, which was recorded as CLS-CCTA.

[0068] Example 2

[0069] 1) Dissolve 1 g of carboxymethylated and sodium trimetaphosphate cross-linked dual-modified starch in 10 ml of normal saline, add 10 μL of thrombin solution (0.3 mg of thrombin) (the amount of thrombin added is 0.03 wt % of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified starch), stir evenly, place in a 4°C environment and stand overnight, and then freeze-dry for 48 hours to obtain a microgel powder.

[0070] 2) The obtained microgel powder was uniformly blended with material B as an assembling agent in a mass ratio of 1:1 to obtain a microgel assembly powder, which was recorded as CLS-CCTA-T1.

[0071] Example 3

[0072] The difference from Example 2 is that the volume of the thrombin solution added in step 1) is 25 μL (thrombin is 0.75 mg), and the amount of thrombin added is 0.075 wt% of the carboxymethylated and sodium trimetaphosphate cross-linked double-modified starch, recorded as CLS-CCTA-T2.

[0073] Example 4

[0074] The difference from Example 2 is that the volume of the thrombin solution added in step 1) is 50 μL (thrombin is 1.5 mg), and the amount of thrombin added is 0.15 wt % of the carboxymethylated and sodium trimetaphosphate cross-linked double-modified starch, recorded as CLS-CCTA-T3.

[0075] Example 5

[0076] The difference from Example 2 is that the volume of the thrombin solution added in step 1) is 100 μL (thrombin is 3 mg), and the amount of thrombin added is 0.3 wt % of the carboxymethylated and sodium trimetaphosphate cross-linked double-modified starch, recorded as CLS-CCTA-T4.

[0077] Comparative Example 1

[0078] The difference from Example 2 is that in step 2), the tannic acid-modified carboxymethyl chitosan powder is not blended, which is recorded as CLS.

[0079] Example 6

[0080] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 2:1.

[0081] Example 7

[0082] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 1:2.

[0083] Example 8

[0084] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 3:1.

[0085] Example 9

[0086] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 1:3.

[0087] Example 10

[0088] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 10:1.

[0089] Example 11

[0090] The difference from Example 2 is that in step 2), the microgel powder and the material B as the assembly agent are uniformly blended in a mass ratio of 1:10.

[0091] Comparative Example 2

[0092] The difference from Example 2 is that the microgel powder in step 1) is unmodified potato starch.

[0093] Comparative Example 3

[0094] The difference from Example 2 is that the assembly agent powder in step 2) is unmodified carboxymethyl chitosan.

[0095] Test Case

[0096] (1) Actual powder and SEM image

[0097] The assembly mechanism of the microgel assembly powder of the present invention upon contact with blood is as follows: Figure 1 As shown, a single microgel powder cannot be assembled into a gel, but the microgel assembly powder can be assembled through hydrogen bonding after swelling. At the same time, hydrogen bonding and hydrophobic interaction are used to attract red blood cells and platelets to participate in the microgel assembly, filling the pores between the microgels to enhance the mechanical properties and wet adhesion of the gel. Figure 4 As shown, the assembly agent powder is light yellow, and the SEM image shows that its particle size is about 10-40 μm, as shown in 4a); the microgel powder is pure white, and the SEM image shows that it is a flake morphology with a particle size of about 100 μm, as shown in 4b).

[0098] (2) Infrared test chart

[0099] like Figure 2 As shown, compared with potato starch, CES has a -1 and 1416cm -1 The obvious enhancement of the carboxylate (-COO-) stretching peak at 1590 cm -1 and 1416cm -1 The carboxylate (-COO-) peak at 1003 cm -1 The POC phosphate bond peak at 802 cm-1 was significantly enhanced, proving that sodium trimetaphosphate was successfully cross-linked with starch, and a double-modified starch CLS with carboxymethylation and sodium trimetaphosphate cross-linking was obtained. -1 The appearance of the NH out-of-plane bending vibration peak at 1260 cm -1 The appearance of the NH in-plane bending vibration peak at α indicates that thrombin was successfully loaded on CLS.

[0100] like Figure 3 As shown in the FT-IR spectrum of CCTA, 1025 cm -1 、1056cm -1 and 3292cm -1The benzene ring peak of tannic acid appears and is significantly enhanced at 1582 cm -1 The increase in the intensity of the carboxylate (-COO-) absorption peak also confirmed that tannic acid was successfully grafted onto carboxymethyl chitosan.

[0101] (3) Liquid absorption, mechanical properties and adhesion properties

[0102] Test method:

[0103] Liquid absorption performance: Add the same mass of the powder to be tested to an excess of PBS solution. After 30 minutes, centrifuge the sample after absorption and weigh it. Each independent experiment is repeated four times. The liquid absorption rate is calculated according to formula (1):

[0104] Liquid absorption rate = (W1-W0) / W0(1)

[0105] The dry weight of the sample is W0, and the weight of the sample after PBS soaking is W1.

[0106] Mechanical Properties: Equal masses of powder were formed into cylinders (5 mm height, 5 mm diameter) by adding dropwise PBS solution. Compression tests were performed on each cylinder using a dynamic thermomechanical analyzer at a strain rate of 0.25 N / min, a maximum strain setting of 90%, and an initial force of 0.010 N. Stress-strain curves were obtained for each experimental group, and their specific compression moduli were calculated. Three replicates were prepared for each group.

[0107] Adhesion Performance: Equal amounts of each powder were added dropwise to form a gel. Two pieces of pigskin measuring 1 cm x 2 cm were bonded with a 0.5 cm x 1 cm gel. Tensile testing was performed using a dynamic thermomechanical analyzer (DMA, Q800DE, TA, USA). The strength at which the two pieces of pigskin completely separated was the adhesion strength of the powder gel. Four replicates were prepared for each group.

[0108] The liquid absorption performance, compression modulus and adhesion strength of the microgel assemblies of Example 2, Example 6 and Example 7 are shown in FIG. Figure 5 、 6 As shown, the microgel assembly exhibits a certain liquid absorption capacity due to the strong water absorption of the microgel powder; at the same time, the ortho-phenolic hydroxyl groups on the assembling agent produce hydrogen bonds with the groups on the microgel and tissue surface, giving the microgel assembly certain mechanical properties and adhesion properties.

[0109] (4) In vitro blood coagulation index and in vitro coagulation time

[0110] Test method:

[0111] In vitro blood coagulation index: Add 15 mg of the powdered sample to 200 μL of recalcified blood (10 μL of 0.2 M calcium chloride per 100 μL of blood) and mix thoroughly. After incubation at 37°C for 3 minutes, add 10 mL of deionized water to dissolve any uncoagulated blood clots. Measure the absorbance of the supernatant at 540 nm using a microplate reader. Set up three replicates for each group. The blood coagulation index is calculated according to formula (2):

[0112] BCI(%)=(Is-I0) / (Ic-I0)×100%(2)

[0113] Where Is represents the absorbance value of the sample, Ic represents the absorbance value of the positive control group (10 mL of deionized water directly dropped into 200 μL of recalcified blood), and I0 represents the absorbance value of the blank well plate.

[0114] In vitro clotting time: Add 15 mg of the powdered sample to 300 μL of recalcified blood (each 100 μL of blood contains 10 μL of 0.2 M calcium chloride) and mix thoroughly. Invert the tube after a period of time. If there is still no blood flow in the EP tube after inversion for 10 seconds, clotting is considered successful. The time at this time is recorded as the clotting time. Three parallel samples are set for each group.

[0115] Red blood cell adhesion: First, collect fresh sodium citrate anticoagulated sheep whole blood, centrifuge at 1200 rpm for 15 minutes, and collect the lower red blood cell suspension (RBC). Take 15 mg of the powder sample to be tested and incubate it with 100 μL of RBC suspension at 37°C for 5 minutes. Then wash the sample five times with DPBS to rinse off the non-adherent red blood cells. Then add 10 ml of deionized water to dissolve the red blood cells adhered to the sample. Use a microplate reader to measure the absorbance value at 540 nm. Set up three parallel samples for each group. The number of adhered red blood cells is calculated as shown in formula (3):

[0116]

[0117] OD sample Represents the absorbance value of the sample, OD control represents the absorbance value of the positive control group (10 mL of deionized water was directly added to 100 μL of red blood cell suspension), OD blank Represents the absorbance value of the blank well plate.

[0118] The in vitro blood coagulation index and in vitro coagulation time of the microgel assemblies of Examples 1 to 5 of the present invention are as follows: Figure 7 、 Figure 8As shown, relying on the coagulation-promoting effect of concentrated blood, aggregated red blood cells / platelets, and the activation of multiple coagulation cascades by thrombin, the microgel assembly group has a significantly lower blood coagulation index and a shorter in vitro coagulation time compared to the pure microgel group and the microgel assembly group without thrombin, showing good in vitro coagulation performance. Figure 9 As shown in the figure, the microgel assembly can adhere to more red blood cells compared with the pure microgel group and the microgel assembly group without thrombin, relying on the attraction of the microgel assembly agent to red blood cells and the capture of red blood cells by thrombin, which reveals one of the sources of its high coagulation performance. Figure 10 As shown in Figures 11 and 12, in three types of animal bleeding models (rat liver, rat femoral artery, and rabbit liver), the microgel assembly can effectively seal the wound and achieve complete hemostasis with minimal bleeding volume and the shortest hemostasis time, demonstrating its efficient and rapid hemostatic performance, which can be applied to a variety of complex bleeding environments.

[0119] (5) Degradation test of microgel assemblies

[0120] Test method: Weigh 0.05g of the powder sample to be tested and place it in 1.5ml of SBF simulated body fluid. After shaking at 37°C for 1, 3, 5, 7, and 14 days, centrifuge at 5000rpm for 10 minutes to collect the precipitate, dry it, and weigh the remaining mass. Set up three replicates on different days. The degradation rate is calculated according to formula (4):

[0121] Degradation rate (%) = (W0-W t ) / W0×100%(4)

[0122] Where W0 is the initial mass of the sample, W t is the remaining mass of the sample after degradation for different days.

[0123] Degradation test of microgel assemblies in Examples 1 and 2 Figure 13 The remaining mass of the microgel assembly of Example 1 on the seventh day was 30.25%, and the remaining mass of the microgel assembly of Example 2 on the seventh day was 28.46%, both of which have excellent biodegradability.

[0124] (6) Hemolysis test

[0125] Test method: After centrifuging the anticoagulated sheep blood at 4000rpm for 10 minutes, remove the upper plasma and obtain the lower layer of dark red red blood cells. Dilute the red blood cells with PBS solution to form a 10% RBC suspension. Mix 5mg of the powder sample to be tested with 0.8mL of PBS solution, then add 0.2mL of RBC solution, incubate on a shaker at 37℃ for 1h, and centrifuge at 3000rpm for 10min. Take 100μL of supernatant in a 96-well plate and use an enzyme reader to measure the absorbance value at a wavelength of 540nm. The control group is CLS and CLS-CCTA group, and three parallel samples are set up in each group. The hemolysis rate is calculated according to formula (5):

[0126]

[0127] OD sample The negative control is a mixed solution of 0.2 ml RBC and 0.8 ml PBC, and the positive control is a mixed solution of 0.2 ml RBC and 0.8 ml deionized water. sample Represents the absorbance value of the sample, OD negative Represents the absorbance value of the negative control group (10 mL PBS added to 100 μL of red blood cell suspension), OD positive Represents the absorbance value of the positive control group (10 mL of deionized water added to 100 μL of red blood cell suspension).

[0128] Hemolysis test of microgel assemblies in Examples 1 and 2 Figure 14 The hemolysis rates of the microgel assemblies of Example 1 and Example 2 were 2.86% and 2.39%, respectively, which were lower than the medical material safety threshold of 5%, demonstrating excellent blood compatibility.

[0129] Table 1

[0130]

[0131] As can be seen from the comparison of Examples 1 to 5 with Comparative Example 1 in Table 1, the microgel assembly group has a lower coagulation index, a shorter coagulation time, and a higher red blood cell adhesion rate than the single microgel group, proving that the addition of the assembly agent gives the microgel assembly self-gel properties. Compared with the single coagulation mechanism of concentrated blood in the microgel, the microgel assembly relies on the coagulation-promoting effect of concentrated blood, aggregated red blood cells / platelets, and thrombin to activate multiple coagulation cascades, showing better coagulation performance. As can be seen from the comparison of Examples 2, 3, 4, and 5 with Example 1 in Table 1, after the addition of thrombin, the microgel assembly loaded with thrombin has a better coagulation effect by activating multiple coagulation cascades and promoting the formation of fibrin networks and blood clots. Comparing Example 2, Example 3, Example 4, and Example 5 in Table 1, the higher the thrombin content, the better the coagulation performance of the microgel assembly. However, the coagulation performance of Example 4 and Example 5 is not much different, which proves that after the amount of thrombin solution added is increased from 50 μL to 100 μL, the effect of thrombin on the coagulation performance of the material becomes smaller.

[0132] Table 2

[0133]

[0134]

[0135] A comparison of Examples 2, 6, 7, 8, 9, 10, and 11 in Table 2 shows that the liquid absorption performance of the microgel assembly is primarily derived from the microgel component; higher microgel component content results in stronger liquid absorption performance. The mechanical and adhesive properties of the microgel assembly are primarily derived from the assembling agent component; higher assembling agent content results in stronger mechanical and adhesive properties. A comparison of Example 2 with Comparative Example 2 in Table 2 shows that carboxymethylated and sodium trimetaphosphate-crosslinked dual-modified starch as the microgel component significantly improves the liquid absorption performance of the microgel assembly. Furthermore, the introduction of carboxyl groups allows for stronger hydrogen bonding with the assembling agent, resulting in stronger mechanical and adhesive properties. A comparison of Example 2 with Comparative Example 3 in Table 2 shows that grafting tannic acid onto carboxymethyl chitosan significantly improves the mechanical and adhesive properties of the microgel assembly through hydrogen bonding between the tannic acid and the microgel.

[0136] The embodiments of the present invention are merely examples for the purpose of illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above embodiments, and it is not necessary or possible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims.

Claims

1. A microgel assembly powder for rapid hemostasis, characterized in that: It is composed of microgel powder and assembly agent powder; wherein the mass ratio of the microgel powder to the assembly agent powder is 1:10 to 10:1; The microgel powder comprises a polymer powder that is double-modified by carboxymethylation and cross-linked with sodium trimetaphosphate, and the assembly agent powder is a polymer powder modified by polyphenol.

2. The rapid hemostatic microgel assembly powder according to claim 1, characterized in that: The polymer in the carboxymethylation and sodium trimetaphosphate cross-linked dual-modified polymer is selected from at least one of starch, chitosan, hyaluronic acid, cellulose, chitin, sodium alginate, and glycogen.

3. The rapid hemostatic microgel assembly powder according to claim 2, characterized in that: The preparation method of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder is as follows: 1) dissolving the polymer in an ethanol solution containing sodium hydroxide, adding chloroacetic acid under condensation reflux at 40-60° C. and reacting for 2-4 hours, filtering the product, washing, and drying to obtain a carboxymethylated modified product; 2) dissolving the carboxymethyl modified product in water with a pH of 9 to 11, adding sodium trimetaphosphate to form an aqueous phase, and emulsifying the aqueous phase with a liquid paraffin oil phase containing an emulsifier for 0.5 to 3 hours, washing, and drying to obtain a carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder.

4. The rapid hemostatic microgel assembly powder according to claim 1, characterized in that: The polymer in the polyphenol-modified polymer is selected from at least one of gelatin, hyaluronic acid, polylysine, carboxymethyl chitosan, collagen, silk fibroin, and polyethyleneimine; The polyphenol group is derived from a polyphenol compound and is selected from at least one of gallic acid, ellagic acid, quercetin, catechin, vanillin, and tannic acid.

5. The rapid hemostatic microgel assembly powder according to claim 4, characterized in that: The preparation method of the polyphenol-modified polymer powder is as follows: The polymer is dissolved in water to obtain a solution, the pH of the solution is adjusted to 8-9, a polyphenol compound is added, and air is introduced to react under a constant pH condition of 50-70°C. After the reaction is completed, the polyphenol-modified polymer powder is obtained after dialysis, freeze-drying and grinding.

6. The rapid hemostatic microgel assembly powder according to claim 1, characterized in that: The mass ratio of the microgel powder to the assembly agent powder is 1:3 to 3:

1.

7. The rapid hemostatic microgel assembly powder according to claim 1, characterized in that: The microgel powder further comprises thrombin, and the amount of thrombin added is 0.03-0.3 wt% of the mass of the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer powder. The particle sizes of the microgel powder and the assembly agent powder are both 1-1000 μm.

8. The method for preparing the rapid hemostatic microgel assembly powder according to any one of claims 1 to 7, characterized in that: The steps include: (1) dissolving the carboxymethylated and sodium trimetaphosphate cross-linked dual-modified polymer in water, or adding thrombin solution, stirring evenly, standing, and freeze-drying to obtain microgel powder; (2) Mixing the microgel powder and the assembly agent powder according to a certain ratio to obtain microgel assembly powder.

9. The method for preparing the rapid hemostatic microgel assembly powder according to claim 8, characterized in that: The thrombin concentration in the thrombin solution is 0.5-20 wt %.

10. Use of the rapid hemostatic microgel assembly powder according to any one of claims 1 to 7 in the preparation of hemostatic materials.

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