Preparation method of adhesive recombinant collagen chitosan composite microspheres capable of efficiently stopping bleeding

Abstract

The invention discloses a preparation method of adhesive recombinant collagen chitosan composite microspheres capable of efficiently stopping bleeding. The preparation method comprises the following steps: 1, preparing chitosan grafted with gallic acid; 2, dispersing the recombinant collagen and the chitosan grafted with gallic acid into deionized water to obtain a water phase; 3, adding a surfactant into petroleum ether to obtain an oil phase; 4, injecting the oil phase into the water phase, emulsifying, and cooling to obtain an emulsion; 5, blending a cross-linking agent, water and absolute ethyl alcohol, and pre-cooling to obtain a stationary liquid; and 6, adding the stationary liquid into the emulsified liquid for curing, washing and freeze-drying to obtain the recombinant collagen chitosan composite microspheres. The prepared composite microspheres are excellent in performance and can be widely applied to the medical instrument industry as styptic powder, and an efficient, safe and reliable styptic material is provided for the medical field.

Description

Technical Field

[0001] The invention belongs to the technical field of hemostatic materials, and in particular relates to a method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding. Background Art

[0002] Uncontrollable bleeding refers to massive bleeding that cannot be effectively controlled by normal hemostasis mechanisms due to ruptured blood vessels caused by trauma, surgery or disease. This type of bleeding is common in patients with traumatic injuries, arterial ruptures or coagulation disorders, and in severe cases may lead to hemorrhagic shock, organ failure or even death. Traditional hemostatic methods such as pressure, suturing or drugs are sometimes difficult to stop bleeding quickly, especially in complex clinical settings. In order to meet this challenge, the development of fast and effective hemostatic materials and technologies, such as biological hemostatic agents and surgical colloids, has become an important direction of current medical research.

[0003] Current hemostatic materials mainly include gauze, thrombin, gelatin sponge, chitosan and absorbable hemostatic agents. These materials accelerate the hemostatic process through physical barriers, promoting the aggregation of coagulation factors or bioactive components. However, traditional hemostatic materials have some defects, such as slow hemostasis, limited effect on severe bleeding, and decreased effect in a wet environment. In addition, some materials may induce immune responses or produce toxicity, leading to tissue irritation or allergic reactions. The degradation rate of some absorbable materials is difficult to control, which may cause inflammation or delay wound healing. Therefore, the development of more efficient, safe and complex environment-adaptable hemostatic materials has become the focus of current research.

[0004] Most hemostatic microsphere particles are mainly made of materials such as chitosan and bentonite, and are widely used in emergency treatment and surgery. These particles achieve hemostasis by quickly absorbing water from the blood, promoting coagulation, and forming a physical barrier. Its advantages are that it is easy to use and stops bleeding quickly, and it is especially suitable for treating large areas or deep wounds. However, hemostatic particles also have some disadvantages. First, their hemostatic effect may be reduced in a wet environment. Second, some granular materials will produce residues, which may cause tissue irritation or allergic reactions. In addition, some particles lack adhesion and are easily washed away by blood, resulting in reduced effectiveness.

[0005] Therefore, in view of the above situation, there is an urgent need to develop a recombinant collagen chitosan composite microsphere that can adhere and effectively stop bleeding, so as to overcome the shortcomings in current practical applications. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing recombinant collagen chitosan composite microspheres that can adhere to and effectively stop bleeding, in view of the deficiencies of the above-mentioned prior art. The method utilizes chitosan grafted with gallic acid, which significantly improves the wet adhesion and antioxidant properties of the microspheres. The gallic acid on the surface of the composite microspheres can achieve strong adhesion to the wound and block the wound, and the recombinant collagen can accelerate the endogenous hemostasis process and improve the hemostatic effect of chitosan; through the synergistic effect of gallic acid, collagen and chitosan, the effect of rapid hemostasis can be achieved.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding, characterized in that it comprises the following steps:

[0008] Step 1, preparing chitosan grafted with gallic acid: dissolving chitosan in deionized water containing glacial acetic acid to obtain a chitosan solution; dispersing gallic acid in a buffer solution, activating it with (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide under the protection of nitrogen, and then adding it to the chitosan solution, and freeze-drying to obtain chitosan grafted with gallic acid;

[0009] Step 2: Dispersing the recombinant collagen and chitosan grafted with gallic acid in deionized water, stirring evenly to obtain an aqueous phase;

[0010] Step 3, adding the surfactant into petroleum ether and stirring evenly to obtain an oil phase;

[0011] Step 4: inject the oil phase in step 3 into the water phase in step 2, and emulsify and stir by a homogenizer. After stirring, lower the system temperature to obtain an emulsion;

[0012] Step 5: Mix the cross-linking agent, water and anhydrous ethanol and pre-cool to obtain a stationary solution;

[0013] Step 6: Add the precooled fixative in step 5 to the emulsion in step 4 for solidification, then wash the solidified microspheres with deionized water, freeze-dry, and obtain recombinant collagen chitosan composite microspheres of uniform size that can adhere and effectively stop bleeding.

[0014] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres that can adhere to high-efficiency hemostasis is characterized in that the mass concentration of glacial acetic acid in the chitosan solution in step one is 0.25% to 3%, the mass concentration of chitosan is 0.1% to 5%, the molecular weight of chitosan is 30,000 to 500,000 Da, the amount of gallic acid is 0.1 to 10 times the amount of chitosan, and the amount of gallic acid, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide The ratio of the amount of substance is 1:1:1; the buffer solution is a phosphate buffer solution, a carbonate buffer solution or a 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

[0015] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is characterized in that the mass percentage of recombinant collagen in the aqueous phase in step 2 is 0.01% to 25%, and the mass percentage of chitosan grafted with gallic acid is 0.1% to 5%.

[0016] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding is characterized in that the recombinant collagen in step 2 is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

[0017] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is characterized in that the mass percentage of the surfactant in the oil phase in step three is 0.1% to 20%.

[0018] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is characterized in that the surfactant in step three is Tween-60, Tween-80, Span 60 or Span 80.

[0019] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is characterized in that the petroleum ether in step three is of pharmaceutical grade with a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

[0020] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres that can adhere to high-efficiency hemostasis is characterized in that the volume ratio of the water phase to the oil phase in step four is 1: (3 to 10), the speed of the homogenizer is 1000rpm to 15000rpm, and the system temperature is reduced to -20°C to -80°C after stirring.

[0021] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres that can adhere to high-efficiency hemostasis is characterized in that the cross-linking agent in step five is glutaraldehyde, genipin, tetrakishydroxymethylphosphonium chloride (THPC) or four-arm PEG succinimidyl carbonate, the volume of anhydrous ethanol is 5 to 50 times the volume of deionized water, the mass concentration of the cross-linking agent in the fixing solution is 0.2% to 3%, and the pre-cooling temperature is -20°C to -80°C.

[0022] The above-mentioned method for preparing recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is characterized in that the volume of the fixing liquid in step six is ​​0.08 to 0.45 times the volume of the emulsion, and the average particle size of the recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding is 40 μm to 600 μm.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The present invention creatively combines chitosan, gallic acid and recombinant collagen to prepare composite microspheres with excellent hemostatic properties and tissue repair functions. Its multiple advantages make it have important application value and development potential in the medical field.

[0025] 2. Improve wet adhesion and antioxidant properties: The present invention significantly improves the wet adhesion and antioxidant properties of the microspheres by grafting chitosan with gallic acid. This enhanced wet adhesion enables the microspheres to adhere more firmly to the moist wound surface to prevent secondary bleeding. At the same time, the improvement of antioxidant properties helps to remove free radicals around the wound surface, reduce the damage of oxidative stress to tissues, and promote wound healing;

[0026] Strong wound adhesion and hemostasis: Gallic acid on the surface of the composite microspheres has excellent adhesion properties, which can achieve strong adhesion to the wound and quickly block the wound. This immediate sealing effect not only helps to stop bleeding, but also forms a protective barrier to prevent the invasion of external pathogens and reduce the risk of infection;

[0027] Accelerate the endogenous hemostasis process: The introduction of recombinant collagen can accelerate the endogenous hemostasis mechanism. As an important matrix for platelet adhesion and aggregation, collagen can promote platelet aggregation and activation at the wound, enhance the coagulation effect, and improve the overall hemostasis efficiency;

[0028] Synergistic effect to achieve rapid hemostasis: The synergistic effect of gallic acid, collagen and chitosan makes the microspheres have multiple hemostatic mechanisms. Gallic acid provides strong adhesion, collagen promotes platelet function, and chitosan itself has hemostatic and antibacterial properties. The combination of the three can achieve rapid and efficient hemostasis.

[0029] Advantages of three-dimensional network structure: The microspheres prepared by emulsification cross-linking technology have a three-dimensional network structure, which increases the specific surface area of ​​the microspheres and is conducive to interaction with biological tissues. At the same time, this structure gives the microspheres good mechanical strength and stability, ensuring that they remain functional during the application process;

[0030] Promote tissue repair and regeneration: Chitosan and recombinant collagen have the functions of immunomodulation and promoting tissue repair. They can promote cell proliferation and migration, accelerate the formation of new tissue, and facilitate rapid wound healing and functional recovery;

[0031] Antibacterial properties: Chitosan has natural antibacterial properties, which can inhibit the growth of a variety of pathogenic microorganisms and reduce the incidence of infection. This is particularly important for the treatment of open wounds and can improve the safety of treatment;

[0032] Biocompatibility and degradability: The materials used are natural or biosynthetic substances with good biocompatibility, which can be gradually degraded in the body, avoiding secondary surgery for removal and reducing the burden on patients;

[0033] The preparation process is highly controllable: the emulsification cross-linking technology is mature, and the size, morphology and function of the microspheres can be precisely controlled by adjusting the process parameters, which is conducive to large-scale production and quality control;

[0034] Wide range of applications: The composite microspheres are not only suitable for hemostasis in surgical operations, but can also be used in trauma emergency care, burn care and other fields, and have broad market prospects;

[0035] Reduce treatment costs: Using cheap and readily available materials such as chitosan and gallic acid, combined with efficient preparation processes, is expected to reduce product costs, making it more cost-effective and benefiting more patients;

[0036] Environmentally friendly: The use of green materials and processes reduces the impact on the environment and conforms to the concept of sustainable development.

[0037] 3. The composite microspheres prepared by the present invention have excellent biocompatibility and strong hemostatic performance, showing broad application prospects. In addition, the composite microspheres also have good dispersibility and uniform size, thanks to the sophisticated preparation process and strict quality control. The characteristics of uniform and controllable size enable the microspheres to play a more precise role in the application and improve the hemostatic effect. In addition, the preparation method of the present invention is simple, environmentally friendly, low in production cost, and has broad application prospects.

[0038] 4. The entire preparation process of the present invention is simple, fast and easy to operate. The prepared composite microspheres have excellent performance and can be widely used in the medical device industry as hemostatic powder, providing an efficient, safe and reliable hemostatic material for the medical field.

[0039] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a SEM image of the recombinant collagen chitosan composite microspheres prepared in Example 1 of the present invention.

[0041] Figure 2 This is the hemostatic effect of the recombinant collagen chitosan composite microspheres prepared in Example 1 of the present invention.

[0042] Figure 3 This is the MTT test result of the recombinant collagen chitosan composite microspheres prepared in Example 1 of the present invention.

[0043] Figure 4 The live and dead cell staining experimental results of the recombinant collagen chitosan composite microspheres prepared in Example 1 of the present invention.

[0044] Figure 5 The red blood cell and platelet adhesion rates of the recombinant collagen chitosan composite microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0046] Example 1

[0047] This embodiment provides a method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding, which specifically comprises the following steps:

[0048] Step 1, preparation of chitosan grafted with gallic acid: dissolve 2g chitosan (100000Da, the molar mass of chitosan is calculated as monomer) in 200mL deionized water containing 1wt% glacial acetic acid to obtain a chitosan solution; disperse 2.4g gallic acid in 40mL MES buffer solution, and under the protection of nitrogen, activate with 2.19g (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and 1.63g N-hydroxysuccinimide (NHS) and then add them to the chitosan solution to obtain chitosan grafted with gallic acid;

[0049] Step 2: Disperse 1 g of recombinant collagen and 2 g of chitosan grafted with gallic acid in 100 mL of deionized water, stir evenly, and obtain a water phase with uniform concentration; the recombinant collagen is a macromolecular type I recombinant collagen, and the recombinant collagen is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, which is repeated multiple times, wherein the short amino acid sequence is GAPGAPGSQGAPGL, and the number of repetitions is 52 times. The collagen has good water solubility and a molecular weight of 78862 Da, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0050] Step 3, add 25 g of Tween-80 into 400 mL of petroleum ether (boiling range 60° C. to 90° C.) and stir evenly to obtain an oil phase;

[0051] Step 4: inject the oil phase in step 3 into the water phase in step 2, and emulsify and stir by a homogenizer. After stirring, lower the system temperature to -40°C to obtain an emulsion; the speed of the homogenizer is 10000 rpm;

[0052] Step 5: 0.5 g of genipin, 5 mL of deionized water and 35 mL of anhydrous ethanol were mixed and precooled to -40°C to obtain a stationary solution;

[0053] Step 6: Add the precooled fixative in step 5 to the emulsion in step 4 for solidification, then wash the solidified microspheres with deionized water, freeze-dry, and obtain recombinant collagen chitosan composite microspheres of uniform size that can adhere and effectively stop bleeding.

[0054] The average particle size of the recombinant collagen chitosan composite microspheres prepared in this example is about 100 μm.

[0055] Example 2

[0056] This embodiment provides a method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding, which specifically comprises the following steps:

[0057] Step 1, preparation of chitosan grafted with gallic acid: dissolving 22.7 g chitosan (500000 Da) in 450 mL deionized water containing 0.25 wt% glacial acetic acid to obtain a chitosan solution; dispersing 2.4 g gallic acid in 30 mL phosphate buffer solution, under the protection of nitrogen, activating with 2.19 g (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and 1.63 g N-hydroxysuccinimide (NHS), and then adding them to the chitosan solution to obtain chitosan grafted with gallic acid;

[0058] Step 2: Disperse 0.01 g of recombinant collagen and 0.1 g of chitosan grafted with gallic acid in 100 mL of deionized water, stir evenly, and obtain a water phase with uniform concentration; the recombinant collagen is a macromolecular type III recombinant collagen, which is composed of a short amino acid sequence from natural human type III collagen as a repeating unit and is repeated multiple times, wherein the short amino acid sequence (GERGAPGFRGPAGPNGIPGEKGPAGER) is repeated 30 times, and the collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0059] Step 3, add 0.3 g of Tween-60 into 300 mL of petroleum ether (boiling range 30°C to 60°C) and stir evenly to obtain an oil phase;

[0060] Step 4: inject the oil phase in step 3 into the water phase in step 2, and emulsify and stir the mixture using a homogenizer. After stirring, lower the system temperature to -80°C to obtain an emulsion. The speed of the homogenizer is 15000 rpm.

[0061] Step 5: Mix 5.4 g of glutaraldehyde (or four-arm PEG succinimidyl carbonate), 30 mL of water and 150 mL of anhydrous ethanol and precool to -80°C to obtain a stationary phase;

[0062] Step 6: Add the precooled fixative in step 5 to the emulsion in step 4 for solidification, then wash the solidified microspheres with deionized water, freeze-dry, and obtain recombinant collagen chitosan composite microspheres of uniform size that can adhere and effectively stop bleeding.

[0063] The average particle size of the recombinant collagen chitosan composite microspheres prepared in this example is about 600 μm.

[0064] Example 3

[0065] This embodiment provides a method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding, which specifically comprises the following steps:

[0066] Step 1, preparation of chitosan grafted with gallic acid: dissolving 1.9 g chitosan (3000 Da) in 1900 mL deionized water containing 3 wt% glacial acetic acid to obtain a chitosan solution; dispersing 20 g gallic acid in 100 mL carbonate buffer solution, under the protection of nitrogen, activating with 18.24 g (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and 13.61 g N-hydroxysuccinimide (NHS), and then adding them to the chitosan solution to obtain chitosan grafted with gallic acid;

[0067] Step 2: Disperse 25g of recombinant collagen and 5g of chitosan grafted with gallic acid in 100mL of deionized water, stir evenly, and obtain a water phase with uniform concentration; the recombinant collagen is a macromolecular type IV recombinant collagen, which is composed of the full-length α1 chain sequence of type IV human collagen, has good water solubility, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0068] Step 3: Add 200 g of Span 60 (or Span 80) into 1000 mL of petroleum ether (boiling range 90° C. to 120° C.) and stir to obtain an oil phase;

[0069] Step 4: inject the oil phase in step 3 into the water phase in step 2, and emulsify and stir by a homogenizer. After stirring, lower the system temperature to -20°C to obtain an emulsion; the speed of the homogenizer is 1000 rpm;

[0070] Step 5: 0.51 g of tetrakishydroxymethylphosphonium chloride (THPC), 5 mL of water and 250 mL of anhydrous ethanol were mixed and precooled to -20°C to obtain a stationary solution;

[0071] Step 6: Add the precooled fixative in step 5 to the emulsion in step 4 for solidification, then wash the solidified microspheres with deionized water, freeze-dry, and obtain recombinant collagen chitosan composite microspheres of uniform size that can adhere and effectively stop bleeding.

[0072] The average particle size of the recombinant collagen chitosan composite microspheres prepared in this example is about 40 μm.

[0073] Comparative Example

[0074] In this comparative example, no recombinant collagen was added, and the other preparation processes were the same as those in Example 1.

[0075] Taking the recombinant collagen chitosan composite microspheres prepared in Example 1 as an example, various properties of the recombinant collagen chitosan composite microspheres with high adhesion and hemostasis prepared by the method of the present invention were measured.

[0076] 1. Morphology measurement

[0077] The recombinant collagen chitosan composite microspheres of Example 1 were photographed using a field emission scanning electron microscope (GeminiSEM 300, Car1 Zeiss, Germany). Figure 1 As shown, the results showed that the diameter of the microspheres was approximately 100 μm.

[0078] 2. Hemostasis

[0079] The hemostatic ability of recombinant collagen chitosan composite microspheres was investigated through a rat liver hemostasis experiment. Figure 2As shown, SD rats were anesthetized and part of the liver was removed with scissors. Then, hemostatic microspheres were sprayed on the bleeding site. The bleeding area in the microsphere group was significantly smaller than that in the blank group. The blood loss of the blank group rats was 450mg. In contrast, the rats in the microsphere group showed significantly less blood loss, which was 110mg respectively.

[0080] 3. Biocompatibility

[0081] The extract was prepared according to the method recommended by GB / T 16886.5-2017 for in vitro cell compatibility testing using MTT and live-dead staining methods. Figure 3 and Figure 4 The results showed that the extract of the recombinant collagen chitosan composite microspheres prepared in Example 1 could effectively promote the proliferation of mouse fibroblasts (L929), and the 24h cell proliferation rate was 129.5%, indicating that it had good biocompatibility.

[0082] 4. Red blood cell adhesion rate and platelet adhesion rate

[0083] Fresh citrate whole blood of SD rats was centrifuged at 400g for 10min to obtain red blood cell suspension (diluted with PBS, 10% v / v PBS, 10% v / v) and platelet-rich plasma (PRP). When conducting the RBCs adhesion test, 100μL was dropped on 10mg recombinant collagen chitosan composite microspheres, incubated at 37℃ for 30min, and then rinsed with PBS to remove non-adherent RBCs, and then deionized water (4mL) was added to dissolve the attached RBCs, and the absorbance value of the supernatant at a wavelength of 540nm was measured. Compared with the control example, the recombinant collagen antibacterial hemostatic powder significantly increased the red blood cell adhesion rate, and its red blood cell adhesion rate was 79.2% ( Figure 5 ).

[0084] 40 μL PRP was dropped on 10 mg sample and then incubated at 37°C for 5 min. Unattached platelets were removed by rinsing with PBS, and then the sample was immersed in 1 mL 1% Triton X-100 to completely dissolve the attached platelets (37°C, 1 h). The released lactate dehydrogenase (LDH) content was measured using an LDH kit. The results showed that recombinant collagen significantly increased the platelet adhesion rate of hemostatic powder, and the platelet adhesion rate of recombinant collagen chitosan composite microspheres was 87.1% ( Figure 5 ).

[0085] The above-mentioned technologies not specifically mentioned are all referred to the prior art.

[0086] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding, characterized in that: The following steps are involved: Step 1, preparing chitosan grafted with gallic acid: dissolving chitosan in deionized water containing glacial acetic acid to obtain a chitosan solution; dispersing gallic acid in a buffer solution, activating it with (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide under the protection of nitrogen, and then adding it to the chitosan solution, and freeze-drying to obtain chitosan grafted with gallic acid; Step 2: Dispersing the recombinant collagen and chitosan grafted with gallic acid in deionized water, stirring evenly to obtain an aqueous phase; Step 3, adding the surfactant into petroleum ether and stirring evenly to obtain an oil phase; Step 4: inject the oil phase in step 3 into the water phase in step 2, and emulsify and stir by a homogenizer. After stirring, lower the system temperature to obtain an emulsion; Step 5: Mix the cross-linking agent, water and anhydrous ethanol and pre-cool to obtain a stationary solution; Step 6: Add the precooled fixative in step 5 to the emulsion in step 4 for solidification, then wash the solidified microspheres with deionized water, freeze-dry, and obtain recombinant collagen chitosan composite microspheres of uniform size that can adhere and effectively stop bleeding.

2. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: In the chitosan solution in step 1, the mass concentration of glacial acetic acid is 0.25% to 3%, the mass concentration of chitosan is 0.1% to 5%, the molecular weight of chitosan is 30000 to 500000 Da, the amount of gallic acid is 0.1 to 10 times the amount of chitosan, and the ratio of the amount of gallic acid, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide is 1:1:1; the buffer solution is a phosphate buffer solution, a carbonate buffer solution or a 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

3. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: In step 2, the mass percentage of the recombinant collagen in the aqueous phase is 0.01% to 25%, and the mass percentage of the chitosan grafted with gallic acid is 0.1% to 5%.

4. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: The recombinant collagen described in step 2 is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen.

5. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: The mass percentage of the surfactant in the oil phase in step three is 0.1% to 20%.

6. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: The surfactant in step 3 is Tween-60, Tween-80, Span 60 or Span 80.

7. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: The petroleum ether described in step 3 is of pharmaceutical grade, with a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

8. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: In step 4, the volume ratio of the water phase to the oil phase is 1:(3-10), the speed of the homogenizer is 1000rpm-15000rpm, and the system temperature is lowered to -20°C-80°C after the stirring is completed.

9. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and efficiently stopping bleeding according to claim 1, characterized in that: The cross-linking agent in step 5 is glutaraldehyde, genipin, tetrakis(hydroxymethyl)phosphine chloride (THPC) or four-arm PEG succinimidyl carbonate, the volume of anhydrous ethanol is 5 to 50 times the volume of deionized water, the mass concentration of the cross-linking agent in the fixing solution is 0.2% to 3%, and the pre-cooling temperature is -20°C to -80°C.

10. The method for preparing the recombinant collagen chitosan composite microspheres capable of adhering and effectively stopping bleeding according to claim 1, characterized in that: In step six, the volume of the fixing liquid is 0.08 to 0.45 times the volume of the emulsion, and the average particle size of the said highly effective hemostatic recombinant collagen chitosan composite microspheres is 40 μm to 600 μm.

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