Efficient gelatin hemostatic microsphere as well as preparation method and application thereof

Abstract

The invention provides efficient gelatin hemostatic microspheres as well as a preparation method and application thereof, the preparation method comprises the following steps: mixing a gelatin solution with a tannic acid solution, and uniformly stirring to obtain a tannic acid modified gelatin aqueous solution; uniformly mixing span-80 with liquid paraffin to obtain an oil phase; adding the tannic acid modified gelatin aqueous solution into an oil phase, and uniformly stirring to obtain a mixed solution; the mixed solution is subjected to a cross-linking reaction under the ice bath condition, after the cross-linking reaction is completed, a reaction solution and acetone are mixed, liquid paraffin is removed after standing, and the tannic acid modified hemostatic microspheres are obtained after drying; mixing the tannic acid modified hemostatic microspheres with an adsorbent, and dehydrating to obtain the gelatin hemostatic microspheres. The prepared gelatin hemostatic microspheres can be attached to the surface of tissue and have the effects of high water absorption and rapid hemostasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an efficient gelatin hemostatic microsphere, a preparation method thereof and an application thereof. Background Art

[0002] At present, massive bleeding caused by traffic accidents, natural disasters and other reasons is still an important cause of death and disability. Many patients are critically ill due to excessive blood loss before being sent to the hospital. Currently, the hemostatic products used clinically mainly include chitosan hemostatic powder, gelatin sponge, and fibrin glue. These products have good hemostatic effects on superficial wounds on the body surface. However, for severe internal bleeding, the adhesiveness and biocompatibility of these products are poor, and it is difficult to stop bleeding quickly. Therefore, hemostatic microspheres have shown great clinical application value. First of all, the powder preparation composed of microspheres is not limited by the size, shape or position of the wound and is suitable for the emergency treatment of irregular wounds. At present, a jet-type hemostatic foam has been developed, which is convenient to use and carry, but it still cannot effectively treat deep bleeding such as internal organs. Microspheres have a large specific surface area and good fluidity, can effectively treat bleeding sites such as internal organs and the inner wall of the peritoneum, and are convenient for storage, transportation, have high safety and are easy to remove, making them more suitable for some specific application scenarios, such as wars, major natural disasters and traffic accidents.

[0003] Due to its excellent biocompatibility and degradability, gelatin microspheres have a wide range of applications in the biomedical field. For example, drug delivery systems, tissue engineering scaffolds, and cell proliferation and culture. In addition, gelatin itself is also a very good hemostatic material and has been widely studied by researchers.

[0004] CN108310448A discloses a preparation method of a fluid gelatin hemostatic material. In this preparation method, a cross-linking agent EDC or TG enzyme that does not participate in the reaction is used. After cross-linking treatment, the gelatin is fully swollen in water, and then crushed into micron-sized particles. Then, through ultrasonic-centrifugal cyclic cleaning operations, the gelatin cross-linking agent can be well removed, achieving a good purification effect of the hemostatic material and effectively avoiding the residue and toxicity of irritating substances. The prepared gelatin hemostatic material has appropriate fluidity and good hemostatic effect, the degradation time is controllable, it can be selectively designed according to the hemostatic position, and it has good biological safety.

[0005] CN109432488A discloses a method for preparing chitosan / gelatin composite hemostatic microspheres, firstly dissolving chitosan and gelatin in an acid solution to obtain a water phase with a total mass fraction of 11%-13%, then preparing an oil phase using liquid paraffin and span-80 emulsifier, dripping the water phase into the oil phase, emulsifying it uniformly under the action of shear force, then cooling and crosslinking, and finally solid-liquid separation, washing and drying. The chitosan / gelatin composite hemostatic microspheres make full use of the good sphericity of gelatin materials, make up for the defect that chitosan is difficult to sphericize, the prepared microspheres have uniform particle size distribution and regular spherical shape, are not affected by the size and position of the wound surface when used for hemostasis, have good hemostatic effect on irregular wounds, and also have good water absorption, can quickly absorb blood and adhere to the wound surface when in contact with the wound, increase the local blood concentration, and accelerate the hemostasis speed.

[0006] CN108187129A discloses an absorbable gelatin hemostatic powder and a preparation method thereof, wherein the gelatin hemostatic powder has a BET specific surface area of ​​30 m 2 / g~70m 2 / g, and its preparation process includes the steps of cross-linking gelatin, water swelling, mechanical crushing, freeze drying and secondary crushing. The hemostatic powder can be well attached to the wound surface, quickly absorb water in the blood, and achieve a good hemostatic effect. Moreover, the preparation method is simple in process, and further, by adopting a cross-linking agent that does not participate in the reaction, and performing mechanical crushing, ultrasonic-centrifugal circulation cleaning or ethanol aqueous solution cleaning operations after gelatin cross-linking and sufficient swelling, the cross-linking agent is effectively removed, so that there is no residue of the cross-linking agent in the hemostatic powder, avoiding the irritation of the cross-linking agent to the organism, improving the safety of the product, and having a good promotion and application value.

[0007] It is known from the existing technology that natural gelatin exhibits weak adhesion strength, and the surface of organs such as viscera often affects the adhesion of hemostatic materials to tissues due to the presence of body fluids and blood. For gelatin microspheres, this is mainly reflected in the low water absorption rate, which cannot quickly absorb water from the tissue surface and blood, thereby weakening the hemostatic effect.

[0008] Therefore, developing a gelatin microsphere powder preparation with high water absorption and rapid in vitro coagulation ability is a research focus in this field. Summary of the invention

[0009] In view of the deficiencies of the prior art, the object of the present invention is to provide a highly efficient gelatin hemostatic microsphere and a preparation method and application thereof, wherein the hemostatic microsphere has high water absorption rate and high in vitro rapid coagulation ability.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for preparing an efficient gelatin hemostatic microsphere, and the preparation method includes the following steps:

[0012] (1) Mix a gelatin solution and a tannic acid solution, and stir evenly to obtain a tannic acid-modified gelatin aqueous solution;

[0013] (2) Mix Span-80 and liquid paraffin evenly to obtain an oil phase; add the tannic acid-modified gelatin aqueous solution to the oil phase and stir evenly to obtain a mixture;

[0014] (3) Carry out a cross-linking reaction on the mixture under ice bath conditions. After completion, mix the reaction solution with acetone, let it stand, remove the liquid paraffin, and dry to obtain tannic acid-modified hemostatic microspheres;

[0015] (4) Mix the tannic acid-modified hemostatic microspheres with an adsorbent and then dehydrate to obtain the gelatin hemostatic microspheres.

[0016] The present invention modifies gelatin through a Michael addition reaction, and adopts a water-in-oil emulsion cross-linking method to prepare solvent-free tannic acid-modified gelatin hemostatic microspheres with high water absorption and rapid hemostasis. Introducing a catechol structure into gelatin can enable the hemostatic dressing to better adhere to the tissue surface, improve water absorption, and further improve the hemostasis efficiency.

[0017] Preferably, the mass percentage content of gelatin in the tannic acid-modified gelatin aqueous solution is 10-30%, for example, it can be 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, etc.

[0018] Preferably, the mass percentage content of tannic acid in the tannic acid-modified gelatin aqueous solution is 0.2-0.4%, for example, it can be 0.25%, 0.3%, 0.35%, etc.

[0019] Preferably, the pH value of the gelatin solution is ≥9.5, for example, it can be 9.8, 10, 10.5, 11, 11.5, 12, etc.

[0020] Preferably, the pH value of the gelatin solution is adjusted with sodium hydroxide.

[0021] Preferably, in step (1), the stirring speed is 200-300 rpm (for example, it can be 220 rpm, 240 rpm, 260 rpm, 280 rpm, etc.), the time is 30-40 min (for example, it can be 32 min, 34 min, 36 min, 38 min, etc.), and the temperature is 55-65 °C (for example, it can be 56 °C, 58 °C, 60 °C, 62 °C, 64 °C, etc.).

[0022] Preferably, the volume ratio of the liquid paraffin to Span-80 is 1:(45 - 55), and for example, it can be 1:46, 1:48, 1:50, 1:52, 1:54, etc.

[0023] Preferably, the volume ratio of the tannic acid-modified gelatin aqueous solution to the oil phase is 1:(3 - 5), and for example, it can be 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0024] Preferably, in step (2), the stirring speed is 600 - 800 rpm (for example, it can be 650 rpm, 700 rpm, 750 rpm, etc.), the time is 30 - 60 min (for example, it can be 35 min, 40 min, 45 min, 50 min, 55 min, etc.), and the temperature is 55 - 65 °C (for example, it can be 56 °C, 58 °C, 60 °C, 62 °C, 64 °C, etc.).

[0025] Preferably, the cross-linking agent used in the cross-linking reaction is an aqueous glutaraldehyde solution.

[0026] Preferably, the mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 20 - 30%, and for example, it can be 22%, 24%, 26%, 28%, etc.

[0027] Preferably, the stirring speed during the cross-linking reaction is 600 - 800 rpm (for example, it can be 650 rpm, 700 rpm, 750 rpm, etc.), the time is 20 - 40 min (for example, it can be 25 min, 30 min, 35 min, etc.), and the temperature is 0 - 4 °C (for example, it can be 1 °C, 2 °C, 3 °C, etc.).

[0028] Preferably, the volume ratio of the reaction solution to acetone is 1:(0.8 - 1.2), and for example, it can be 1:0.9, 1:1, 1:1.1, etc.

[0029] Preferably, the standing time is 5 - 20 min, and for example, it can be 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, etc.

[0030] Preferably, the drying temperature is 20 - 30 °C (for example, it can be 22 °C, 24 °C, 26 °C, 28 °C, etc.), and the time is 12 - 24 h (for example, it can be 14 h, 16 h, 18 h, 20 h, 22 h, etc.).

[0031] Preferably, the adsorbent includes a chitosan quaternary ammonium salt solution or a calcium chloride solution.

[0032] Preferably, the mass percentage content of the chitosan quaternary ammonium salt in the chitosan quaternary ammonium salt solution is 0.05 - 0.3%, for example, it can be 0.1%, 0.15%, 0.2%, 0.25%, etc.

[0033] Preferably, the concentration of calcium chloride in the calcium chloride solution is 0.05 - 1 mol / L, for example, it can be 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, etc.

[0034] Preferably, the mass - to - volume ratio of the tannic acid - modified hemostatic microspheres to the adsorbent is 1:(0.1 - 4), for example, it can be...

[0035] Preferably, the preparation method includes the following steps:

[0036] (1) Mix a gelatin solution with a pH value ≥ 9.5 and a tannic acid solution, and stir at 55 - 65 °C and 200 - 300 rpm for 30 - 40 min until uniform to obtain a tannic acid - modified gelatin aqueous solution;

[0037] The mass percentage content of gelatin in the tannic acid - modified gelatin aqueous solution is 10 - 30%, and the mass percentage content of tannic acid is 0.2 - 0.4%;

[0038] (2) Mix Span - 80 and liquid paraffin in a volume ratio of 1:(45 - 55) to obtain an oil phase; add the tannic acid - modified gelatin aqueous solution to the oil phase in a volume ratio of 1:(3 - 5), and stir at 55 - 65 °C and 600 - 800 rpm for 30 - 60 min until uniform to obtain a mixture;

[0039] (3) Carry out a cross - linking reaction on the mixture with an aqueous glutaraldehyde solution with a mass percentage content of 20 - 30% under ice - bath conditions, stir at 0 - 4 °C and 600 - 800 rpm for 20 - 40 min. After cross - linking, mix the reaction solution with acetone in a volume ratio of 1:(0.8 - 1.2), let it stand, remove the liquid paraffin, and dry at 20 - 30 °C for 12 - 24 h to obtain tannic acid - modified hemostatic microspheres;

[0040] (4) Mix the tannic acid - modified hemostatic microspheres and the adsorbent in a volume ratio of 1:(0.1 - 4), and dehydrate to obtain the gelatin hemostatic microspheres;

[0041] The adsorbent includes a chitosan quaternary ammonium salt solution with a mass percentage content of 0.05 - 0.3% or a calcium chloride solution with a concentration of 0.05 - 1 mol / L.

[0042] In a second aspect, the present invention provides an efficient gelatin hemostatic microsphere, and the gelatin hemostatic microsphere is prepared by using the preparation method described in the first aspect.

[0043] In a third aspect, the present invention provides an application of the high-efficiency gelatin hemostatic microspheres as described in the second aspect in the preparation of a hemostatic material for treating bleeding of body surface wounds and / or internal organ bleeding.

[0044] The numerical ranges described in the present invention not only include the above-listed point values, but also include any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. In the present invention, gelatin is modified with tannic acid through Michael addition reaction, and then a catechol structure is introduced into the gelatin through cross-linking reaction. The hemostatic microspheres are prepared by a water-in-oil emulsion cross-linking method and adsorbed, so that the hemostatic dressing can better adhere to the tissue surface, having the effects of high water absorption rate and rapid hemostasis.

[0047] 2. The preparation method of the high-efficiency gelatin hemostatic microspheres provided by the present invention is simple, easy to operate, control and mass-produce industrially; the raw materials are easily available and all come from nature, with high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Electron micrograph of the gelatin hemostatic microspheres prepared in Example 1;

[0049] Figure 2 Electron micrograph of the gelatin hemostatic microspheres prepared in Example 2;

[0050] Figure 3 Electron micrograph of the gelatin hemostatic microspheres prepared in Comparative Example 1;

[0051] Figure 4 Electron micrograph of the gelatin hemostatic microspheres prepared in Comparative Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0053] As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may also include other elements not explicitly listed or elements inherent to such composition, step, method, article or device.

[0054] "Optional" or "any one" means that the matters or events described thereafter may or may not occur, and such description includes the cases where the events occur and the cases where the events do not occur.

[0055] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirement (i.e., the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the singular forms of the elements or components also include the plural forms, unless the quantity clearly refers only to the singular form.

[0056] The descriptions of terms such as "one embodiment", "some embodiments", "exemplarily", "specific examples", or "some examples" described in the present invention mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this article, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example.

[0057] In the present invention, unless otherwise clearly stated, the percentages and percentage contents are by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased from commercial channels.

[0058] Example 1

[0059] This example provides a preparation method of gelatin hemostatic microspheres, and the preparation method includes the following steps:

[0060] (1) Dissolve 2 g of gelatin in 10 mL of deionized water, add sodium hydroxide to adjust the pH value to 10 to obtain a gelatin solution;

[0061] (2) Dissolve 40 mg of tannic acid in 2 mL of deionized water to obtain a tannic acid solution;

[0062] (3) Mix the tannic acid solution and the gelatin solution, stir at 60 °C and 250 rpm for 35 min until the solution is uniform, and keep it exposed to air to obtain a tannic acid-modified gelatin aqueous solution;

[0063] (4) Mix 1 mL of Span-80 and 50 mL of liquid paraffin, stir well and heat to 60 °C to obtain an oil phase;

[0064] (5) Slowly drop the tannic acid-modified gelatin aqueous solution into the oil phase, stir at 60 °C and 700 rpm for 45 min until uniform to obtain a mixed solution;

[0065] (6) Transfer the mixed solution to an ice-water bath, add 2 mL of 25% glutaraldehyde aqueous solution for crosslinking, and continuously stir well at 700 rpm for 30 min to obtain a reaction solution;

[0066] (7) Mix the reaction solution with an equal volume of acetone, let it stand for 10 min, then remove the supernatant liquid paraffin, wash it three times repeatedly with acetone, and then dry it at room temperature for 20 h to obtain tannic acid-modified hemostatic microspheres;

[0067] (8) Place the tannic acid-modified hemostatic microspheres in a solution of 0.2% chitosan quaternary ammonium salt with a volume twice that of the microspheres, stir well, and dehydrate with ethanol to obtain gelatin hemostatic microspheres.

[0068] Example 2

[0069] This example provides a method for preparing gelatin hemostatic microspheres, and the preparation method includes the following steps:

[0070] (1) Dissolve 1.5 g of gelatin in 10 mL of deionized water, add sodium hydroxide to adjust the pH value to 10 to obtain a gelatin solution;

[0071] (2) Dissolve 60 mg of tannic acid in 2.5 mL of deionized water to obtain a tannic acid solution;

[0072] (3) Mix the tannic acid solution and the gelatin solution, stir at 55 °C and 300 rpm for 30 min until the solution is uniform, and keep it exposed to air to obtain a tannic acid-modified gelatin aqueous solution;

[0073] (4) Mix 1 mL of Span-80 with 45 mL of liquid paraffin, stir well and heat to 60 °C to obtain an oil phase;

[0074] (5) Slowly drop the tannic acid-modified gelatin aqueous solution into the oil phase, stir at 55 °C and 800 rpm for 30 min until uniform to obtain a mixed solution;

[0075] (6) Transfer the mixed solution to an ice-water bath, add 2 mL of 20% glutaraldehyde aqueous solution for crosslinking, and continuously stir vigorously at 800 rpm for 20 min to obtain a reaction solution;

[0076] (7) Mix the reaction solution with 0.8 times the volume of acetone, let it stand for 20 min, then remove the supernatant liquid paraffin, wash it three times repeatedly with acetone, and then dry it at room temperature for 24 h to obtain tannic acid-modified hemostatic microspheres;

[0077] (8) Place the tannic acid-modified hemostatic microspheres in a solution of 0.1 times the volume of 0.5 mol / L calcium chloride solution, stir well, and dehydrate with ethanol to obtain gelatin hemostatic microspheres.

[0078] Example 3

[0079] This example provides a method for preparing gelatin hemostatic microspheres, and the preparation method includes the following steps:

[0080] (1) Dissolve 3.5 g of gelatin in 8 mL of deionized water, add sodium hydroxide to adjust the pH value to 11 to obtain a gelatin solution;

[0081] (2) Dissolve 30 mg of tannic acid in 2 mL of deionized water to obtain a tannic acid solution;

[0082] (3) Mix the tannic acid solution and the gelatin solution, stir at 65 °C and 200 rpm for 40 min until the solution is uniform, and keep it exposed to air to obtain a tannic acid-modified gelatin aqueous solution;

[0083] (4) Mix 1 mL of Span-80 and 55 mL of liquid paraffin, stir well and heat to 60 °C to obtain an oil phase;

[0084] (5) Slowly drop the tannic acid-modified gelatin aqueous solution into the oil phase, stir at 65 °C and 600 rpm for 60 min until uniform to obtain a mixed solution;

[0085] (6) Transfer the mixed solution to an ice-water bath, add 2 mL of 20% glutaraldehyde aqueous solution for crosslinking, and continuously stir well at 800 rpm for 20 min to obtain a reaction solution;

[0086] (7) Mix the reaction solution with 1.2 times the amount of acetone, let it stand for 8 min, then remove the supernatant liquid paraffin, wash it three times with acetone, and then dry it at room temperature for 15 h to obtain tannic acid-modified hemostatic microspheres;

[0087] (8) Place the tannic acid-modified hemostatic microspheres in 4 times the amount of 0.3% chitosan quaternary ammonium salt solution, stir well, and dehydrate with ethanol to obtain gelatin hemostatic microspheres.

[0088] Example 4

[0089] This example provides a method for preparing gelatin hemostatic microspheres, which is only different from Example 1 in that the acetone in step (7) is adjusted to an equal amount of absolute ethanol, and other raw materials, dosages and preparation methods refer to Example 1.

[0090] Comparative Example 1

[0091] This comparative example provides a method for preparing gelatin hemostatic microspheres, and the preparation method includes the following steps:

[0092] (1) Dissolve 2 g of gelatin in 10 mL of deionized water, add sodium hydroxide to adjust the pH value to 10 to obtain a gelatin solution;

[0093] (2) Mix 1 mL of Span-80 and 50 mL of liquid paraffin, stir well and heat to 60 °C to obtain an oil phase;

[0094] (3) Slowly add the gelatin aqueous solution to the oil phase, stir at 60 °C and 700 rpm for 45 min until homogeneous to obtain a mixed solution;

[0095] (4) Transfer the mixed solution to an ice-water bath, add 2 mL of 25% glutaraldehyde aqueous solution for cross-linking, and continuously stir vigorously at 700 rpm for 30 min to obtain a reaction solution;

[0096] (5) Mix the reaction solution with an equal volume of acetone, let it stand for 10 min, then remove the supernatant liquid paraffin, wash it three times repeatedly with acetone, and then dry it at room temperature for 20 h to obtain tannic acid-modified hemostatic microspheres;

[0097] (6) Place the tannic acid-modified hemostatic microspheres in 2 times the amount of 0.2% chitosan quaternary ammonium salt solution, stir well, and dehydrate with ethanol to obtain gelatin hemostatic microspheres.

[0098] Comparative Example 2

[0099] This comparative example provides a method for preparing gelatin hemostatic microspheres, which is only different from Example 1 in that step (8) is not carried out, and the product prepared in step (7) is the gelatin hemostatic microspheres; other raw materials, dosages and preparation methods refer to Example 1.

[0100] Comparative Example 3

[0101] This comparative example provides a method for preparing gelatin hemostatic microspheres, and the preparation method includes the following steps:

[0102] (1) Dissolve 2 g of gelatin in 10 mL of deionized water, add sodium hydroxide to adjust the pH value to 10 to obtain a gelatin solution;

[0103] (2) Dissolve 40 mg of tannic acid in 2 mL of deionized water to obtain a tannic acid solution;

[0104] (3) Mix the tannic acid solution and the gelatin solution, stir at 60 °C and 250 rpm for 35 min until the solution is homogeneous, and keep it exposed to air to obtain a tannic acid-modified gelatin aqueous solution;

[0105] (4) Transfer the tannic acid-modified gelatin aqueous solution to an ice-water bath, add 2 mL of 25% glutaraldehyde aqueous solution for cross-linking, and continuously stir vigorously at 700 rpm for 30 min to obtain a reaction solution;

[0106] (5) Mix the reaction solution with an equal volume of acetone, let it stand for 10 min, then remove the supernatant liquid paraffin, wash it three times repeatedly with acetone, and then dry it at room temperature for 20 h to obtain tannic acid-modified hemostatic microspheres;

[0107] (6) Place the tannic acid-modified hemostatic microspheres in 2 times the amount of 0.2% chitosan quaternary ammonium salt solution, stir well, and dehydrate with ethanol to obtain gelatin hemostatic microspheres.

[0108] Comparative Example 4

[0109] This comparative example provides a method for preparing gelatin hemostatic microspheres, which is only different from Example 1 in that step (6) cross-linking reaction is not carried out, and the mixture obtained in step (5) is directly subjected to step (7), and other raw materials, dosages and preparation methods refer to Example 1.

[0110] Test Example 1

[0111] The gelatin hemostatic microspheres prepared in the examples and comparative examples were subjected to electron microscopy detection. Exemplarily, the electron micrograph of the gelatin hemostatic microspheres prepared in Example 1 is as Figure 1 shown, the electron micrograph of the gelatin hemostatic microspheres prepared in Example 2 is as Figure 2 shown, the electron micrograph of the gelatin hemostatic microspheres prepared in Comparative Example 1 is as Figure 3 shown, the electron micrograph of the gelatin hemostatic microspheres prepared in Comparative Example 2 is as Figure 4 shown. It can be seen from the figure that the gelatin hemostatic microspheres prepared in Examples 1-2 can effectively adsorb salt crystals on the surface. In Comparative Example 1, the gelatin was not modified with tannic acid, and the obtained gelatin hemostatic microspheres could not adsorb chitosan quaternary ammonium salt. In Comparative Example 2, the last step of adsorption was not carried out, and the obtained gelatin hemostatic microspheres had high dispersion and smooth surface, and poor hemostatic effect.

[0112] Test Example 2

[0113] Water absorption rate test

[0114] Test method for water absorption rate: Place the gelatin hemostatic microsphere samples (m1) prepared in the examples and comparative examples with a certain mass in a petri dish, weigh the mass m2 of the sample plus the petri dish, and then add 0.9% normal saline preheated to (37±1)°C. The mass of the normal saline is 20 times the mass of the test material. Transfer the petri dish into an oven and keep it at (37±1)°C for 60 min. Take out the petri dish, carefully tilt it to pour out the excess unabsorbed water until no water droplets fall within 30 s, and then accurately weigh the mass m3 of the sample plus the petri dish with an electronic balance, and measure it in parallel 3 times. The water absorption rate (X) can be obtained by calculation. The calculation formula for the water absorption rate (X) is: X = (m3 - m2) / m1×100%. The test results are shown in Table 1.

[0115] Test Example 3

[0116] Coagulation test

[0117] (1) Experimental design

[0118] Twenty healthy New Zealand rabbits were selected and divided into six groups. Among them, 18 groups were experimental groups, and the gelatin hemostatic microspheres prepared in the examples and comparative examples were used respectively. The other 2 groups were control groups (gauze control groups).

[0119] (2) Experimental method

[0120] The specific experimental method is as follows:

[0121] 1. After the rabbits were anesthetized, they were fixed on their backs with their abdomens facing up. The abdominal cavity was opened to fully expose the liver. A wound about 1 cm × 1 cm was made on the surface of the liver with a scalpel to create a liver trauma bleeding model;

[0122] 2. After the model was established, the pre-weighed gauze (w0) was taken and quickly covered the wound surface. Press it slightly, and start timing at the same time. After 10 s, remove the gauze and weigh it (w1). The value of w1 - w0 is the bleeding volume;

[0123] 3. After removing the gauze, quickly cover the hemostatic material and cover the gauze on the hemostatic material. Press the gauze hard. During the pressing, remove the gauze every 5 s to observe the bleeding condition of the wound surface. When there is no bleeding, stop timing and record the hemostasis time.

[0124] (3) Experimental results

[0125] During the experiment, if the hemostasis time exceeded 5 min, it was judged as hemostasis failure. Each group was tested five times respectively, and the average value of each group was calculated. The hemostasis time is shown in Table 1.

[0126] Table 1

[0127] Sample Water Absorption Rate (%) Coagulation Time (s) Example 1 1197.44 175 Example 2 360.23 154 Example 3 1395.91 163 Example 4 1152.51 178 Comparative Example 1 / ＞300 Comparative Example 2 415.02 265 Comparative Example 3 / ＞300 Comparative Example 4 / ＞300

[0128] According to the data in the table, it can be seen that the gelatin hemostatic microspheres obtained by the preparation methods provided in Examples 1 - 3 of the present invention have a high water absorption rate, a short hemostasis time, and high - efficiency hemostasis; from Examples 1 and 4, it can be known that when absolute ethanol is used to wash the liquid paraffin by suction, the water absorption rate and coagulation effect are slightly worse than those of acetone. The main influence is that the drying efficiency will decrease when absolute ethanol is used in the preparation process, affecting the preparation efficiency; from Examples 1 and Comparative Example 1, it can be known that when gelatin is not modified with tannic acid, the obtained hemostatic microspheres cannot adsorb chitosan quaternary ammonium salt, so the water absorption effect and hemostasis effect are almost lost; from Examples 1 and Comparative Example 2, it can be known that when the prepared hemostatic microspheres are not adsorbed with chitosan quaternary ammonium salt or calcium chloride, the water absorption effect and hemostasis effect of the obtained gelatin hemostatic microspheres are greatly reduced; from Examples 1 and Comparative Example 3, it can be known that when the tannic acid - modified gelatin aqueous solution is directly cross - linked without cross - linking in the water - in - oil emulsion state, the water absorption effect and hemostasis effect of the prepared product are almost lost; from Examples 1 and Comparative Example 4, it can be known that if gelatin is not cross - linked and directly adsorbed, the obtained product cannot maintain the microsphere structure, and then loses the water absorption and hemostasis effects.

[0129] The applicant declares that the process of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A preparation method of an efficient gelatin hemostatic microsphere, characterized in that The preparation method comprises the following steps: (1) Mix the gelatin solution and the tannic acid solution, and stir evenly to obtain a tannic acid-modified gelatin aqueous solution; (2) Mix Span-80 and liquid paraffin evenly to obtain an oil phase; add the tannic acid-modified gelatin aqueous solution to the oil phase and stir evenly to obtain a mixture; (3) Carry out a cross-linking reaction on the mixture under ice bath conditions. After completion, mix the reaction solution with acetone, let it stand, remove the liquid paraffin, and dry to obtain tannic acid-modified hemostatic microspheres; (4) Mix the tannic acid-modified hemostatic microspheres with an adsorbent and dehydrate to obtain the gelatin hemostatic microspheres.

2. The preparation method according to claim 1, wherein The mass percentage content of gelatin in the tannic acid-modified gelatin aqueous solution is 10-30%; Preferably, the mass percentage content of tannic acid in the tannic acid-modified gelatin aqueous solution is 0.2-0.4%.

3. The preparation method according to claim 1 or 2, characterized in that The pH value of the gelatin solution ≥ 9.5; Preferably, in step (1), the stirring speed is 200-300 rpm, the time is 30-40 min, and the temperature is 55-65 °C.

4. The preparation method according to any one of claims 1-3, characterized in that, The volume ratio of the liquid paraffin to Span-80 is 1:(45-55); Preferably, the volume ratio of the tannic acid-modified gelatin aqueous solution to the oil phase is 1:(3-5); Preferably, in step (2), the stirring speed is 600-800 rpm, the time is 30-60 min, and the temperature is 55-65 °C.

5. The preparation method according to any one of claims 1-4, characterized in that, The cross-linking agent used in the cross-linking reaction is an aqueous glutaraldehyde solution; Preferably, the mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 20-30%; Preferably, the stirring speed during the cross-linking reaction is 600-800 rpm, the time is 20-40 min, and the temperature is 0-4 °C.

6. The preparation method according to any one of claims 1-5, characterized in that, The volume ratio of the reaction solution to acetone is 1:(0.8-1.2); Preferably, the standing time is 5-20 min; Preferably, the drying temperature is 20-30 °C and the time is 12-24 h.

7. The preparation method according to any one of claims 1-6, characterized in that, The adsorbent includes a chitosan quaternary ammonium salt solution or a calcium chloride solution; Preferably, the mass percentage content of chitosan quaternary ammonium salt in the chitosan quaternary ammonium salt solution is 0.05-0.3%; Preferably, the concentration of calcium chloride in the calcium chloride solution is 0.05-1 mol / L; Preferably, the mass-to-volume ratio of the tannic acid-modified hemostatic microspheres to the adsorbent is 1:(0.1-4).

8. The preparation method according to any one of claims 1-7, characterized in that, The preparation method comprises the following steps: (1) Mix the gelatin solution with a pH value ≥ 9.5 and the tannic acid solution, and stir at 55-65 °C and 200-300 rpm for 30-40 min until uniform to obtain a tannic acid-modified gelatin aqueous solution; The mass percentage content of gelatin in the tannic acid-modified gelatin aqueous solution is 10-30%, and the mass percentage content of tannic acid is 0.2-0.4%; (2) Mix Span-80 and liquid paraffin in a volume ratio of 1:(45-55) evenly to obtain an oil phase; add the tannic acid-modified gelatin aqueous solution in a volume ratio of 1:(3-5) to the oil phase, and stir at 55-65 °C and 600-800 rpm for 30-60 min until uniform to obtain a mixture; (3) Crosslink the mixture with an aqueous glutaraldehyde solution with a mass percentage of 20 - 30% under ice bath conditions, stir at 0 - 4°C and 600 - 800 rpm for 20 - 40 min. After crosslinking is completed, mix the reaction solution with acetone in a volume ratio of 1:(0.8 - 1.2), let it stand for 5 - 20 min, then remove liquid paraffin, and dry at 20 - 30°C for 12 - 24 h to obtain tannic acid modified hemostatic microspheres; (4) Mix the tannic acid modified hemostatic microspheres with an adsorbent in a volume ratio of 1:(0.1 - 4), and then dehydrate to obtain the gelatin hemostatic microspheres; The adsorbent includes a chitosan quaternary ammonium salt solution with a mass percentage of 0.05 - 0.3% or a calcium chloride solution with a concentration of 0.05 - 1 mol / L.

9. An efficient gelatin hemostatic microsphere, characterized in that, The gelatin hemostatic microspheres are prepared by the preparation method described in any one of claims 1 - 8.

10. Use of an efficient gelatin hemostatic microsphere as described in claim 9 in the preparation of a hemostatic material for treating bleeding of body surface wounds and / or internal organs.

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